Disaster: The Nepal Earthquake

Disaster: The Nepal Earthquake Major Event: Disaster The Nepal Earthquake ; Figure 1: Mounting damage Presented by: Steven Eu Su Shian   PLAN INTRODUCTION A tragedy happened two years ago on April 25th, 2015 in an Asian country called Nepal. It also; known; as The Nepal Earthquake. It caused a lot of difficulties to the Nepalese citizens and also it drew a lot of attention from the people around the world. The purpose of this report is to provide; information about The Nepal Earthquake. This report will pay particular attention to the effects after Nepal Earthquake happened, the economic impact and also the effects on the Nepalese citizens. METHODOLOGY The information for this report were all found from the Internet. Besides, there will be some recommendations in the concluding part. MAIN POINTS A) The effects after the earthquake happened After the earthquake happened, Revd Lewis Lew described that the effects of the tremendous 7.9 magnitude that happened in the 80 km far in northwest of the capital city, Kathmandu and the after-effect after an hour were shocking. Besides, according to the National Emergency Operation Centre, at the minimum 3617 people were killed by the earthquake, and also above 6500 people were injured during the earthquake. On the other hand, more than hundreds people from the neighbouring countries such as China, India and Bangladesh were also killed by the earthquake. Moreover, huge damage to the assets and the substructures such as banks has been reported in Kathmandu and the distant villages, access to which has been split by the mudslides. Furthermore, according to Dean Revd Lewis Lew, plenty of buildings collapsed, particularly those in old Kathmandu City. And also, a lot of survivors were in bad shape, they were all waiting for the medical treatment. ; Figure 2: Hospitals in the street B) The economic impact The early evaluations of the economic impact caused by The Nepal Earthquake were astonishing. The total of the economic impact in Nepal is evaluated to be above $10 billion USD, which is also 50 percent of the countrys gross domestic product-GDP (https://qz.com/409848), according to the Nepalese government. Next, the Nepalese government could also spend as much as 5 billion USD or even more than that as the budget for the different kind of infrastructures such as houses, highways or bridges. On the other hand, the damages and destruction of the apartment buildings and houses caused by the earthquake has had a serious impact. The rentals, housing price and the land price could possibly affected by; the earthquake . For the examples the house prices might increase, and also the rental distension could also happen in Nepal. It might also increase the burden of the citizens in the future. C) The effects on the citizens After the tremendous earthquake that happened on April 25th, 2015 in Nepal, according to the United Nations (www.bbc.com/news/world-asia-32492232), eight million Nepalese citizens were affected, which was more than 25 percent of the countrys population. Besides, eight million people from 39 different areas were affected, and the major problems were lack of food, water and electricity supplies. Furthermore, according to an American doctor Rebecca McAteer (www.bbc.com/news/world-asia-32492232) majority of houses were destroyed. Not only that but also most of the citizens were being displaced and the young people had to leave to find the work. Moreover, the camps were also set up on a park, with the rubbish around such as paper plates, packages, and plastic glasses. One of the men who was living there with his spouse and his four children said that they were not going home even though they were still able to live in their house. He explained the reason why they refused to live in their house was because of they have heard the stories about more earthquake and after-effects. In addition, the hospitals were also not able to handle the massive numbers of the citizens who were in need of the medical treatment. And this caused some of them started to complain on the medical service. ; Figure 3: Cremating victims CONCLUSION In conclusion, it would be useful if we could know more about the Nepal Earthquake that happened on 25th, 2015. As the report mentioned earlier, Nepal Earthquake has caused a lot of troubles and difficulties to the Nepalese citizens. For example it caused massive numbers of death, food, water and electricity supplies shortage, economic impact and also the displaced problems. From the information, perhaps the Nepalese government can begin to implement some methods to prevent the next potential earthquake .For instance the government can organise a conference about an earthquake such as what should the citizens do during the earthquake, or government can also begin to plan a better evacuation plan for the citizens so that the government and the citizens can be well prepared for the next earthquake and minimize the effection after an earthquake. Bibliography Effect of earthquake devastating report Anglican Deanery of Nepal. Retrieved March 7, 2017. From www.anglicannews.org/news 2015/04/effects-of-earthquake-devastating-report-anglican-deanery-of-nepal.aspx Karnik,(2015,May). The economic damage from the Nepal earthquake is almost half of the countrys GDP. Retrieved March 7,2017. From https://qz.com/409848 Nepal earthquake: Eight million people affected, UN says. Retrieved March 7, 2017. From www.bbc.com/news /world-asia-32492232 Figure 1. Damaged houses. Adapted from: ,20 Pictures reveal Nepals heartbreaking Earthquake devastation, By Nicole Werbeck, and Mallory Benedict, 2015, April 26th, National Geographic, p.1. Copyright 2015 by National Geographic. Figure 2. Survivor after the medical treatment. Adapted from: 20 Pictures reveal Nepals heartbreaking Earthquake devastation, By Nicole Werbeck and Mallory Benedict, 2015, April 26th, National Geographic, p.19. Copyright 2015 by National Geographic. Figure 3. Heartbreaking moment for the victims family. Adapted from: 20 Pictures reveal Nepals heartbreaking Earthquake devastation, By Nicole Werbeck and Mallory Benedict, 2015, April 26th, National Geographic, p.8. Copyright 2015 by National Geographic. Saudi Pak Commercial Bank: Analysis Saudi Pak Commercial Bank: Analysis THE VISION To transform the Bank into a modern, dynamic and premier service oriented institution. THE MISSION â€Å"To turnaround the Bank into a leading commercial bank by ensuring transparency, promoting technology, improving quality of human resource, providing premium services to customers, and adding value for all stakeholders.† Statement Of Ethics And Business Practices: The code of ethics established by Saudi Pak Commercial Bank Limited sets forth the guiding principles through which we operate and conduct our daily business with our shareholders, customers, vendors and with other group companies. These principles also apply to the officers and employees of the Bank Our Customers: We operate as customer oriented Bank and serve our customers with the highest standard of skill and service In dealing with our customers, we comply with legal, statutory and regulatory requirements We ensure transparency in operations. Our Shareholders We strive to maximize the value for our shareholders and minimize the risk of dilution in the value of shareholders through adequate risk management. Our Staff Our staff observes a high standard of integrity and demonstrates befitting conduct and behavior. In performing their duties, our staff shows sense of responsibility and team spirit. Concerted efforts are made to promote congenial corporate culture in the Bank. Credit Operations. We conduct the credit operations under clearly spelt out policies and procedures and review the policies and procedures periodically to suit changing conditions. Our credit operations are transparent and the credit decisions are made with prudence. We keep adequate provisioning against doubtful loans as per State Bank of Pakistan guidelines and ensure that the internal controls are in place and are observed in right earnest. Risk Management: We have designed and placed a proper system to identigy, measure and control the material risks. Social Responsibility: We recognize our wider social responsibility in terms of the contribution we can make to the well being of the communities in which we operate. We do not make donations to or extend any financing to or take any exposure towards any political parties. Maintaining Confidentiality: We attach great importance to safeguarding the confidentiality of data concerning the its customers and other business relationships. We do not disclose information about our customers to third parties without the customers consent unless there is a clear legal or regulatory obligation to do so. CHAPTER # 2 REVIEW OF DIFFERENT DEPARTMENT Car Financing Scheme As all banks are moving towards consumer Banking. Saudi Pak Commercial moves towards consumer Banking through Car Financing Scheme. They start Car financing Scheme in the month of June to Satisfy the needs of the customer. Competitor: As Mirpur (AJK) is the only well flourished industrial city of Azad Kashmir, so there is large variety of Financial Institute and Banks and the changing attitude of banks towards consumer banking is one of the major reason of competition. Some of the major competitors are Bolan Bank Citi Bank PICIC Commercial Bank Faysal Bank Prime Bank MCB HBL Out of these banks major threat was from HBL, Bolan Bank and Faysal Bank. Usually the customers look forward for low Markup rate, so these banks were giving low markup rates, but they were also charging the hidden charges. Saudi Pak Commercial Bank Car Financing Scheme: As the rising demand in the market, Saudi Pak Commercial Bank Limited start the car financing scheme in month of June. Down Payment Options: Saudi Pak Commercial Bank Limited gives the large variety of down payments. The range of down payments varies from 10% to 60% to original value of the car. Secondly the loan amortization schedule can vary between 12 months and five years time period. So you has the large variety of options to pay the installments according to your convenience. One of the most important and plus point of Saudi Pak Commercial Bank car financing scheme is that they offer the variety of cars which are available in the market such as: Toyota Honda Suzuki Hyundai Fiat Nissan Saudi Pak Commercial Bank Limited offers the free insurance to their customer Age Limit: The age limit to apply for car financing Scheme is between 21 and 50. this is only for individuals. Eligibility Basically Saudi Pak Commercial Bank divides its customer in three groups on the basis of probability of the consumer to pay back the installments within time Group A: Group A usually include the executive class or regular premium customer of the banks and well known organizations. In business class the customer who are including in Group A are working in the specific profession or kind of business from the last five years and their mostly income is at least Rs. 30,000/-. Instead of business class we also include the high authority Government servants in this class. Those whose scale is above eighteen. The people who are including in Group A usually get loan more easily because they are consider the most reliable customer. Group B: Group B includes the customer who are running the individual business and small organization and they are working in that field from the last two years. In that group usually includes the shopkeepers and small organization such as cable operator Group C: Group C is the group which we usually dont prefer to give the loan and the chances of recommendation of their loans are very low, usually customers who belongs to that group are businessman having income less than Twelve thousands and organization such as Rent A Car Services. Documentations: The following documents are required Application Form ID Card photo copy Bank Statements Three years proof of tax return Salary Slip ( For Salaried Person) Process: Step 1: Filling Application Form: The first step involve in applying for Car Financing Scheme is to fill the application form along with the documents mentioned above. Customer should fill the application form very carefully. They should mentioned right data about themselves, any wrong or incorrect data may reject the application form. Step 2: Approval of Application Form from the Branch: Second step is the approval of application form from the branch. Bank officer examine the application form. If the customer fulfill the requirements mentioned above bank accept the application and send this application to the head office for further approval. Step 3: Delivery of Car: If the head office find no objection in application form and eligibility criteria of car financing. They send approval letter to the bank. After the approval car delivered on the basis of availability of car in the showroom. If the customer financed the car on the market price basis than the bank deliver the car just after the approval otherwise on the availability. Deposits Department Deposit is the main functional unit of any commercial bank. It is the primary function of commercial bank. Main function of commercial bank is to get money from the customer and give some markup on that amount. Two types of deposits are offered by the Saudi Pak Commercial Bank. Call Deposits: These are payable on demand. They include current account, sundry deposit (e.g. margin account) and call deposit receipt. No profit is given on demand deposits. Time Deposit: Payable on demand with certain maturity. Attracts profit with respect to time. TYPE OF ACCOUNTS: Let us now turn to procedures to be followed in cases of each type of account. Individual Account: Such accounts may be classified and gentlemen Account of literate ladies and gentlemen. Account of illiterate ladies and gentlemen. Account of Parda observing ladies. Joint Account. Minor Account 1: In case of illiterate ladies and gents, the following precautions are observed in addition to those provided in the above guidelines. Two photographs are to be obtained. One to be pasted on account opening form and the other on specimen signature card Instead of signatures, left had thumb impression to be obtained on the specimen card from gents and right hand thumb impression from the ladies. Each time such customer should attend the bank personally and will put their thumb impression on the cheques before the passing officer. Such customers should be advised not to issue cheques payable to 3rd parties. Cheque should be marked â€Å"Payment in person† to ensure even if the cheque is presented through clearing that particular cheque can only be paid in person. 2: the problems arise particularly in case of parda observing ladies. Some serious complication are involved in this concern. As for as possible, they should be encouraged to open a joint account with their close family members. 3: when more than one person opens account but the relationship between them is neither of trustees nor partners, it would be termed as joint account. Whenever such account is opened, definite instructions regarding operations on the accounts and payment of balance in case of death of any one of them should be obtained. In absence of any instructions for the accounts operations, all the joint account holders should sign the cheques for withdrawal of amount from their accounts. Likewise, definite instructions must also be obtain for payment of balance in the account in the event of death of any of the joint account holders. For this purpose all the joint account holders are required to sign account opening form as well as either or survivorship declaration. In operation of joint accounts, following important points are required to be remembered: Any member of the joint account may lodge stop payment instruction of any cheque with the bank and the bank shall honor such instructions. However, all the members must sign removal of these instruction. The member of joint account may wish to delegate authority to any third person to operate upon the accounts. However such a mandate is necessarily to be signed by all the members. Any mandate, reference to which is given herein 2ne above, becomes automatically rescinded or cancelled when the bank come to know of death, insolvency or insanity of any of the members In case, any of the members of the joint account becomes insolvent or insane operation on the account should be stopped and instruction to be required for payment of the balance amount from the remaining solvent and same members. In case, any of the member of the joint account dies, operation on the account must be stopped and balance in account is to be paid as per instructions recorded with the bank. Accounts of Partnership Firm: While opening accounts of the partnership firm, the partnership deed from registered firms is required to be obtained in addition to account opening form and specimen signature card. The partnership letter is incorporated in the account opening form , which must also be signed by all the partners of the firm weather registered or un-registered. In these accounts, the following points are required to be remembered. For Example: The account opening form must be signed by all the partners. The names of persons authorized to operate the account must neatly and correctly given in the account opening form. For partnership concern carrying on the business under impersonal names, it is generally described that the title of account should show name of the partners of Managing Partners. CASH DEPARTMENT Two most important tasks of bank are performed in the cash department and they are Payments Receipts In Payments cheques are presented to the bank against which payment is made. In Receipts, money is deposited with the bank and the bank issues a receipt against it. PROCEDURE Step I Customer presents his cheque over the counter, the person receiving it checks whether it is Drawn on a particular branch Date is correctly written on the cheque it should be neither stale nor post-dated. Amounts in words figures match. Duly signed by the payee on the front and back of the cheque. Step II After going through step `1 the cheque is sent to another person who verifies the signatures of the customer with his/her signatures on `SS card. Step III The cheque is sent to another officer who checks the account to see whether the account has sufficient balance to meet the payment or not. If the amount is more than Rs.10,000 then the cheque is approved by two persons they are Department Incharge Operational Manager If the amount is more than Rs.1000000 then the cheque is authenticated by three person they are Department Incharge Operational Manager Chief Manager Crossed Cheque These cheques are stamped with payees account only. This cheque is not directly paid on the counter, rather the payment is made through account of the customer to the one whose name is mentioned on the cheque. SCHOOL COMPANY BILLS Fees and dues submitted by companies and schools are in the form of bills. All these bills are credited to the bank. POSITION SLIP Some companies school have been given overdrawn facility. This slip is attached to those cheques of those schools and companies when officer has a doubt about paying those cheques. This position slip is attached to these cheques to inform the manager about their current position situation. Then if chief manager approves it then the payment is made otherwise not. CHEQUE RETURNED MEMO If the cheque presented by customer is returned because of the reason that the account does not has the required balance then the customer has this facility that he can submit cheque returned memo. But this memo is given to him on his own request. INCIDENTAL CHARGES `CD account should have a minimum balance of Rs.10,000. If in the account during six months at any time the balance of account goes below Rs.10,000. Then incidental charges of Rs.200 are charged from the customer. RETURN OF A CHEQUE A cheque is returned to the customer if the account balance is less than the amount of money demanded. ISSUANCE OF BANK STATEMENT The bank statement shows the overall position of an account at a particular date. Bank statements are issued to customers as per their request. For this customer has to give his account number and specific period for which he wants to have this statement. CASH INSURANCE All the cash on the counter and in the locker is insured one of the most important responsibilities of cash department is to manage liquidity, but not to keep idle cash with it. Cash is kept according to branch requirement, which in Saudi Pak Commercial Bank MirPur is Rs.10,000,000. Amount of money exceeding it is sent to main branch which given interest on it. This interest is added to branchs profit. Here `2 to `3 million is sent to the head office. A large number of customers come to this department during working hours. Therefore staff should be cooperative and helpful. REMITTANCE DEPARTMENT â€Å"It is a process by which the amount of cash is transferred from one place to another either in or out of the city but not in the form of cash but through the involvement of two banks† Demand Draft: It is the request of one bank to other bank to pay a certain sum of money to or to the order of the person whose name is mentioned in DD. It is used for funds transferred outside the city. Telegraphic Transferred: It is also a request b one bank to another bank to pay a certain sum of money to or to the order of the certain person it is used for funds transferred out of city. Pay Order: It is a request which is made to the other bank to pay the amount of the person whose name is mentioned there. It is within the city. When the person has transfer funds to another person in the city or out of city then he comes to the bank and gives the application on the specified form called Remittance From. This form contains name of branch, date, bank branch name beneficiarys name, account number, bank branch name, city, mode of payment in which the payment is received by other option given for DD, TT, PLO or SBP cheque the particulars of the purchaser i.e sender and his address is also written. Then on the lower side a column for instruments number (which is to be issued, its amount and the rate is there. Then another column is for cost, telex changes, commission, postage, excise duty, with holding tax, and total is there and in the last applicants acknowledge is received. And the signatures of two authorized persons are there. After this an entry is made in the system and is authenticated by another person. After that for TT or DD advices is printed by the printer and with the form it is sent to the beneficiary bank and incase of pay order a specified form is for this purpose which is used and on this the print is made and is handled over to the customer and an advice is sent to the beneficiary bank and the customer can take money from that bank. The account of the beneficiarys banks is settled through entries in books with SBP. CLEARING It is process through which one may receive the amount of a cheque presented to the bank while the cheque is drawn on any other bank. Procedure: First of all in the morning the banks representative goes to the NIFT office andreceives the cheques which are drawn upon the branch but are presented in any other branch of the same bank of the same bank or any other bank. The main Branch receives cheques of other branches, which are in the Lahore City.Then in the branch, these cheques are sorted out on the basis of parties and the cheques of parties who are the credit arrangements, are sent to the Credit Deptt. for their confirmation or cancellation. But before sending to this, their signatures, amounts in words and figures are verified and dates are checked and the clearing stamps of the bank in which they were presented in seen and checked. The clearing stamps bears the date which is to come next date because the cheque are presents next day on the payee bank. In which these cheques are deposited and his signatures are seen there. Then the cheques which have no mistakes are entered in the system. The cheques which have an error i .e. , not having sufficient balance, post dated, out of date, or due to any other reason i.e. the sending or presenting bank may not properly present the cheques, the return slips are made for these cheques and on the slip, the main Branch name, Cheque #, and amount in works and figures in written. Then the entry in the return register is made and the signatures of the authorized persons are affixed on the register and on the return slip. In the register the reason of the cheque is also given and the slip contains number wise reason which are also marked like this (X) then the cheques which are sends Accounts department in the forms of supplies and the treatment with thee cheques is made here. The cheques, which are returned due to any reason, are returned to the presenting bank which returns these to the customers. Above is the procedure of cheques of main branch which are presented in other branches, now I come to the point where the cheques of other branches a presented in our branch. OUTWARD CLEARING: When one of our customers receives cheques of another bank or branch, but does not have any account in that branch. Then he deposits the cheque in his account through a credit slips and receiving the slip and cheques of other person, an entry in the system is made and stamps containing payees account only and clearing stamps bearing the next coming date is affixed on both the slip and on the cheques and, on the back the stamps of authorized person and his initials are made then all thee cheques are presented to another person for slip authenticated and verification it is because the error chances may be made minimum and at the time of closing the cheques are separated form the credit slips and, are attached with the photocopies of the cheques and are kept for banks records.And the cheques are handed over to the SBP through NIFT. And on the next daythe cheques go to their respective banks and if those banks are not satisfied then the cheques are come back and again are entered in the register on which, the bank in which these were drawn and these are presented is written and reason for their return is mentioned and are handed over to the customers by taking their signatures. INWARD CLEARING Signatures are necessary in case if less than 10000 one authentication less than 500,000 double by BOSS more than 500,000 third authentication which is by branch manager. The total of these cheques are entered in the suspense A/C debit the A/C of our customer the amount in the suspense A/C goes on reducing and at the end it shows the zero balance when the returned cheques are also dealt with the returned memos are made for cheques which are to be entered in the registered, date, name of presenting bank, cheques number, account number and amount is written and are signed by person memo is also signed and the total of those is made and the summary is made and these cheques are send to shift office with proper seal. When the inward clearing comes the SBP gives debit to our bank and when the cheques are returned dishonored a credit is given. The reverse is the case with outward clearing. Rs.250/- are received as charges on same day clearing and it is for the cheques of more than or equal to Rs.500,000/- and the cheque must be deposited before 10:00 am. Foreign Currency Department The department which makes the transactions of foreign currency and all related matters which are dealt in local currency. A form named as form M is used to maintain the record of the foreign currency dealt by this department because it is the requirement of SBP on monthly, quarterly, half yearly and on yearly basis. Here is a list of functions and -activities of the department; Functions: Foreign Currency Account Opening Government securities Issuance of Exchange Entry to Daily Exchange Rates Foreign Currency FDR/NDR/NDR-III Activities: Foreign Currency Account Opening: Checking of Documents before Account Opening Dispatching Letter of Thanks to Account Holders Dispatching Letter of Thanks to Introducer Issuance of FCY Cheque Book Recovering Provincial Tax on Cheque Book Issuance Account Closing Government Securities: Issuance: Special U.S. Dollar Bonds Receiving Application Verifying Signature and Checking Balance To Debit the Account Stock Out Entry in Stock Register Preparation of Balance Certificates Delivery to Customer after Affixing Required Stamps Sending Sale Statement to SBP through Karachi Branch Encashment: Special Original Instrument Checking of Instrument Affixing Encashment Stamps Preparing Debit Cash Voucher for Payment to Customer Making Payment from Suspense Account Reversing Suspense Account on Receiving Credit from SBP Issuing Encashment Certificates FEBCs (Foreign Exchange Bearer Certificate): Receiving Original Instrument Checking of Instrument Affixing Encashment Stamps Preparation of Debit Cash Voucher for Payment Making Payment from Suspense Account Deducting Challan Forms Surrendering Tax to SBP Preparing Encashment Reports Reversal of Suspense Entry on Receiving Credit from SBP Issuance of Encashemnt Certificate FCBCs (Foreign Currency Bearer Certificates): Same Steps followed as described above. Special U.S. Dollar Bonds: Preparation of Profit Coupon Affixing Stamps required by SBP Payment of Debit Cash Voucher Preparation of Profit Certificate for SBP Preparation of Profit Payment Report Reversal of Suspense Account on Receiving Credit from SBP Foreign Currency Bearer Certificates: Same Procedure followed as described Supply of Stock: Supply of Stock to Branches Supply of Profit Coupon Books to Branches Foreign Exchange Issuance: Checking of Documents Making Photocopies of ID Card, Ticket Passport Affixing Stamps on Ticket and Passport Obtaining Signature of Customer on TCs Receiving Payment to Debit the Account Delivery to Customer Entry in Stock-out Register Reversing Contra Liability Foreign Currency FDR/NDR/NDRP-III Encashment of FDR/NDR/NDRP-III Quarterly Payment of Profit on NDRP-III CUSTOMER SERVICE DEPARMENT Customer services department is the department which keeps the customer needs fulfilled. Because the customers may feel problems in doing transactions with bank. So the customer service people keeps them aware of the customer needs.Two special posts of CRM and CRO are there and they all time ready to serve the people who come to bank. Personal Bankers are also there who ready to free the customers from their problems.CRM and CRO provides the people facility to know their account balances and to know the comments of the people about the performance of other departments. Special comment forms are used to know the views of the people about the performance of various personals of other departments. Another comment form is used to check the time which is spent on the encashment of a cheque through the cash department. The time starts when the token is received by the customer and ends when the payment is received. Daily 10 such forms are prepared and sent to management to know the efficiency of cash department people. Arrangements are made regarding the availability of written and printed material to the people who come in the bank. Daily reports of the accounts opened with the branch is made and is delivered to branch manager and efforts are made to increase the balance. Frequent meetings are held with Branch Manager to take fresh instructions and to get feed back to the management about the performance of personal bankers. For the service of people there is free offer of local phone calls to the customers. And their problems regarding their balances, Cheque Books, various documents and such other matters. In order to further reinforce our commitment towards priority service to our customers we have decided on the following: CREATION OF A PRIORITY BANKING AREA: The area currently occupied by the CRM, Account opening and personal bankers will be designated as the â€Å"Priority Banking Area†.CRM, Account opening and one priority banker will occupy this area. The Priority Banking Area will be exclusively for Priority Customers who not only hold a substantial amount in terms of deposit with the bank but also demand individual attention. PURPOSE OF PRIORITY BANKING: The basic purpose of priority banking is to provide a valued customer with a pleasant atmosphere in which all his banking requirements are met in the shortest time span possible. WHO IS A PRIORITY CUSTOMER? Individuals who retain an average deposit of 0.5M or above qualify as being a priority customer. However department Heads of various companies who have their corporate accounts with us also qualify. LEVEL OF SERVICE TO BE PROVIDED: As the name depicts the utmost level of priority is to be provided to priority cases. The code for Priority Customers is â€Å"PC† and its notification should ensure the highest level of priority, no matter which department it involves. ROLE OF BOSS, CRM CRO: Initially the CRM along with the Priority Banker are responsible for providing the priority customer with quality service. If another department is involved the Priority Banker will inform the head of the department or Boss with the status of the customer and what is needed. Whoever it is will then help and try to complete the process in the least possible time. RESPONSIBILITIES OF PRIORITY BANKER: Being on the look out for priority customers who enter. Approaching a customer and being the first to initiate a conversation by inquiring about his reason of visit. Handling of Priority Calls. Entertaining the customer with drinks while his visit. Assisting the CRM in his tasks of customer services. Providing the customer with all relevant information regarding his status with the bank. Making sure the Priority Area is clean at all times. Giving each and every customer individual attention. Maintaining a register of all complaints and reporting them to the Branch Manager. Providing the customer with several kinds of reading material to pass his time. Informing a personal banker if a specific client wants to meet him. GENERAL CUSTOMER SERVICE STANDARDS Always be courteous to the customer. Imagine yourself in the customers position and then start to service him the way you would want to be. Give full attention to the person sitting across your table. Do not engage in personal conversation over the phone or with another staff member while dealing with a customer. The job at hand must be dealt with utmost efficiency. If the need to leave a customer arises, explain to him why and return as soon as possible. Atleast one staff member should be present in all the departments at all times, even during lunch hour to accommodate a customer. Talking to a customer or staff member in a loud tone is to be avoided at all costs. If a certain staff member is to be called calling him out loud should be avoided, instead calling at his or at a nearby extension should be done. Be sure of what you are saying. Make sure that you know what you can and what you cant do for a customer. Stick by the commitments you have made to a customer. Give the customer a magazine or newspaper to kill time while he is waiting. Make sure that the customer knows that his work is being taken seriously. Is there such thing as complete synonymy? Is there such thing as complete synonymy? Complete synonymy is rare, and absolute synonymy hardly exists. Lyons (1981:148). Fromkin et al. (2003: 181) state that no two words ever have exactly the same meaning.. These quotations seem odd and unfamiliar to many people in general and I in particular. It is conventionally known that there are many synonyms in the lexicon sharing the same meaning. If a teacher asks one of his students what the opposite of the adjective big is, the student, based on his previous knowledge, will directly answers large. Languages in general- as to speak- have many synonyms, particularly the English language. It is rich in many examples such as plentiful and rich, pretty and attractive, combine and mix, student and pupil, sick and ill, happiness and joy and many others, just to name a few. These words share the same denotation- literal meaning which makes them synonyms and can be used as substitutes for each others to avoid repetition in writing and speaking. As to the complexity of meaning, a perso n looking for replacing a word with another word must choose a precise and accurate synonym. In this regard, many semanticists have presented studies on synonymy from different perspectives. Thus, there is a consensus regarding the difficulty of finding two perfect, absolute or complete words sharing the same synonymy. Semanticists have attacked the translation of words in two different languages as these words cannot mean exactly the same because of the different linguistic and social contexts they occur. But what about two synonyms in the same language?. The rarity or impossibility of perfect synonymy can clearly be discussed through the definition of synonymy, types- scale of synonymy and conditions of perfect synonymy, substitution tests and reasons of rarity. Defining synonymy is a difficult process. Maja, (2009) has argued that when it comes to giving a clear, precise and correct definition of synonymy, many difficulties arise. There are many approaches with many definitions of synonymy and types of synonyms because there are different ways in which synonyms may differ. Maja, (2009) has defined synonymy as the phenomenon of two or more different linguistic forms with the same meaning. Those linguistic forms are called synonyms, e.g. danger and risk can be substituted with one another in certain contexts. Synonymy in semantics is an inter-lexical sense relation. Synonymy is sameness of meaning (Palmer F. R. 1996:88, Lyons John 1996:60). Fromkin et al. (2003:181) has stated that: there are words that sound different but have the same or nearly the same meaning, such words are called synonyms.. John (1995) has also presented a definition indicating that expressions with the same meaning are synonyms. Two important points should be noted abo ut the definition. Firstly, it does not restrict relation of synonymy to lexemes; it allows for the possibility that lexically simple expressions may have the same meaning as lexically complex expressions. Secondly, it makes identity, not merely similarity, of meaning the criterion of synonymy. It is noteworthy that all linguists and semanticists such as Palmer, Lyons and Fromkin agree that synonymy means two words with the same meaning. I completely agree with these definitions from the perspective of sameness. However, I feel that such synonyms may resemble in meaning but they would differ in formality, style, or of some other aspects of connotations. All in all, the definition of synonymy is still a controversial subject among semanticists and difficult to find a specific definition for synonymy. The scale of synonymy is important for all to figure out the relationship between two synonyms. Cruse (2000:157) claims that a scale of synonymy can be established. The scale consists of absolute synonymy, cognitive synonymy and near-synonymy. First, absolute synonymy is set as the complete identity of all meanings of two or more lexemes in all contexts. However, it is unnatural for a language to have absolute synonyms, or lexemes with exactly the same meaning. It is generally accepted that absolute synonymy is impossible or non-existent. It is regarded only as a referential point on the alleged scale of synonymy or the initial criterion for the defining of synonymy (Cruse, 2000, 157). Second, as there are no two lexemes with absolutely the same meaning and no real synonyms, cognitive synonymy is what most semanticists would regard as synonymy. Lyons (1996:63) claims that many theories of semantics would restrict the notion of synonymy to what he calls descriptive or cognitive synony my, which is the identity of descriptive meaning. Third, near-synonyms are lexemes whose meaning is relatively close or more or less similar (mist/fog, stream/brook, dive/plunge). However, the given definition of near-synonymy is vague, because there isnt a precise correlation between synonymy and semantic similarity. Near-synonymy is associated with overlapping of meaning and senses. The senses of near-synonyms overlap to a great degree, but not completely (Murphy, 2003, 155). Moreover, unlike cognitive synonyms, near-synonyms can contrast in certain contexts: He was killed, but I can assure you he was NOT murdered, madam (Cruse, 2000, 159). Near-synonymy is regularly found in dictionaries of synonyms or thesauri where most of the terms listed under a single dictionary entry are not considered to be cognitive synonyms (e.g. govern direct, control, determine, require). The scale presented by Cruse is the most general. There are also other views. Lyons (1981:148) claims that there are absolute synonymy, complete synonymy, descriptive synonymy and near-synonymy. Noticeably, there is a new type compared to Cruse. According to Lyons (1981), complete synonyms must have the identity of all descriptive, social and expressive meaning in all contexts. Since most lexemes are polysemous- have different senses in different contexts, Murphy (2004:146) introduces logical synonyms- which include full synonyms and sense synonyms and near-synonyms. Denotationally equivalent words, whose all senses are identical such as (toilet/john), are called full synonyms, whereas sense synonyms share one or more senses, but differ in others, i.e. they have at least one identical sense (sofa/couch). Near-synonyms, as words with similar senses, are context-dependent. Cognitive synonyms are arguably what Murphy (2003) regards as sense synonyms. At last, there are many types of syn onyms proposed by linguists and semanticists regarding the types of synonymy. By now, it is almost true that absolute synonymy is extremely rare- at least a relation between lexemes- in natural languages. According to John (1995), two or more expressions are perfectly or absolutely anonymous if, and only if, they satisfy three conditions. First, all their meanings are identical. In other words, standard dictionaries of English treat the adjectives big and large as polysemous. For instance, they live in a big/large house. The two words would generally be regarded as synonymous. However, it is easy to show that these adjectives are not synonymous in all their meanings: i.e., that they fail to satisfy condition (1) and so are only partially, not absolutely or perfectly. I will tell my big sister is lexically ambiguous, by virtue of big; in a way that I will tell my large sister is not. All three sentences are well-formed and interpretable. They show that big has at least one meaning which it does not share with large. Second, they are synonymous in all contexts. The main issue here is what we call collocations- a set of contexts where an expression can occur. It might be thought that the collocational range of an expression is wholly determined by its meaning, so that synonyms must of necessity have the same collocational range. But this does not seem to be so. For example, big and large can be used as a good example. There are many contexts in which big cannot be substituted for large (in the meaning which big shares with large) without violating the collocational restrictions of the one or the other. For example, large is not interchangeable with big in: you are making a big mistake. The sentence you are making a large mistake is not only grammatically well-formed, but also meaningful. It is however collocationally unacceptable or unidiomatic. And yet big seems to have the same meaning in you are making a big mistake as it does in phrases such as a big house, for which we could, as we have seen, substitute a large house. It is attempting to argue, in cases like this, that there must be some subtle difference of lexical meaning which accounts for the collocational differences, such that it is not synonymy, but near-synonymy, that is involved. Third, they are semantically equivalent i.e., their meaning or meanings are identical on all dimensions of meaning, descriptive and non-descriptive. The most widely recognized dimension of meaning that is relevant to this condition is descriptive or propositional meaning. I think it is sufficient to say that two expressions have the same descriptive meaning if propositions containing the one necessarily imply otherwise identical propositions containing the other, and vice versa. By this criterion, big and large are descriptively synonymous (in one of their meanings and over a certain range of contexts). For instance, one cannot assert that someone lives in a big house and deny that they live in a large house. Another example is between the words bachelor and unma rried. Some people deny that these two expressions are descriptively synonymous on the grounds that a divorced man who is not married is not a bachelor. As for expressive or socio-expressive meaning, in order to determine that two or more descriptively synonymous expressions differ in respect of the degree or nature of their expressive meaning, it is obvious that a whole set of words including huge, enormous, gigantic and colossal are more expressive of their speakers feelings towards what they are describing than very big or very large, with which they are perhaps descriptively synonymous. It is difficult to compare huge, enormous, gigantic and colossal in terms of their degree of expressivity. But speakers may have clear intuitions about two or more of them. In the end, such conditions must be used to identify whether the two lexemes are synonyms or not and the three conditions have proved that perfect synonyms are not available in any language. Palmer (1981) differentiates between synonyms in terms of dialects, styles, emotive and evaluative values, collocational constraints and overlap of meanings of words. First, some synonyms go with different dialects of the language. For instance, the word movie is used in the United States and film is used in Britain. Second, some synonyms are used in different styles based on formality; colloquial, formal. For instance, depart (formal), go (informal). Third, some words differ only in their emotive or evaluative values but their cognitive meaning is the same. For instance, hide, conceal. Fourth, some words are subject to collocational restraints, i.e. they occur only with specific words. For instance, rancid occurs with butter, addled with eggs. Fifth, the meanings of some words overlap. For instance, mature, adult, ripe. If we take each of these words, we will have a larger set of synonyms. Palmer suggests a substitution test for judging whether two linguistic items are synonyms or n ot. Because perfect synonyms are mutually interchangeable in all contexts, it is rare to find perfect synonyms in a specific language. Anonyms are another way of testing synonymy. For instance, superficial is the opposite of deep and profound, while shallow is the opposite of deep only. Briefly, the true test of synonymy is substitutability: the ability of two words to be substituted for one another without a change in meaning. For instance, the example below contains the verb assist. The research assistant was available to assist patients completing the survey. If help is a synonym of assist, then it should be able to be substituted for assist in the above example without a change in meaning: The research assistant was available to help patients completing the survey. Help and assist can be considered as absolute synonyms, because the two sentences are identical in meaning, at least in the above contexts. Linguists and semanticists have extensively studied synonymy. Consequently, many reasons have been suggested regarding the impossibility of finding perfect synonyms. Firstly, Maja (2009) argued that the function or use of one of the two lexemes would gradually become unnecessary or unmotivated and, as a result, it would soon be abandoned or dropped. Secondly, their interchangeability in all the contexts can neither be demonstrated nor proved, for, on one hand, the number of contexts is infinite, and, on the other hand, the exceptions from absolute interchangeability are inevitable. Therefore, the lexicons of natural languages do not have absolute synonymy. Thirdly, Edmonds and Hirst (2002) also argued that if words were truly synonymous, they would need to be able to be substituted one for the other in any context in which their common sense is denoted with no change to truth value, communicative effect, or à ¢Ãƒ ¢Ã¢â‚¬Å¡Ã‚ ¬Ãƒâ€¹Ã…“meaningà ¢Ãƒ ¢Ã¢â‚¬Å¡Ã‚ ¬Ãƒ ¢Ã¢â‚¬Å¾Ã‚ ¢. Fourt hly, each linguistic form is polysemous so that it is difficult to two lexemes sharing whose all meanings are identical in all contexts. In conclusion, there is a consensus among linguists and semanticists about the impossibility of finding two perfect linguistic forms in any language. They have attributed the impossibility to many reasons. Some semanticists tried to simplify the matter of types of synonymy by classifying synonyms based on their own perspectives. Therefore, there are many types suggested by them so that it is difficult to find a specific definition set by them. All studies conducted on synonymy have proved that no perfect synonyms are found in a language.

Enzyme Biocatalysis

Enzyme Biocatalysis Andr? s Illanes e Editor Enzyme Biocatalysis Principles and Applications 123 Prof. Dr. Andr? s Illanes e School of Biochemical Engineering Ponti? cia Universidad Cat? lica o de Valpara? so ? Chile [email  protected] cl ISBN 978-1-4020-8360-0 e-ISBN 978-1-4020-8361-7 Library of Congress Control Number: 2008924855 c 2008 Springer Science + Business Media B. V. No part of this work may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, micro? ming, recording or otherwise, without written permission from the Publisher, with the exception of any material supplied speci? cally for the purpose of being entered and executed on a computer system, for exclusive use by the purchaser of the work. Printed on acid-free paper. 9 8 7 6 5 4 3 2 1 springer. com Contents Foreword . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix 1 Introdu ction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Andr? s Illanes e 1. 1 Catalysis and Biocatalysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. 2 Enzymes as Catalysts. Structure–Functionality Relationships . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. 3 The Concept and Determination of Enzyme Activity . . . . . . . . . . . . . . 1. 4 Enzyme Classes. Properties and Technological Signi? cance . . . . . . . 1. 5 Applications of Enzymes. Enzyme as Process Catalysts . . . . . . . . . . . 1. 6 Enzyme Processes: the Evolution from Degradation to Synthesis. Biocatalysis in Aqueous and Non-conventional Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Enzyme Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Andr? s Illanes e 2. 1 Enzyme Sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. 2 Production of Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. 2. 1 Enzyme Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. 2. 2 Enzyme Recovery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. 2. 3 Enzyme Puri? cation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. 2. 4 Enzyme Formulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 4 8 16 19 31 39 57 57 60 61 65 74 84 89 2 3 Homogeneous Enzyme Kinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107 Andr? s Illanes, Claudia Altamirano, and Lorena Wilson e 3. 1 General Aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107 3. 2 Hypothesis of Enzyme Kinetics. Determination of Kinetic Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108 3. 2. 1 Rapid Equilibrium and Steady-State Hypothesis . . . . . . . . . . . 108 v vi Contents Determination of Kinetic Parameters for Irreversible and Reversible One-Substrate Reactions . . . . . . . . . . . . . . . . . . . . . 112 3. 3 Kinetics of Enzyme Inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116 3. 3. 1 Types of Inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116 3. 3. Development of a Generalized Kinetic Model for One-Substrate Reactions Under Inhibition . . . . . . . . . . . . . . . . 117 3. 3. 3 Determination of Kinetic Parameters for One-Substrate Reactions Under Inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120 3. 4 Reactions with More than One Substr ate . . . . . . . . . . . . . . . . . . . . . . . . 124 3. 4. 1 Mechanisms of Reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124 3. 4. 2 Development of Kinetic Models . . . . . . . . . . . . . . . . . . . . . . . . 125 3. 4. 3 Determination of Kinetic Parameters . . . . . . . . . . . . . . . . . . . 131 3. 5 Environmental Variables in Enzyme Kinetics . . . . . . . . . . . . . . . . . . . . 133 3. 5. 1 Effect of pH: Hypothesis of Michaelis and Davidsohn. Effect on Enzyme Af? nity and Reactivity . . . . . . . . . . . . . . . . 134 3. 5. 2 Effect of Temperature: Effect on Enzyme Af? nity, Reactivity and Stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140 3. 5. 3 Effect of Ionic Strength . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151 4 Heterogeneous Enzyme Kinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155 Andr? s Illanes, Roberto Fern? ndez-Lafuente, Jos? M. Guis? n, e a e a and Lorena Wilson 4. 1 Enzyme Immobilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155 4. 1. 1 Methods of Immobilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156 4. 1. 2 Evaluation of Immobilization . . . . . . . . . . . . . . . . . . . . . . . . . . 166 4. 2 Heterogeneous Kinetics: Apparent, Inherent and Intrinsic Kinetics; Mass Transfer Effects in Heterogeneous Biocatalysis . . . . . . . . . . . . . 169 4. 3 Partition Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171 4. 4 Diffusional Restrictions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172 4. 4. 1 External Diffusional Restrictions . . . . . . . . . . . . . . . . . . . . . . . 173 4. 4. 2 Internal Diffusional Restrictions . . . . . . . . . . . . . . . . . . . . . . . . 181 4. 4. 3 Combined Effect of E xternal and Internal Diffusional Restrictions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197 Enzyme Reactors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205 Andr? s Illanes and Claudia Altamirano e 5. 1 Types of Reactors, Modes of Operation . . . . . . . . . . . . . . . . . . . . . . . . . 205 5. 2 Basic Design of Enzyme Reactors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207 5. 2. 1 Design Fundamentals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207 5. 2. 2 Basic Design of Enzyme Reactors Under Ideal Conditions. Batch Reactor; Continuous Stirred Tank Reactor Under Complete Mixing; Continuous Packed-Bed Reactor Under Plug Flow Regime . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209 3. 2. 2 5 Contents vii Effect of Diffusional Restrictions on E nzyme Reactor Design and Performance in Heterogeneous Systems. Determination of Effectiveness Factors. Batch Reactor; Continuous Stirred Tank Reactor Under Complete Mixing; Continuous Packed-Bed Reactor Under Plug Flow Regime . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223 5. 4 Effect of Thermal Inactivation on Enzyme Reactor Design and Performance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224 5. 4. 1 Complex Mechanisms of Enzyme Inactivation . . . . . . . . . . . 225 5. 4. 2 Effects of Modulation on Thermal Inactivation . . . . . . . . . . . . 231 5. 4. 3 Enzyme Reactor Design and Performance Under Non-Modulated and Modulated Enzyme Thermal Inactivation . . . . . . . . . . . . . . . . . . . . . . . . . . 234 5. 4. 4 Operation of Enzyme Reactors Under Inactivation and Thermal Optimization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 240 5. 4. 5 Enzyme Reactor Design and Performance Under Thermal Inactivation an d Mass Transfer Limitations . . . . . . . . . . . . . . . 245 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 248 6 Study Cases of Enzymatic Processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253 6. 1 Proteases as Catalysts for Peptide Synthesis . . . . . . . . . . . . . . . . . . . . . 253 Sonia Barberis, Fanny Guzm? n, Andr? s Illanes, and a e Joseph L? pez-Sant? n o ? 6. 1. 1 Chemical Synthesis of Peptides . . . . . . . . . . . . . . . . . . . . . . . . . 254 6. 1. 2 Proteases as Catalysts for Peptide Synthesis . . . . . . . . . . . . . . 257 6. 1. 3 Enzymatic Synthesis of Peptides . . . . . . . . . . . . . . . . . . . . . . . . 258 6. 1. 4 Process Considerations for the Synthesis of Peptides . . . . . . . 263 6. 1. Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 268 6. 2 Synthesis of ? -Lactam Antibiotics with Penicillin Acylases . . . . . . . 273 Andr? s Illanes and Lorena Wilson e 6. 2. 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 274 6. 2. 2 Chemical Versus Enzymatic Synthesis of Semi-Synthetic ? -Lactam Antibiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 274 6. 2. 3 Strategies of Enzymatic Synthesis . . . . . . . . . . . . . . . . . . . . . . 276 6. 2. 4 Penicillin Acylase Biocatalysts . . . . . . . . . . . . . . . . . . . . . . . . . 277 6. 2. 5 Synthesis of ? -Lactam Antibiotics in Homogeneous and Heterogeneous Aqueous and Organic Media . . . . . . . . . . . . . . 279 6. 2. 6 Model of Reactor Performance for the Production of Semi-Synthetic ? -Lactam Antibiotics . . . . . . . . . . . . . . . . . . . 282 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285 6. 3 Chimiosel ective Esteri? cation of Wood Sterols with Lipases . . . . . . . 292 ? Gregorio Alvaro and Andr? Illanes e 6. 3. 1 Sources and Production of Lipases . . . . . . . . . . . . . . . . . . . . . . 293 6. 3. 2 Structure and Functionality of Lipases . . . . . . . . . . . . . . . . . . . 296 5. 3 viii Contents Improvement of Lipases by Medium and Biocatalyst Engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 299 6. 3. 4 Applications of Lipases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 304 6. 3. 5 Development of a Process for the Selective Transesteri? cation of the Stanol Fraction of Wood Sterols with Immobilized Lipases . . . . . . . . . . . . . . . . . . . . . . 308 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 315 6. 4 Oxidoreductases as Powerful Biocatalysts for Green Chemistry . . . . 323 Jos? M. Guis? n, Roberto Fern? ndez-Lafuente, Lorena Wilson, and e a a C? sar Mateo e 6. 4. 1 Mild and Selective Oxidations Catalyzed by Oxidases . . . . . . 324 6. 4. 2 Redox Biotransformations Catalyzed by Dehydrogenases . . . 326 6. 4. 3 Immobilization-Stabilization of Dehydrogenases . . . . . . . . . . 329 6. 4. 4 Reactor Engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 330 6. 4. Production of Long-Chain Fatty Acids with Dehydrogenases 331 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 332 6. 5 Use of Aldolases for Asymmetric Synthesis . . . . . . . . . . . . . . . . . . . . . 333 ? Josep L? pez-Sant? n, Gregorio Alvaro, and Pere Clap? s o ? e 6. 5. 1 Aldolases: De? nitions and Classi? cation . . . . . . . . . . . . . . . . . 334 6. 5. 2 Preparation of Aldolase Biocatalysts . . . . . . . . . . . . . . . . . . . . 335 6. 5. 3 Reaction Performance: Medium Engineering and Kinetics . . 339 6. 5. 4 Synthetic Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 346 6. 5. 5 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 352 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 352 6. 6 Application of Enzymatic Reactors for the Degradation of Highly and Poorly Soluble Recalcitrant Compounds . . . . . . . . . . . . . . . . . . . . 355 o Juan M. Lema, Gemma Eibes, Carmen L? pez, M. Teresa Moreira, and Gumersindo Feijoo 6. 6. 1 Potential Application of Oxidative Enzymes for Environmental Purposes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 355 6. 6. 2 Requirements for an Ef? cient Catalytic Cycle . . . . . . . . . . . . . 357 6. 6. 3 Enzymatic Reactor Con? gurations . . . . . . . . . . . . . . . . . . . . . . 358 6. 6. 4 Modeling of Enzymatic Reactors . . . . . . . . . . . . . . . . . . . . . . . 364 6. 6. 5 Case Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365 6. 6. 6 Conclusions and Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . 374 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 375 Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 379 6. 3. 3 Foreword This book was written with the purpose of providing a sound basis for the design of enzymatic reactions based on kinetic principles, but also to give an updated vision of the potentials and limitations of biocatalysis, especially with respect to recent applications in processes of organic synthesis. The ? rst ? ve chapters are structured in the form of a textbook, going from the basic principles of enzyme structure and function to reactor design for homogeneous systems with soluble enzymes and heterogeneous systems with immobilized enzymes.The last chapter of the book is divided into six sections that represe nt illustrative case studies of biocatalytic processes of industrial relevance or potential, written by experts in the respective ? elds. We sincerely hope that this book will represent an element in the toolbox of graduate students in applied biology and chemical and biochemical engineering and also of undergraduate students with formal training in organic chemistry, biochemistry, thermodynamics and chemical reaction kinetics. Beyond that, the book pretends also to illustrate the potential of biocatalytic processes with case studies in the ? ld of organic synthesis, which we hope will be of interest for the academia and professionals involved in R&D&I. If some of our young readers are encouraged to engage or persevere in their work in biocatalysis this will certainly be our more precious reward. ? a Too much has been written about writing. Nobel laureate Gabriel Garc? a M? rquez wrote one of its most inspired books by writing about writing (Living to Tell the Tale). There he wrote â€Å"life is not what one lived, but what one remembers and how one remembers it in order to recount it†. This hardly applies to a scienti? book, but certainly highlights what is applicable to any book: its symbiosis with life. Writing about biocatalysis has given me that privileged feeling, even more so because enzymes are truly the catalysts of life. Biocatalysis is hardly separable from my life and writing this book has been certainly more an ecstasy than an agony. A book is an object of love so who better than friends to build it. Eleven distinguished professors and researchers have contributed to this endeavor with their knowledge, their commitment and their encouragement. Beyond our common language, I share with all of them a view and a life-lasting friendship.That is what lies behind this book and made its construction an exciting and rewarding experience. ix x Foreword Chapters 3 to 5 were written with the invaluable collaboration of Claudia Altamirano and Lorena Wil son, two of my former students, now my colleagues, and my bosses I am afraid. Chapter 4 also included the experience of Jos? Manuel Guis? n, e a Roberto Fern? ndez-Lafuente and C? sar Mateo, all of them very good friends who a e were kind enough to join this project and enrich the book with their world known expertise in heterogeneous biocatalysis. Section 6. is the result of a cooperation sustained by a CYTED project that brought together Sonia Barberis, also a former graduate student, now a successful professor and permanent collaborator and, beyond that, a dear friend, Fanny Guzm? n, a reputed scientist in the ? eld of peptide a synthesis who is my partner, support and inspiration, and Josep L? pez, a well-known o scientist and engineer but, above all, a friend at heart and a warm host. Section 6. 3 was the result of a joint project with Gregorio Alvaro, a dedicated researcher who has been a permanent collaborator with our group and also a very special friend and kind host. Secti on 6. is the result of a collaboration, in a very challenging ? eld of applied biocatalysis, of Dr. Guisan’s group with which we have a long-lasting academic connection and strong personal ties. Section 6. 5 represents a very challengo e ing project in which Josep L? pez and Gregorio Alvaro have joined Pere Clap? s, a prominent researcher in organic synthesis and a friend through the years, to build up an updated review on a very provocative ? eld of enzyme biocatalysis. Finally, section 6. 6 is a collaboration of a dear friend and outstanding teacher, Juan Lema, and his research group that widens the scope of biocatalysis to the ? ld of environmental engineering adding a particular ? avor to this ? nal chapter. A substantial part of this book was written in Spain while doing a sabbatical in the o Universitat Aut` noma de Barcelona, where I was warmly hosted by the Chemical Engineering Department, as I also was during short stays at the Institute of Catalysis and Petroleum Ch emistry in Madrid and at the Department of Chemical Engineering in the Universidad de Santiago de Compostela. My recognition to the persons in my institution, the Ponti? cia Universidad Cat? lica de Valpara? so, that supported and encouraged this project, particularly to o ? the rector Prof.Alfonso Muga, and professors Atilio Bustos and Graciela Mu? oz. n Last but not least, my deepest appreciation to the persons at Springer: Marie Johnson, Meran Owen, Tanja van Gaans and Padmaja Sudhakher, who were always delicate, diligent and encouraging. Dear reader, the judgment about the product is yours, but beyond the product there is a process whose beauty I hope to have been able to transmit. I count on your indulgence with language that, despite the effort of our editor, may still reveal our condition of non-native English speakers. Andr? s Illanes e Valpara? so, May 15, 2008 ? Chapter 1 Introduction Andr? s Illanes e . 1 Catalysis and Biocatalysis Many chemical reactions can occur sponta neously; others require to be catalyzed to proceed at a signi? cant rate. Catalysts are molecules that reduce the magnitude of the energy barrier required to be overcame for a substance to be converted chemically into another. Thermodynamically, the magnitude of this energy barrier can be conveniently expressed in terms of the free-energy change. As depicted in Fig. 1. 1, catalysts reduce the magnitude of this barrier by virtue of its interaction with the substrate to form an activated transition complex that delivers the product and frees the catalyst.The catalyst is not consumed or altered during the reaction so, in principle, it can be used inde? nitely to convert the substrate into product; in practice, however, this is limited by the stability of the catalyst, that is, its capacity to retain its active structure through time at the conditions of reaction. Biochemical reactions, this is, the chemical reactions that comprise the metabolism of all living cells, need to be catalyze d to proceed at the pace required to sustain life. Such life catalysts are the enzymes. Each one of the biochemical reactions of the cell metabolism requires to be catalyzed by one speci? enzyme. Enzymes are protein molecules that have evolved to perform ef? ciently under the mild conditions required to preserve the functionality and integrity of the biological systems. Enzymes can be considered then as catalysts that have been optimized through evolution to perform their physiological task upon which all forms of life depend. No wonder why enzymes are capable of performing a wide range of chemical reactions, many of which extremely complex to perform by chemical synthesis. It is not presumptuous to state that any chemical reaction already described might have an enzyme able to catalyze it.In fact, the possible primary structures of an enzyme protein composed of n amino acid residues is 20n so that for a rather small protein molecule containing 100 amino acid residues, there are 201 00 or 10130 possible School of Biochemical Engineering, Ponti? cia Universidad Cat? lica de Valpara? so, Avenida Brasil o ? 2147, Valpara? so, Chile. Phone: 56-32-273642, fax: 56-32-273803; e-mail: [email  protected] cl ? A. Illanes (ed. ), Enzyme Biocatalysis. c Springer Science + Business Media B. V. 2008 1 2 Trasition State A. Illanes Catalyzed Path Uncatalyzed PathFree Energy Ea Ea’ Reactans ? G Products Reaction Progress Fig. 1. 1 Mechanism of catalysis. Ea and Ea are the energies of activation of the uncatalyzed and catalyzed reaction. ?G is the free energy change of the reaction amino acid sequences, which is a fabulous number, higher even than the number of molecules in the whole universe. To get the right enzyme for a certain chemical reaction is then a matter of search and this is certainly challenging and exciting if one realizes that a very small fraction of all living forms have been already isolated.It is even more promising when one considers the possibility of obtaining DNA pools from the environment without requiring to know the organism from which it comes and then expressed it into a suitable host organism (Nield et al. 2002), and the opportunities of genetic remodeling of structural genes by site-directed mutagenesis (Abi? n et al. 2004). a Enzymes have been naturally tailored to perform under physiological conditions. However, biocatalysis refers to the use of enzymes as process catalysts under arti? cial conditions (in vitro), so that a major challenge in biocatalysis is to transform these hysiological catalysts into process catalysts able to perform under the usually tough reaction conditions of an industrial process. Enzyme catalysts (biocatalysts), as any catalyst, act by reducing the energy barrier of the biochemical reactions, without being altered as a consequence of the reaction they promote. However, enzymes display quite distinct properties when compared with chemical catalysts; most of these properties are a consequence of their complex molecular structure and will be analyzed in section 1. 2.Potentials and drawbacks of enzymes as process catalysts are summarized in Table 1. 1. Enzymes are highly desirable catalysts when the speci? city of the reaction is a major issue (as it occurs in pharmaceutical products and ? ne chemicals), when the catalysts must be active under mild conditions (because of substrate and/or product instability or to avoid unwanted side-reactions, as it occurs in several reactions of organic synthesis), when environmental restrictions are stringent (which is now a 1 Introduction Table 1. 1 Advantages and Drawbacks of Enzymes as Catalysts Advantages High speci? ity High activity under moderate conditions High turnover number Highly biodegradable Generally considered as natural products Drawbacks High molecular complexity High production costs Intrinsic fragility 3 rather general situation that gives biocatalysis a distinct advantage over alternative technologies) or when the l abel of natural product is an issue (as in the case of food and cosmetic applications) (Benkovic and Ballesteros 1997; Wegman et al. 2001). However, enzymes are complex molecular structures that are intrinsically labile and costly to produce, which are de? ite disadvantages with respect to chemical catalysts (Bommarius and Broering 2005). While the advantages of biocatalysis are there to stay, most of its present restrictions can be and are being solved through research and development in different areas. In fact, enzyme stabilization under process conditions is a major issue in biocatalysis and several strategies have been developed (Illanes 1999) that include ? chemical modi? cation (Roig and Kennedy 1992; Ozturk et al. 2002; Mislovi? ov? c a et al. 2006), immobilization to solid matrices (Abi? n et al. 2001; Mateo et al. 2005; a Kim et al. 2006; Wilson et al. 006), crystallization (H? ring and Schreier 1999; Roy a and Abraham 2006), aggregation (Cao et al. 2003; Mateo et al. 2004 ; Schoevaart et al. 2004; Illanes et al. 2006) and the modern techniques of protein engineering (Chen 2001; Declerck et al. 2003; Sylvestre et al. 2006; Leisola and Turunen 2007), namely site-directed mutagenesis (Bhosale et al. 1996; Ogino et al. 2001; Boller et al. 2002; van den Burg and Eijsink 2002; Adamczak and Hari Krishna 2004; Bardy et al. 2005; Morley and Kazlauskas 2005), directed evolution by tandem mutagenesis (Arnold 2001; Brakmann and Johnsson 2002; Alexeeva et al. 003; Boersma et al. 2007) and gene-shuf? ing based on polymerase assisted (Stemmer 1994; Zhao et al. 1998; Shibuya et al. 2000; Kaur and Sharma 2006) and, more recently, ligase assisted recombination (Chodorge et al. 2005). Screening for intrinsically stable enzymes is also a prominent area of research in biocatalysis. Extremophiles, that is, organisms able to survive and thrive in extreme environmental conditions are a promising source for highly stable enzymes and research on those organisms is very active at present (Adams and Kelly 1998; Davis 1998; Demirjian et al. 001; van den Burg 2003; Bommarius and Riebel 2004; Gomes and Steiner 2004). Genes from such extremophiles have been cloned into suitable hosts to develop biological systems more amenable for production (Halld? rsd? ttir et al. 1998; o o Haki and Rakshit 2003; Zeikus et al. 2004). Enzymes are by no means ideal process catalysts, but their extremely high speci? city and activity under moderate conditions are prominent characteristics that are being increasingly appreciated by different production sectors, among which the pharmaceutical and ? ne-chemical industry (Schmid et al. 001; Thomas et al. 2002; Zhao et al. 2002; Bruggink et al. 2003) have added to the more traditional sectors of food (Hultin 1983) and detergents (Maurer 2004). 4 Fig. 1. 2 Scheme of peptide bond formation between two adjacent ? -amino acids R1 + H3N CH C OH O A. Illanes H R2 + H N CH COO? H2O R1 H2O H R2 H3N CH C N CH COO? O + 1. 2 Enzymes as Cataly sts. Structure–Functionality Relationships Most of the characteristics of enzymes as catalysts derive from their molecular structure. Enzymes are proteins composed by a number of amino acid residues that range from 100 to several hundreds.These amino acids are covalently bound through the peptide bond (Fig. 1. 2) that is formed between the carbon atom of the carboxyl group of one amino acid and the nitrogen atom of the ? -amino group of the following. According to the nature of the R group, amino acids can be non-polar (hydrophobic) or polar (charged or uncharged) and their distribution along the protein molecule determines its behavior (Lehninger 1970). Every protein is conditioned by its amino acid sequence, called primary structure, which is genetically determined by the deoxyribonucleotide sequence in the structural gene that codes for it.The DNA sequence is ? rst transcribed into a mRNA molecule which upon reaching the ribosome is translated into an amino acid sequence a nd ? nally the synthesized polypeptide chain is transformed into a threedimensional structure, called native structure, which is the one endowed with biological functionality. This transformation may include several post-translational reactions, some of which can be quite relevant for its functionality, like proteolytic cleavage, as it occurs, for instance, with Escherichia coli penicillin acylase (Schumacher et al. 986) and glycosylation, as it occurs for several eukaryotic enzymes (Longo et al. 1995). The three-dimensional structure of a protein is then genetically determined, but environmentally conditioned, since the molecule will interact with the surrounding medium. This is particularly relevant for biocatalysis, where the enzyme acts in a medium quite different from the one in which it was synthesized than can alter its native functional structure. Secondary three-dimensional structure is the result of interactions of amino acid residues proximate in the primary structure, ma inly by hydrogen bonding of the amide groups; for the ase of globular proteins, like enzymes, these interactions dictate a predominantly ribbon-like coiled con? guration termed ? -helix. Tertiary three-dimensional structure is the result of interactions of amino acid residues located apart in the primary structure that produce a compact and twisted con? guration in which the surface is rich in polar amino acid 1 Introduction 5 residues, while the inner part is abundant in hydrophobic amino acid residues. This tertiary structure is essential for the biological functionality of the protein.Some proteins have a quaternary three-dimensional structure, which is common in regulatory proteins, that is the result of the interaction of different polypeptide chains constituting subunits that can display identical or different functions within a protein complex (Dixon and Webb 1979; Creighton 1993). The main types of interactions responsible for the three-dimensional structure of proteins are (Haschemeyer and Haschemeyer 1973): †¢ Hydrogen bonds, resulting from the interaction of a proton linked to an electronegative atom with another electronegative atom.A hydrogen bond has approximately one-tenth of the energy stored in a covalent bond. It is the main determinant of the helical secondary structure of globular proteins and it plays a signi? cant role in tertiary structure as well. †¢ Apolar interactions, as a result of the mutual repulsion of the hydrophobic amino acid residues by a polar solvent, like water. It is a rather weak interaction that does not represent a proper chemical bond (approximation between atoms exceed the van der Waals radius); however, its contribution to the stabilization of the threedimensional structure of a protein is quite signi? ant. †¢ Disulphide bridges, produced by oxidation of cysteine residues. They are especially relevant in the stabilization of the three-dimensional structure of low molecular weight extracellular protein s. †¢ Ionic bonds between charged amino acid residues. They contribute to the stabilization of the three-dimensional structure of a protein, although to a lesser extent, because the ionic strength of the surrounding medium is usually high so that interaction is produced preferentially between amino acid residues and ions in the medium. Other weak type interactions, like van der Waals forces, whose contribution to three-dimensional structure is not considered signi? cant. Proteins can be conjugated, this is, associated with other molecules (prosthetic groups). In the case of enzymes which are conjugated proteins (holoenzymes), catalysis always occur in the protein portion of the enzyme (apoenzyme). Prosthetic groups may be organic macromolecules, like carbohydrates (in the case of glycoproteins), lipids (in the case of lipoproteins) and nucleic acids (in the case of nucleoproteins), or simple inorganic entities, like metal ions.Prosthetic groups are tightly bound (usually covale ntly) to the apoenzyme and do not dissociate during catalysis. A signi? cant number of enzymes from eukaryotes are glycoproteins, in which case the carbohydrate moiety is covalently linked to the apoenzyme, mainly through serine or threonine residues, and even though the carbohydrate does not participate in catalysis it confers relevant properties to the enzyme. Catalysis takes place in a small portion of the enzyme called the active site, which is usually formed by very few amino acid residues, while the rest of the protein acts as a scaffold.Papain, for instance, has a molecular weight of 23,000 Da with 211 amino acid residues of which only cysteine (Cys 25) and histidine (His 159) 6 A. Illanes are directly involved in catalysis (Allen and Lowe 1973). Substrate is bound to the enzyme at the active site and doing so, changes in the distribution of electrons in its chemical bonds are produced that cause the reactions that lead to the formation of products. The products are then rele ased from the enzyme which is ready for the next catalytic cycle.According to the early lock and key model proposed by Emil Fischer in 1894, the active site has a unique geometric shape that is complementary to the geometric shape of the substrate molecule that ? ts into it. Even though recent reports provide evidence in favor of this theory (Sonkaria et al. 2004), this rigid model hardly explains many experimental evidences of enzyme biocatalysis. Later on, the induced-? t theory was proposed (Koshland 1958) according to which he substrate induces a change in the enzyme conformation after binding, that may orient the catalytic groups in a way prone for the subsequent reaction; this theory has been extensively used to explain enzyme catalysis (Youseff et al. 2003). Based on the transition-state theory, enzyme catalysis has been explained according to the hypothesis of enzyme transition state complementariness, which considers the prefc erential binding of the transition state rather than the substrate or product (Benkovi? and Hammes-Schiffer 2003).Many, but not all, enzymes require small molecules to perform as catalysts. These molecules are termed coenzymes or cofactors. The term coenzyme is used to refer to small molecular weight organic molecules that associate reversibly to the enzyme and are not part of its structure; coenzymes bound to enzymes actually take part in the reaction and, therefore, are sometime called cosubstrates, since they are stoichiometric in nature (Kula 2002). Coenzymes often function as intermediate carriers of electrons (i. e. NAD+ or FAD+ in dehydrogenases), speci? c atoms (i. e. oenzyme Q in H atom transfer) or functional groups (i. e. coenzyme A in acyl group transfer; pyridoxal phosphate in amino group transfer; biotin in CO2 transfer) that are transferred in the reaction. The term cofactor is commonly used to refer to metal ions that also bind reversibly to enzymes but in general are not chemically altered during the reaction; c ofactors usually bind strongly to the enzyme structure so that they are not dissociated from the holoenzyme during the reaction (i. e. Ca++ in ? -amylase; Co++ or Mg++ in glucose isomerase; Fe+++ in nitrile hydratase).According to these requirements, enzymes can be classi? ed in three groups as depicted in Fig. 1. 3: (i) those that do not require of an additional molecule to perform biocatalysis, (ii) those that require cofactors that remain unaltered and tightly bound to the enzyme performing in a catalytic fashion, and (iii) those requiring coenzymes that are chemically modi? ed and dissociated during catalysis, performing in a stoichiometric fashion. The requirement of cofactors or coenzymes to perform biocatalysis has profound technological implications, as will be analyzed in section 1. 4.Enzyme activity, this is, the capacity of an enzyme to catalyze a chemical reaction, is strictly dependent on its molecular structure. Enzyme activity relies upon the existence of a proper str ucture of the active site, which is composed by a reduced number of amino acid residues close in the three-dimensional structure of 1 Introduction Fig. 1. 3 Enzymes according to their cofactor or coenzyme requirements. 1: no requirement; 2: cofactor requiring; 3: coenzyme requiring S 1 7 P E E CoE 2 S E-CoE P E CoE 3 E CoE’ E P S E-CoE the protein but usually far apart in the primary structure.Therefore, any agent that promotes protein unfolding will move apart the residues constituting the active site and will then reduce or destroy its biological activity. Adverse conditions of temperature, pH or solvent and the presence of chaotropic substances, heavy metals and chelating agents can produce this loss of function by distorting the proper active site con? guration. Even though a very small portion of the enzyme molecule participates in catalysis, the remaining of the molecule is by no means irrelevant to its performance.Crucial properties, like enzyme stability, are very muc h dependent on the enzyme three-dimensional structure. Enzyme stability appears to be determined by unde? ned irreversible processes governed by local unfolding in certain labile regions denoted as weak spots. These regions prone to unfolding are the determinants of enzyme stability and are usually located in or close to the surface of the protein molecule, which explains why the surface structure of the enzyme is so important for its catalytic stability (Eijsink et al. 2004). These regions have been the target of site-speci? c mutations for increasing stability.Though extensively studied, rational engineering of the enzyme molecule for increased stability has been a very complex task. In most cases, these weak spots are not easy to identify so it is not clear to what region of the protein molecule should one be focused on and, even though properly selected, it is not clear what is the right type of mutation to introduce (Gaseidnes et al. 2003). Despite the impressive advances in th e ? eld and the existence of some experimentally based rules (Shaw and Bott 1996), rational improvement of the stability is still far from being well established.In fact, the less rational approaches of directed evolution using error-prone PCR and gene shuf? ing have been more successful in obtaining more stable mutant enzymes (Kaur and Sharma 2006). Both strategies can combine using a set of rationally designed mutants that can then be subjected to gene shuf? ing (O’F? g? in 2003). a a A perfectly structured native enzyme expressing its biological activity can lose it by unfolding of its tertiary structure to a random polypeptide chain in which the amino acids located in the active site are no longer aligned closely enough to perform its catalytic function.This phenomenon is termed denaturation and it may be reversible if the denaturing in? uence is removed since no chemical changes 8 A. Illanes have occurred in the protein molecule. The enzyme molecule can also be subjected to chemical changes that produce irreversible loss of activity. This phenomenon is termed inactivation and usually occurs following unfolding, since an unfolded protein is more prone to proteolysis, loss of an essential cofactor and aggregation (O’F? g? in 1997). These phenomena de? e what is called thermodynamic or cona a formational stability, this is the resistance of the folded protein to denaturation, and kinetic or long-term stability, this is the resistance to irreversible inactivation (Eisenthal et al. 2006). The overall process of enzyme inactivation can then be represented by: N U ? > I where N represents the native active conformation, U the unfolded conformation and I the irreversibly inactivated enzyme (Klibanov 1983; Bommarius and Broering 2005). The ? rst step can be de? ned by the equilibrium constant of unfolding (K), while the second is de? ed in terms of the rate constant for irreversible inactivation (k). Stability is not related to activity and in many cases they have opposite trends. It has been suggested that there is a trade-off between stability and activity based on the fact that stability is clearly related to molecular stiffening while conformational ? exibility is bene? cial for catalysis. This can be clearly appreciated when studying enzyme thermal inactivation: enzyme activity increases with temperature but enzyme stability decreases. These opposite trends make temperature a critical variable in any enzymatic process and make it prone to optimization.This aspect will be thoroughly analyzed in Chapters 3 and 5. Enzyme speci? city is another relevant property of enzymes strictly related to its structure. Enzymes are usually very speci? c with respect to its substrate. This is because the substrate is endowed with the chemical bonds that can be attacked by the functional groups in the active site of the enzyme which posses the functional groups that anchor the substrate properly in the active site for the reaction to take p lace. Under certain conditions conformational changes may alter substrate speci? city.This has been elegantly proven by site-directed mutagenesis, in which speci? c amino acid residues at or near the active site have been replaced producing an alteration of substrate speci? city (Colby et al. 1998; diSioudi et al. 1999; Parales et al. 2000), and also by chemical modi? cation (Kirk Wright and Viola 2001). K k 1. 3 The Concept and Determination of Enzyme Activity As already mentioned, enzymes act as catalysts by virtue of reducing the magnitude of the barrier that represents the energy of activation required for the formation of a transient active complex that leads to product formation (see Fig. . 1). This thermodynamic de? nition of enzyme activity, although rigorous, is of little practical signi? cance, since it is by no means an easy task to determine free energy changes for molecular structures as unstable as the enzyme–substrate complex. The direct 1 Introduction 9 conseq uence of such reduction of energy input for the reaction to proceed is the increase in reaction rate, which can be considered as a kinetic de? nition of enzyme activity. Rates of chemical reactions are usually simple to determine so this de? nition is endowed with practicality.Biochemical reactions usually proceed at very low rates in the absence of catalysts so that the magnitude of the reaction rate is a direct and straightforward procedure for assessing the activity of an enzyme. Therefore, for the reaction of conversion of a substrate (S) into a product (P) under the catalytic action of an enzyme (E): S ? > P v=? ds dp = dt dt (1. 1) E If the course of the reaction is followed, a curve like the one depicted in Fig 1. 4 will be obtained. This means that the reaction rate (slope of the p vs t curve) will decrease as the reaction proceeds.Then, the use of Eq. 1. 1 is ambiguous if used for the determination of enzyme activity. To solve this ambiguity, the reasons underlying this beh avior must be analyzed. The reduction in reaction rate can be the consequence of desaturation of the enzyme because of substrate transformation into product (at substrate depletion reaction rate drops to zero), enzyme inactivation as a consequence of the exposure of the enzyme to the conditions of reaction, enzyme inhibition caused by the products of the reaction, and equilibrium displacement as a consequence of the law of mass action.Some or all of these phenomena are present in any enzymatic reaction so that the catalytic capacity of the enzyme will vary throughout the course of the reaction. It is customary to identify the enzyme activity with the initial rate of reaction (initial slope of the â€Å"p† versus â€Å"t† curve) where all the above mentioned Product Concentration e e 2 e 4 Time Fig. 1. 4 Time course of an enzyme catalyzed reaction: product concentration versus time of reaction at different enzyme concentrations (e) 10 A. Illanes phenomena are insigni? a nt. According to this: a = vt>0 = ? ds dt = t>0 dp dt (1. 2) t>0 This is not only of practical convenience but fundamentally sound, since the enzyme activity so de? ned represents its maximum catalytic potential under a given set of experimental conditions. To what extent is this catalytic potential going to be expressed in a given situation is a different matter and will have to be assessed by modulating it according to the phenomena that cause its reduction. All such phenomena are amenable to quanti? ation as will be presented in Chapter 3, so that the determination of this maximum catalytic potential is fundamental for any study regarding enzyme kinetics. Enzymes should be quanti? ed in terms of its catalytic potential rather than its mass, since enzyme preparations are rather impure mixtures in which the enzyme protein can be a small fraction of the total mass of the preparation; but, even in the unusual case of a completely pure enzyme, the determination of activity is unavoida ble since what matters for evaluating the enzyme performance is its catalytic potential and not its mass.Within the context of enzyme kinetics, reaction rates are always considered then as initial rates. It has to be pointed out, however, that there are situations in which the determination of initial reaction rates is a poor predictor of enzyme performance, as it occurs in the determination of degrading enzymes acting on heterogeneous polymeric substrates. This is the case of cellulase (actually an enzyme complex of different activities) (Montenecourt and Eveleigh 1977; Illanes et al. 988; Fowler and Brown 1992), where the more amorphous portions of the cellulose moiety are more easily degraded than the crystalline regions so that a high initial reaction rate over the amorphous portion may give an overestimate of the catalytic potential of the enzyme over the cellulose substrate as a whole. As shown in Fig. 1. 4, the initial slope o the curve (initial rate of reaction) is proportio nal to the enzyme concentration (it is so in most cases). Therefore, the enzyme sample should be properly diluted to attain a linear product concentration versus time relationship within a reasonable assay time.The experimental determination of enzyme activity is based on the measurement of initial reaction rates. Substrate depletion or product build-up can be used for the evaluation of enzyme activity according to Eq. 1. 2. If the stoichiometry of the reaction is de? ned and well known, one or the other can be used and the choice will depend on the easiness and readiness for their analytical determination. If this is indifferent, one should prefer to measure according to product build-up since in this case one will be determining signi? ant differences between small magnitudes, while in the case of substrate depletion one will be measuring small differences between large magnitudes, which implies more error. If neither of both is readily measurable, enzyme activity can be determine d by coupling reactions. In this case the product is transformed (chemically or enzymatically) to a ? nal analyte amenable for analytical determination, as shown: E S P A X B Y C Z 1 Introduction 11 In this case enzyme activity can be determined as: a = vt>0 = ? ds dt = t>0 dp dt = t>0 dz dt (1. 3) t>0 rovided that the rate limiting step is the reaction catalyzed by the enzyme, which implies that reagents A, B and C should be added in excess to ensure that all P produced is quantitatively transformed into Z. For those enzymes requiring (stoichiometric) coenzymes: E S CoE CoE P activity can be determined as: a = vt>0 = ? dcoe dt = t>0 dcoe dt (1. 4) t>0 This is actually a very convenient method for determining activity of such class of enzymes, since organic coenzymes (i. e. FAD or NADH) are usually very easy to determine analytically. An example of a coupled system considering coenzyme determination is the assay for lactase (? galactosidase; EC 3. 2. 1. 23). The enzyme catalyzes the hydrolysis of lactose according to: Lactose + H2 O > Glucose + Galactose Glucose produced can be coupled to a classical enzymatic glucose kit, that is: hexoquinase (Hx) plus glucose 6 phosphate dehydrogenase (G6PD), in which: Glucose + ATP ? > Glucose 6Pi + ADP Glucose 6Pi + NADP+ ? ? ? ?> 6PiGluconate + NADPH where the initial rate of NADPH (easily measured in a spectrophotometer; see ahead) can be then stoichiometrically correlated to the initial rate of lactose hydrolysis, provided that the auxiliary enzymes, Hx and G6PD, and co-substrates are added in excess.Enzyme activity can be determined by a continuous or discontinuous assay. If the analytical device is provided with a recorder that register the course of reaction, the initial rate could be easily determined from the initial slope of the product (or substrate, or coupled analyte, or coenzyme) concentration versus time curve. It is not always possible or simple to set up a continuous assay; in that case, the course of react ion should be monitored discontinuously by sampling and assaying at predetermined time intervals and samples should be subjected to inactivation to stop the reaction.This is a drawback, since the enzyme should be rapidly, completely and irreversibly inactivated by subjecting it to harsh conditions that can interfere with the G6PD Hx 12 A. Illanes analytical procedure. Data points should describe a linear â€Å"p† versus â€Å"t† relationship within the time interval for assay to ensure that the initial rate is being measured; if not, enzyme sample should be diluted accordingly. Assay time should be short enough to make the effect of the products on the reaction rate negligible and to produce a negligibly reduction in substrate concentration. A major issue in enzyme activity determination is the de? ition of a control experiment for discriminating the non-enzymatic build-up of product during the assay. There are essentially three options: to remove the enzyme from the r eaction mixture by replacing the enzyme sample by water or buffer, to remove the substrate replacing it by water or buffer, or to use an enzyme placebo. The ? rst one discriminates substrate contamination with product or any non-enzymatic transformation of substrate into product, but does not discriminate enzyme contamination with substrate or product; the second one acts exactly the opposite; the third one can in rinciple discriminate both enzyme and substrate contamination with product, but the pitfall in this case is the risk of not having inactivated the enzyme completely. The control of choice depends on the situation. For instance, when one is producing an extracellular enzyme by fermentation, enzyme sample is likely to be contaminated with substrate and or product (that can be constituents of the culture medium or products of metabolism) and may be signi? ant, since the sample probably has a low enzyme protein concentration so that it is not diluted prior to assay; in this ca se, replacing substrate by water or buffer discriminates such contamination. If, on the other hand, one is assaying a preparation from a stock enzyme concentrate, dilution of the sample prior to assay makes unnecessary to blank out enzyme contamination; replacing the enzyme by water or buffer can discriminate substrate contamination that is in this case more relevant.The use of an enzyme placebo as control is advisable when the enzyme is labile enough to be completely inactivated at conditions not affecting the assay. An alternative is to use a double control replacing enzyme in one case and substrate in the other by water or buffer. Once the type of control experiment has been decided, control and enzyme sample are subjected to the same analytical procedure, and enzyme activity is calculated by subtracting the control reading from that of the sample, as illustrated in Fig. . 5. Analytical procedures available for enzyme activity determinations are many and usually several alternati ves exist. A proper selection should be based on sensibility, reproducibility, ? exibility, simplicity and availability. Spectrophotometry can be considered as a method that ful? ls most, if not all, such criteria. It is based on the absorption of light of a certain wavelength as described by the Beer–Lambert law: A? = ?  · l  · c where: A? = log I I0 (1. 5) (1. 6) The value of ? an be experimentally obtained through a calibration curve of absorbance versus concentration of analyte, so that the reading of A? will allow the determination of its concentration. Optical path width is usually 1 cm. The method is based on the differential absorption of product (or coupling analyte or modi? ed 1 Introduction 13 Fig. 1. 5 Scheme for the analytical procedure to determine enzyme activity. S: substrate; P: product; P0 : product in control; A, B, C: coupling reagents; Z: analyte; Z0 : analyte in control; s, p, z are the corresponding molar concentrations oenzyme) and substrate (or co enzyme) at a certain wavelength. For instance, the reduced coenzyme NADH (or NADPH) has a strong peak of absorbance at 340 nm while the absorbance of the oxidized coenzyme NAD+ (or NADP+ ) is negligible at that wavelength; therefore, the activity of any enzyme producing or consuming NADH (or NADPH) can be determined by measuring the increase or decline of absorbance at 340 nm in a spectrophotometer. The assay is sensitive, reproducible and simple and equipment is available in any research laboratory.If both substrate and product absorb signi? cantly at a certain wavelength, coupling the detector to an appropriate high performance liquid chromatography (HPLC) column can solve this interference by separating those peaks by differential retardation of the analytes in the column. HPLC systems are increasingly common in research laboratories, so this is a very convenient and ? exible way for assaying enzyme activities. Several other analytical procedures are available for enzyme activity determination.Fluorescence, this is the ability of certain molecules to absorb light at a certain wavelength and emit it at another, is a property than can be used for enzymatic analysis. NADH, but also FAD (? avin adenine dinucleotide) and FMN (? avin mononucleotide) have this property that can be used for those enzyme requiring that molecules as coenzymes (Eschenbrenner et al. 1995). This method shares some of the good properties of spectrophotometry and can also be integrated into an HPLC system, but it is less ? exible and the equipment not so common in a standard research laboratory.Enzymes that produce or consume gases can be assayed by differential manometry by measuring small pressure differences, due to the consumption of the gaseous substrate or the evolution of a gaseous product that can be converted into substrate or product concentrations by using the gas law. Carboxylases and decarboxylases are groups of enzymes that can be conveniently assayed by differential manomet ry in a respirometer. For instance, the activity of glutamate decarboxylase 14 A. Illanes (EC 4. 1. 1. 15), that catalyzes the decarboxylation of glutamic acid to ? aminobutyric acid and CO2 , has been assayed in a differential respirometer by measuring the increase in pressure caused by the formation of gaseous CO2 (O’Learys and Brummund 1974). Enzymes catalyzing reactions involving optically active compounds can be assayed by polarimetry. A compound is considered to be optically active if polarized light is rotated when passing through it. The magnitude of optical rotation is determined by the molecular structure and concentration of the optically active substance which has its own speci? rotation, as de? ned in Biot’s law: ? = ? 0  · l  · c (1. 7) Polarimetry is a simple and accurate method for determining optically active compounds. A polarimeter is a low cost instrument readily available in many research laboratories. The detector can be integrated into an HPL C system if separation of substrates and products of reaction is required. Invertase (? -D-fructofuranoside fructohydrolase; EC 3. 2. 1. 26), a commodity enzyme widely used in the food industry, can be conveniently assayed by polarimetry (Chen et al. 2000), since the speci? optical rotation of the substrate (sucrose) differs from that of the products (fructose plus glucose). Some depolymerizing enzymes can be conveniently assayed by viscometry. The hydrolytic action over a polymeric substrate can produce a signi? cant reduction in kinematic viscosity that can be correlated to the enzyme activity. Polygalacturonase activity in pectinase preparations (Gusakov et al. 2002) and endo ? 1–4 glucanase activity in cellulose preparations (Canevascini and Gattlen 1981; Illanes and Schaffeld 1983) have been determined by measuring the reduction in viscosity of the corresponding olymer solutions. A comprehensive review on methods for assaying enzyme activity has been recently published ( Bisswanger 2004). Enzyme activity is expressed in units of activity. The Enzyme Commission of the International Union of Biochemistry recommends to express it in international units (IU), de? ning 1 IU as the amount of an enzyme that catalyzes the transformation of 1  µmol of substrate per minute under standard conditions of temperature, optimal pH, and optimal substrate concentration (International Union of Biochemistry).Later on, in 1972, the Commission on Biochemical Nomenclature recommended that, in order to adhere to SI units, reaction rates should be expressed in moles per second and the katal was proposed as the new unit of enzyme activity, de? ning it as the catalytic activity that will raise the rate of reaction by 1 mol/second in a speci? ed assay system (Anonymous 1979). This latter de? nition, although recommended, has some practical drawbacks. The magnitude of the katal is so big that usual enzyme activities expressed in katals are extremely small numbers that are har d to appreciate; the de? ition, on the other hand, is rather vague with respect to the conditions in which the assay should be performed. In practice, even though in some journals the use of the katal is mandatory, there is reluctance to use it and the former IU is still more widely used. 1 Introduction 15 Going back to the de? nition of IU there are some points worthwhile to comment. The magnitude of the IU is appropriate to measure most enzyme preparations, whose activities usually range from a few to a few thousands IU per unit mass or unit volume of preparation.Since enzyme activity is to be considered as the maximum catalytic potential of the enzyme, it is quite appropriate to refer it to optimal pH and optimal substrate concentration. With respect to the latter, optimal is to be considered as that substrate concentration at which the initial rate of reaction is at its maximum; this will imply reaction rate at substrate saturation for an enzyme following typical Michaelis-Mente n kinetics or the highest initial reaction rate value in the case of inhibition at high substrate concentrations (see Chapter 3).With respect to pH, it is straightforward to determine the value at which the initial rate of reaction is at its maximum. This value will be the true operational optimum in most cases, since that pH will lie within the region of maximum stability. However, the opposite holds for temperature where enzymes are usually quite unstable at the temperatures in which higher initial reaction rates are obtained; actually the concept of â€Å"optimum† temperature, as the one that maximizes initial reaction rate, is quite misleading since that value usually re? cts nothing more than the departure of the linear â€Å"p† versus â€Å"t† relationship for the time of assay. For the de? nition of IU it is then more appropriate to refer to it as a â€Å"standard† and not as an â€Å"optimal† temperature. Actually, it is quite dif? cult to de? ne the right temperature to assay enzyme activity. Most probably that value will differ from the one at which the enzymatic process will be conducted; it is advisable then to obtain a mathematical expression for the effect of temperature on the initial rate of reaction to be able to transform the units of activity according to the temperature of operation (Illanes et al. 000). It is not always possible to express enzyme activity in IU; this is the case of enzymes catalyzing reactions that are not chemically well de? ned, as it occurs with depolymerizing enzymes, whose substrates have a varying and often unde? ned molecular weight and whose products are usually a mixture of different chemical compounds. In that case, units of activity can be de? ned in terms of mass rather than moles. These enzymes are usually speci? c for certain types of bonds rather than for a particular chemical structure, so in such cases it is advisable to express activity in terms of equivalents of bonds b roken.The choice of the substrate to perform the enzyme assay is by no means trivial. When using an enzyme as process catalyst, the substrate can be different from that employed in its assay that is usually a model substrate or an analogue. One has to be cautious to use an assay that is not only simple, accurate and reproducible, but also signi? cant. An example that illustrates this point is the case of the enzyme glucoamylase (exo-1,4-? -glucosidase; EC 3. 2. 1. 1): this enzyme is widely used in the production of glucose syrups from starch, either as a ? al product or as an intermediate for the production of high-fructose syrups (Carasik and Carroll 1983). The industrial substrate for glucoamylase is a mixture of oligosaccharides produced by the enzymatic liquefaction of starch with ?-amylase (1,4-? -D-glucan glucanohydrolase; EC 3. 2. 1. 1). Several substrates have been used for assaying enzyme activity including high molecular weight starch, small molecular weight oligosaccharid es, maltose and maltose synthetic analogues (Barton et al. 1972; Sabin and Wasserman 16 A. Illanes 1987; Goto et al. 1998). None of them probably re? cts properly the enzyme activity over the real substrate, so it will be a matter of judgment and experience to select the most pertinent assay with respect to the actual use of the enzyme. Hydrolases are currently assayed with respect to their hydrolytic activities; however, the increasing use of hydrolases to perform reactions of synthesis in non-aqueous media make this type of assay not quite adequate to evaluate the synthetic potential of such enzymes. For instance, the protease subtilisin has been used as a catalyst for a transesteri? cation reaction that produces thiophenol as one of the products (Han et al. 004); in this case, a method based on a reaction leading to a ? uorescent adduct of thiophenol is a good system to assess the transesteri? cation potential of such proteases and is to be preferred to a conventional protease as say based on the hydrolysis of a protein (Gupta et al. 1999; Priolo et al. 2000) or a model peptide (Klein et al. 1989). 1. 4 Enzyme Classes. Properties and Technological Signi? cance Enzymes are classi? ed according to the guidelines of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUBMB) (Anonymous 1984) into six families, based on the type of chemical reaction catalyzed.A four digit number is assigned to each enzyme by the Enzyme Commission (EC) of the IUBMB: the ? rst one denotes the family, the second denotes the subclass within a family and is related to the type of chemical group upon which it acts, the third denotes a subgroup within a subclass and is related to the particular chemical groups involved in the reaction and the forth is the correlative number of identi? cation within a subgroup. The six families are: 1. Oxidoreductases. Enzymes catalyzing oxidation/reduction reactions that involve the transfer of electrons, hydroge n or oxygen atoms.There are 22 subclasses of oxido-reductases and among them there are several of technological signi? cance, such as the dehydrogenases that oxidize a substrate by transferring hydrogen atoms to a coenzyme (NAD+ , NADP+ ,