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Design problem No: 2 Course Instructor: Himanshu, sir D.O.

A: 30/10/10

Course code: BTY-461 Course Tutor: NA D.O.S: 19/11/10

Student’s Roll No: RA7801A09

Section No: A7801

Declaration I declare that this homework is my individual work. I have not copied from any other students work or from any other source except where due acknowledgment is made explicitly in the text, nor has any part been written for me by another person. Students Signature: Gurbakhshish Singh

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Content of Design Problem should start from this page only.
Design Problem Statement: Select any enzyme of your interest and specify the considerations you will use for its isolation from specific micro-organisms and what are the improvements techniques or metabolic changes you can induce in microbe to enhance the production of your chosen enzyme. Basic Implications: 1. Isolation from Extreme environments. 2. Microorganism needs cheap medium for growth. 3. Site Directed Mutagenesis. 4. rDNA Technology/ Metabolic Engineering

the use of enzymes in industrial sector is increasing due to the increase of industries especially in food. The enzyme production process can be divided into following phases: 1. it is important to develop a technique of enzyme isolation and purification in order to obtain enzyme which posses’ not only high activity but also high grade purified enzyme. glycogen and oligosaccharide.INTRODUCTION Nowadays. textile. Most of the enzymes are. Optimization of recovery process (and purification if needed) 6.4-glucan-4-glucanohydrolase.4)-glucosidic bond in amylum. however. leather and paper industries.1) which catalyses the breakdown of α-D-(1.2.1. Bacillus and flavobacterium are used. Construction of an overproducing strain by genetic engineering 4. produced by microorganisms in submerged cultures in large reactors called fermentors. Selection of a production strain 3. EC 3. Selection of an enzyme 2. It is because they are cheap and readily available. One of the enzymes widely used in industrial sectors is α-Amylase enzyme (α-1. enzymes have been used in industry for the treatment of industrial waste. strains of Aspergillus. In this highly commercialized time. such as cellulase which is able to convert cellulose of wood and paper wastes to ethanol. beverage.M. Optimization of culture medium and production conditions 5. Enzymes are produced at industrial scale by using over producing strains which are genetically modified (G.) to give the maximum production rate of desired enzyme. Formulation of a stable enzyme product . Commonly. Besides it.

It is also present in seeds containing starch as a food reserve. We choose Bacillus subtilis as a production strain for α-Amylase as genetic modification can easily been done and it has given higher yield of α-Amylase after genetic modification by recombinant DNA technology. It is the major form of amylase found in humans and other mammals. Selection of an Enzyme We have selected our enzyme that we are interested in producing i. and is secreted by many fungi.4)-glucosidic bond in amylum. Thus genetic material derived from one species may be incorporated into another where it may be expressed. It catalyses the breakdown of α-D-(1. 3. Construction of an Overproducing Strain by Genetic Engineering Following methods have been applied for Genetic Modification of microbe for strain improvement • Using recombinant DNA technology • Protoplast fusion • Selection of Auxotrophic mutants • By site directed mutagenesis  Recombinant DNA Technology Transfer of DNA between different species of bacteria can be done using in vivo and in vitro techniques. Selection of a Production Strain α-Amylase is best produced from Bacillus and Aspergillus species.e. helping in increase of product formation i. yielding glucose and maltose. αAmylase in this case. glycogen and oligosaccharide.e. . hence.ENZYME PRODUCTION PROCESS 1. 2. α-Amylase Alpha (α)-Amylase α-Amylase is an enzyme that hydrolyses alpha-bonds of large alpha-linked polysaccharides such as starch and glycogen.

It quantitatively increases the product formation. It is a technique of amplifying gene expression by transferring the gene from one host cell to another. 1. Also this can act as plasmid vector which is small in size. E. we will use in vitro techniques as it helps producing multiple copies of the product in lesser time and in more efficient way. Identify and isolate the gene expressing protein for production of α-Amylase 2. easy to handle and easy to replicate inside host cells. For strain improvement of Bacillus subtilis. recombinant DNA can be inserted into desired appropriate host which can increase the production of α-Amylase as the gene will replicate inside host cell(Bacillus subtilis) Fig: formation of chimeric (Recombinant) DNA Gene Amplification Gene amplification involves same technique involved in formation of chimeric DNA discussed above. .coli is best suited for this purpose as the DNA has to be expressed in bacterial species. 3. infect other bacterial species and in so doing introduce the genetic information from the first host and express it in the second host. After successful ligation. Insertion can be done by digesting both insert DNA and vector with same Restriction Endonuclease enzyme 4.In vivo techniques make use of phage particles which will pick up genetic information from the chromosome of one bacterial species. Insert it in the suitable vector (plasmid or phage).

During washing with PEG solution. This increased α-Amylase production by 10 folds. during fusion two or more protoplasts come in contact and adhere with one another either spontaneously or in presence of fusion inducing agents. By protoplast fusion it is possible to transfer some useful genes such as disease resistance. Then High voltage current pulses are given to fuse those protoplasts . protein quality. nitrogen fixation. gene amplification is done by transferring amyE gene responsible for production of α-Amylase from Bacillus subtilis host cell to Bacillus amyloliquefaciens host cell.In the case of α-Amylase. more product formation rate. Briefly. Electrofusion Polyethylene glycol (PEG) treatment: PEG mediated protoplast fusion is best suited for efficient protoplast fusion as it is less toxic. over the other two methods of chemical fusion of protoplasts. PEG with a highly alkaline solution (pH 9-10) containing a high Ca2+ ion concentration (50 mM CaC12. It creates disturbance in the plasma membrane of protoplast. herbicide resistance. Protoplast fusion is a physical phenomenon. Another merit of PEG-induced fusion.  Protoplast Fusion Protoplasts are the cells of which cell walls are removed and cytoplasmic membrane is the outermost layer in such cells. Chemical fusion • NaNO3 treatment • High pH and high Ca2+ treatment • PEG (Polyethylene Glycol) treatment 2.2H20) leads to a higher frequency of fusion than washing with the culture medium. rapid growth rate.r. hence results in fusion of protoplast placed nearby w. Protoplasts are pushed with gentle pace and are made to come close to each other. Electrofusion: Low voltage electric current is used to align the protoplast. each other. frost hardiness. is the formation of a high proportion of binucleate heterokaryons. the freshly isolated protoplasts from the two selected parents are mixed in appropriate proportions and treated with 15-45% PEG (1500-6000 MW) solution for 15-30 min followed by gradual washing of the protoplasts with the culture medium. Protoplast can be fused in the following ways: 1. drought resistance. heat and cold resistance from one species to another hence giving improved strain(hybrid).t. PEG pulls the components of plasma lama. Protoplast can be obtained by specific lytic enzymes to remove cell wall.

to threefold over. was ligated into pAMY10 linearized with EcoRI. carrying the amyR2 locus and part of the amyE gene. The basic procedure requires the synthesis of a short DNA primer containing the desired base change. yielded the increased amylase activity in the presence of glucose that is characteristic of the amyR2 strain. Site Directed Mutagenesis in α-Amylase The amyR2 allele of the Bacillus subtilis ox-amylase cis-regulatory region enhances production of amylase and transcription of amyE. The amylase gene bearing each of these alleles is cloned on plasmids of about 10 to 15 copies per chromosome. Site-directed mutagenesis is used to change bases in the promoter-operator region of amyRl to their amyR2 counterparts. Fig: Construction of pAR2. . E. In general. is a molecular biology technique in which a mutation is created at a defined site in a DNA molecule. and cells containing the amyR2-bearing plasmid. by two. which copies the rest of the gene. pAR2 produced two. An EcoRI fragment from plasmid pEATalW15. The single stranded fragment is then extended using a DNA polymerase. this form of mutagenesis requires that the wild type gene sequence be known. The amount of amylase produced is roughly proportional to the copy number of the plasmid. mutants are selected. coli JM109 was transformed to Cm" Amy'. Site Directed Mutagenesis Site-directed mutagenesis. next to the linker insertion of 3' deletion site. This synthetic primer has to hybridize with a single-stranded DNA containing the gene of interest. but only the change at +7. The double stranded molecule thus obtained is then introduced into a host cell and cloned. the structural gene. Transcription of the cloned amylase gene by each amyR allele is activated at the end of exponential growth and was subject to catabolite repression by glucose. Either change alone increases amylase production twofold.to threefold more amylase. also called site-specific mutagenesis or oligonucleotidedirected mutagenesis. Finally.

Production of α-Amylase α-Amylase was produced in fermentation media containing:  Amylum 0. 4. and fermentation duration of 72 hours. Optimization of Culture Medium and Production Conditions Once the biological production organism has been genetically engineered to overproduce the desired products.1% insoluble starch) and nitrogen (0. a production process has to be developed.2H2O 0.6.  Carbon source (Sucrose) This is a cost effective and cheap media. In some cases.6.01% and pH 6.  Yeast extract 0. Reversion mutants of appropriate auxotrophs may often be high producers. The optimisation of a fermentation process for amylase includes • • • Media composition: discussed below Cultivation type : Fed batch culture is optimum as it prevents substrate inhibition and catabolite repression Process conditions like pH= 6.2% sodium nitrate) for maximum production of alpha-amylase are established. selection for resistance to the antibiotic produced by the organism itself may lead to increased yields.Plasmids were isolated and screened by restriction digests for the replacement of the pAMY10 amyRI allele by amyR2. It is shown that optimum parameters of the producer cultivation are • Temperature 42 degrees C • Carbon nitrogen ratio 1:2 • pH 7. They regulate and control the feedback inhibition for product formation hence increasing the amount of product formed.  KH2PO4 0. and the black box is amnyR.5%. The fermentation temperature of 37°C is used. The amyE is the structural gene for a-amylase. Optimization of culture conditions and Media The optimum sources of carbon (0.  Auxotrophic Mutants Auxotropic mutants are a mutant strain of microorganism that will proliferate only when the medium is supplemented with some specific substance not required by wild-type organisms.0 .5%.05% and  CaCl2. temp= 37°C as described below.

Optimization of Recovery Process (and purification if needed) Isolation of α-amylase α-Amylase in fermentation media was separated from cell of locale bacteria isolate B. Formulation of a Stable Enzyme Product The recovered enzyme solution can be treated with low concentration of boric acid or calcium chloride to stabilize its structure. . Ion exchange column chromatography is performed 3. Purification of α-Amylase The purification of enzyme can be in a few steps. If needed the purification is carried out by ion exchange or gel filtration. As a result the enzyme activity is observed to be increased by 14 times. subtilis by cold centrifuge with speed of 6000 rpm for 30 minutes as to obtain raw extract enzyme. And in the end. • • 5.Volume of a nutritious medium 100 ml Rotation rate 220 rev/min during 4-6 days. 6. Molecule filtration column chromatography to get the purified enzyme. separators or microfiltration) by ultrafiltration. This will ensure minimum degradation of enzyme and will enhance its activity. Amylase can be recovered after cell removal (by vacuum drum filtration. Fractionation of raw extract with ammonium sulphate salt in a variety of saturated degree 2. Calcium chloride basically strengthens and stabilizes the polypeptide sequences The final product is either a concentrated liquid with necessary preservatives like salts or polyols or alternatively can be granulated to a non-dusty dry product. namely: 1.

α-Amylase is best produced from Bacillus and Aspergillus species The enzyme production process can be divided into following phases: 1. it is important to develop a technique of enzyme isolation and purification in order to obtain enzyme which posses’ not only high activity but also high grade purified enzyme. α-Amylase(α-1. Selection of an enzyme 2. It quantitatively increases the product formation. Optimization of culture medium and production conditions 5.2. we will use in vitro techniques as it helps producing multiple copies of the product in lesser time and in more efficient way. Either change alone increases amylase production twofold.SUMMARY & CONCLUSION In this highly commercialized time. EC 3. . glycogen and oligosaccharide. which is then incubated and inserted within suitable host cell. Formulation of a stable enzyme product Following methods have been applied for Genetic Modification of microbe for strain improvement • Using recombinant DNA technology • Protoplast fusion • Selection of Auxotrophic mutants • By site directed mutagenesis We conclude that for strain improvement of Bacillus subtilis.4-glucan-4-glucanohydrolase.1) is widely used enzyme in industrial sectors which catalyses the breakdown of α-D-(1. Briefly. Site-directed mutagenesis in Bacillus subtilis is used to change bases in the promoteroperator region of amyRl to their amyR2 counterparts. in vitro technique involve insertion of gene of interest into vector to create recombinant DNA. Optimization of recovery process (and purification if needed) 6. where it replicates and expresses the gene.1. Selection of a production strain 3.4)-glucosidic bond in amylum. Construction of an overproducing strain by genetic engineering 4.

Amylase can be recovered after cell removal (by vacuum drum filtration. separators or microfiltration) by ultrafiltration Fractionation of raw extract with ammonium sulphate salt in a variety of saturated degree and Ion exchange column chromatography is performed for purification of α-Amylase. The final product is either a concentrated liquid with necessary preservatives like salts or polyols or alternatively can be granulated to a non-dusty dry product. . Yeast extract 0. α-Amylase in fermentation media was separated from cell by cold centrifuge with speed of 6000 rpm for 30 minutes as to obtain raw extract enzyme.2% sodium nitrate)  Carbon nitrogen ratio 1:2. Nitrogen (0. pH 7.5%.2H2O 0.1% insoluble starch).0  Volume of a nutritious medium 100 ml  Rotation rate 220 rev/min during 4-6 days. This will ensure minimum degradation of enzyme and will enhance its activity.05% and CaCl2.01% As a result the enzyme activity is observed to be increased by 14 times.5%.  KH2PO4 0.  Amylum 0.It is shown and hence concluded that optimum parameters for the cultivation for maximum production of alpha-amylase are:  Temperature 42 degrees C  Carbon (0. The recovered enzyme solution can be treated with low concentration of boric acid or calcium chloride to stabilize its structure.