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

Students 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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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

INTRODUCTION
Nowadays, the use of enzymes in industrial sector is increasing due to the increase of industries especially in food, beverage, textile, leather and paper industries. Besides it, enzymes have been used in industry for the treatment of industrial waste, such as cellulase which is able to convert cellulose of wood and paper wastes to ethanol. In this highly commercialized time, 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. One of the enzymes widely used in industrial sectors is -Amylase enzyme (-1,4-glucan-4-glucanohydrolase, EC 3.2.1.1) which catalyses the breakdown of -D-(1,4)-glucosidic bond in amylum, glycogen and oligosaccharide. Enzymes are produced at industrial scale by using over producing strains which are genetically modified (G.M.) to give the maximum production rate of desired enzyme. Commonly, strains of Aspergillus, Bacillus and flavobacterium are used. It is because they are cheap and readily available. Most of the enzymes are, however, produced by microorganisms in submerged cultures in large reactors called fermentors. The enzyme production process can be divided into following phases: 1. Selection of an enzyme 2. Selection of a production strain 3. Construction of an overproducing strain by genetic engineering 4. Optimization of culture medium and production conditions 5. Optimization of recovery process (and purification if needed) 6. Formulation of a stable enzyme product

ENZYME PRODUCTION PROCESS

1. Selection of an Enzyme
We have selected our enzyme that we are interested in producing i.e. -Amylase

Alpha ()-Amylase
-Amylase is an enzyme that hydrolyses alpha-bonds of large alpha-linked polysaccharides such as starch and glycogen, yielding glucose and maltose. It catalyses the breakdown of -D-(1,4)-glucosidic bond in amylum, glycogen and oligosaccharide. It is the major form of amylase found in humans and other mammals. It is also present in seeds containing starch as a food reserve, and is secreted by many fungi.

2. Selection of a Production Strain

-Amylase is best produced from Bacillus and Aspergillus species. We choose Bacillus subtilis as a production strain for -Amylase as genetic modification can easily been done and it has given higher yield of -Amylase after genetic modification by recombinant DNA technology.

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. Thus genetic material derived from one species may be incorporated into another where it may be expressed, hence, helping in increase of product formation i.e. Amylase in this case.

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

In the case of -Amylase, gene amplification is done by transferring amyE gene responsible for production of -Amylase from Bacillus subtilis host cell to Bacillus amyloliquefaciens host cell. This increased -Amylase production by 10 folds.

Protoplast Fusion
Protoplasts are the cells of which cell walls are removed and cytoplasmic membrane is the outermost layer in such cells. Protoplast can be obtained by specific lytic enzymes to remove cell wall. Protoplast fusion is a physical phenomenon, 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, nitrogen fixation, rapid growth rate, more product formation rate, protein quality, frost hardiness, drought resistance, herbicide resistance, heat and cold resistance from one species to another hence giving improved strain(hybrid). Protoplast can be fused in the following ways: 1. Chemical fusion NaNO3 treatment High pH and high Ca2+ treatment PEG (Polyethylene Glycol) treatment 2. Electrofusion Polyethylene glycol (PEG) treatment: PEG mediated protoplast fusion is best suited for efficient protoplast fusion as it is less toxic. Another merit of PEG-induced fusion, over the other two methods of chemical fusion of protoplasts, is the formation of a high proportion of binucleate heterokaryons. Briefly, 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. PEG with a highly alkaline solution (pH 9-10) containing a high Ca2+ ion concentration (50 mM CaC12.2H20) leads to a higher frequency of fusion than washing with the culture medium. During washing with PEG solution, PEG pulls the components of plasma lama. It creates disturbance in the plasma membrane of protoplast, hence results in fusion of protoplast placed nearby w.r.t. each other.

Electrofusion: Low voltage electric current is used to align the protoplast. Protoplasts
are pushed with gentle pace and are made to come close to each other. Then High voltage current pulses are given to fuse those protoplasts

 Site Directed Mutagenesis

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

Fig: Construction of pAR2. An EcoRI fragment from plasmid pEATalW15, carrying the amyR2 locus and part of the amyE gene, was ligated into pAMY10 linearized with EcoRI. E. coli JM109 was transformed to Cm" Amy'.

Plasmids were isolated and screened by restriction digests for the replacement of the pAMY10 amyRI allele by amyR2. The amyE is the structural gene for a-amylase, and the black box is amnyR.

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. They regulate and control the feedback inhibition for product formation hence increasing the amount of product formed. Reversion mutants of appropriate auxotrophs may often be high producers. In some cases, selection for resistance to the antibiotic produced by the organism itself may lead to increased yields.

4. Optimization of Culture Medium and Production Conditions

Once the biological production organism has been genetically engineered to overproduce the desired products, a production process has to be developed. 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= 6.6; temp= 37C as described below.

Production of -Amylase -Amylase was produced in fermentation media containing: Amylum 0.5%; Yeast extract 0.5%; KH2PO4 0.05% and CaCl2.2H2O 0.01% and pH 6.6. Carbon source (Sucrose) This is a cost effective and cheap media. The fermentation temperature of 37C is used; and fermentation duration of 72 hours. Optimization of culture conditions and Media The optimum sources of carbon (0.1% insoluble starch) and nitrogen (0.2% sodium nitrate) for maximum production of alpha-amylase are established. It is shown that optimum parameters of the producer cultivation are Temperature 42 degrees C Carbon nitrogen ratio 1:2 pH 7.0

Volume of a nutritious medium 100 ml Rotation rate 220 rev/min during 4-6 days. As a result the enzyme activity is observed to be increased by 14 times.

5. Optimization of Recovery Process (and purification if needed)

Isolation of -amylase -Amylase in fermentation media was separated from cell of locale bacteria isolate B. subtilis by cold centrifuge with speed of 6000 rpm for 30 minutes as to obtain raw extract enzyme. Amylase can be recovered after cell removal (by vacuum drum filtration, separators or microfiltration) by ultrafiltration. If needed the purification is carried out by ion exchange or gel filtration. Purification of -Amylase The purification of enzyme can be in a few steps, namely: 1. Fractionation of raw extract with ammonium sulphate salt in a variety of saturated degree 2. Ion exchange column chromatography is performed 3. And in the end, Molecule filtration column chromatography to get the purified enzyme.

6. 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. This will ensure minimum degradation of enzyme and will enhance its activity. 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.

SUMMARY & CONCLUSION

In this highly commercialized time, 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. -Amylase(-1,4-glucan-4-glucanohydrolase, EC 3.2.1.1) is widely used enzyme in industrial sectors which catalyses the breakdown of -D-(1,4)-glucosidic bond in amylum, glycogen and oligosaccharide. -Amylase is best produced from Bacillus and Aspergillus species The enzyme production process can be divided into following phases: 1. Selection of an enzyme 2. Selection of a production strain 3. Construction of an overproducing strain by genetic engineering 4. Optimization of culture medium and production conditions 5. Optimization of recovery process (and purification if needed) 6. 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, we will use in vitro techniques as it helps producing multiple copies of the product in lesser time and in more efficient way. It quantitatively increases the product formation. Briefly, in vitro technique involve insertion of gene of interest into vector to create recombinant DNA, which is then incubated and inserted within suitable host cell, where it replicates and expresses the gene. Site-directed mutagenesis in Bacillus subtilis is used to change bases in the promoteroperator region of amyRl to their amyR2 counterparts. Either change alone increases amylase production twofold.

It is shown and hence concluded that optimum parameters for the cultivation for maximum production of alpha-amylase are: Temperature 42 degrees C Carbon (0.1% insoluble starch); Nitrogen (0.2% sodium nitrate) Carbon nitrogen ratio 1:2; pH 7.0 Volume of a nutritious medium 100 ml Rotation rate 220 rev/min during 4-6 days. Amylum 0.5%; Yeast extract 0.5%; KH2PO4 0.05% and CaCl2.2H2O 0.01% As a result the enzyme activity is observed to be increased by 14 times. -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. 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 -Amylase. The recovered enzyme solution can be treated with low concentration of boric acid or calcium chloride to stabilize its structure. This will ensure minimum degradation of enzyme and will enhance its activity. 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.