Title of Invention	"GENES, VECTORS AND PRODUCTION OF STABLE LIPASE"
Abstract	The present invention relates to novel gene variants of lipase enzyme as developed through site directed muatgenesis. These gene variants are highly thermostable, resistant against strong organic solvents and can tolerate high pH. The thermostability of the developed gene variants was as high 200 fold in the temperature range of 50 to 90°. The developed gene variants due to high thermostability, specific activity and tolerance due to high pH have application in household detergents and laundry industry.

Title of Invention

"GENES, VECTORS AND PRODUCTION OF STABLE LIPASE"

Abstract

The present invention relates to novel gene variants of lipase enzyme as developed through site directed muatgenesis. These gene variants are highly thermostable, resistant against strong organic solvents and can tolerate high pH. The thermostability of the developed gene variants was as high 200 fold in the temperature range of 50 to 90°. The developed gene variants due to high thermostability, specific activity and tolerance due to high pH have application in household detergents and laundry industry.

Full Text	Field of Invention: The present invention relates to creation, optimization and production of a lipase. More specifically, the invention relates to the generation and production of lipase variants that are thermostable and also stable in organic solvents. The invention also relates to methods of selecting lipase variants for high temperatures and for their purification. BACKGROUND OF THE INVENTION Enzymes are the workhorses of a cell that affect virtually every biological process that characterizes a living organism. They catalyze chemical reactions with remarkable specificity and rate enhancements. The awesome catalytic power and versatility of enzymes has long been recognized and enzymes have proved to be very useful outside the living system as well. Enzymes today have widespread application in industry and are seen as environment friendly alternatives to chemical reagents because enzymatic reactions require milder conditions and tend to be cleaner with lesser byproduct and waste generation. Enzymes are being used in numerous new applications in the food, feed, agriculture, paper, leather, and textiles industries, resulting in significant cost reductions and environment-friendly operations. Enzymes have evolved to function best under the physiological conditions of the parent organism. In vitro applications often call upon enzymes to work under non-physiological conditions or to perform functions they have not evolved for. For example, enzymes may have to catalyze reactions involving novel substrates; they may have to work under extreme conditions of salt, temperature, pH etc., or in the presence of potentially inhibiting or denaturing chemicals. Such applications have brought to light the severe disabilities of enzymes to function as industrial catalysts. In order to extract optimum performance from enzymes in the test-tube and in industrial reactors, these biocatalysts need to be tailored to suit specific applications. The commercial success of these enzymes can be attributed to their ease of use. In addition to functioning at high temperatures, thermostable enzymes generally posses an increased shelf life which markedly improves handling conditions. If enzymes are to play a significant role in large scale processing of chemicals, they must be able to endure the harsh conditions associated with these processes. Thermostable enzymes are easier to handle, last longer, and given the proper immobilization support should be reusable for multiple applications In obtaining thermostable enzymes the conventional approach is to screen the microbial collections collected from extremephillic environments1. The promising candidate enzymes are further investigated for suitability for a specific process. For example, for applications requiring thermostable or salt stable enzymes, enzymes from thermophilic or halophilic organisms were used, respectively. However, such an approach severely restricted the use of enzymes because enzymes for all applications cannot be found in nature. There may not be a natural enzyme for many kinds of transformations. Moreover, enzyme usage is often restricted by undesirable properties of enzymes like product inhibition, low stability etc. Very often an enzyme is required to have a combination of several properties that may be impossible to find in a natural enzyme. Another approach to obtain thermostable enzymes is based on the current knowledge on the protein structures (crystal structures) of homologous enzymes from mesophiles and thermophiles 2-3. Such comparisons yielded information on the probable interactions that enhance thermostability. Using such information efforts were made to incorporate these changes in mesophillic enzymes to improve their thermostability. Such approaches have not been very successful since interactions that improve thermostability in a protein are many and each protein acquires, over evolutionary times, those interactions that are best suited for its sequence and the mileu in which it functions. Though structural determinants of protein stability have been objects of numerous studies on model proteins, no universal stabilization mechanism has yet been found 4. The most obvious conclusion that can be drawn from the literature is that different proteins have adapted to different thermal environments by a variety of evolutionary devices. The lack of understanding of the structural features leading to protein thermostability has been partly due to a scarcity of data because experimental studies comparing homologous proteins from psychrophilic, mesophilic and thermophilic organisms have been limited to only a few proteins. Moreover, inability to form definite rules for improving protein thermostability is due to the large number and complexity of possible contributing factors 4'7. Based on comparisons between mesophiles and thermophiles, the main mechanisms responsible for increased thermostability have been identified as increase in the number of hydrogen bonds and salt bridges, increased core hydrophobicity, better packing efficiency, a-helix and loop stabilization and resistance to covalent destruction. Often it becomes difficult to delineate protein interactions that contribute to thermostability from other selection pressures such as salt, pH etc. Other strategies adapted to increase the thermostability was based on the observation that immobilized enzymes acquire thermostability to some extent8. Hence, several solid supports were tried to immobilize proteins. And also recent observations made with enzymes in organic solvents indicated that in organic solvents enzymes acquire thermostability 9. The advent of recombinant DNA techniques has greatly facilitated protein engineering by allowing facile mutagenesis and production of proteins. The term protein thermostability refers to the preservation of the unique chemical and spatial structure of a polypeptide chain under extremes of temperature conditions 4. In general, the higher the temperature to which the enzyme is exposed, the shorter the half-life of the enzyme (i.e., the shorter the enzyme retains its activity). Similarly, the greater levels of organic solvent to which said enzymes are exposed, the shorter the half-life of the enzyme. The phrase "catalytic activity" or simply "activity," means an increase in the k.sub.cat or a decrease in the K.sub.M for a given substrate, reflected in an increase in the k.sub.catt /K.sub.M ratio.The structural basis of protein thermostability has been an actively pursued area of research for at least two decades 10 . However, enzymes lifted out of the context of living organisms do not always function as well as they do in their natural milieu. For example, they have optimum activity at the physiological temperature of the organism and tend to denature at higher temperatures leading to drop in activity. Thermostable enzymes are important as they can be used at high temperatures and harsher conditions required in industrial contexts. Also they generally have higher storage stabilities and bring down costs by obliviating the need for low temperature storage and decreasing the loss due to denaturation on storage and handling. Moreover reactions carried out at higher temperatures generally proceed at higher rates further bringing down operation times. In view of the environmental safety reasons, there is a constant pressure to reduce the use of environmentally polluting processes in industry. Enzymes are increasingly used to replace chemical processes in leather, food, and pharmaceutical industries. Comparison of protein structures from extremeophiles demonstrated that protein structural plasticity is enormous and is resident in the primary structure. This lent considerable support to strategies that alter the primary structure of the proteins at the genetic level and screen for the variants with special properties such as thermostability. The tremendous success in handling the genes and developing protocols to alter it at will, has allowed to evolve proteins with special functions. The strategy relies in generating variation in gene sequences by molecular biology methods and screening the variants by expressing them and screening the mutant population n-13. The screening protocols are based on the property of interest, e.g., activity at high temperature or activity in the presence of organic solvents. The present invention encompasses methods for generating variation in gene sequences, protocols for screening the enzymes with higher thermostability and also protocols for sequencing and expression. Lipases (triacylglycerol acylhydrolases, E.G. 3.1.1.3) are water-soluble enzymes that catalyze the hydrolysis of ester bonds in triacylglycerols and often also exhibit phospholipase, cutinase and amidase activities 14. They are used for the production of detergents, pharmaceuticals, perfumes, flavour enhancers and texturising agents in consmetic products. Lipases are crucial for the production of a wide variety of foods, especially for products from milk, fat and oil. Lipases are ubiquitous enzymes of considerable physiological significance and industrial potential. Lipases catalyze the hydrolysis of triacylglycerols to glycerol and free fatty acids. In contrast to esterases, lipases are activated only when adsorbed to an oil-water interface and do not hydrolyze dissolved substrates in the bulk fluid. A true lipase will split emulsified esters of glycerine and long-chain fatty acids such as triolein and tripalmitin. Lipases are serine hydrolases. Commercially useful lipases are usually obtained from microorganisms that produce a wide variety of extracellular lipases 15 . Many lipases are active in organic solvents where they catalyze a number of useful reactions including esterification transesterification, regioselective acylation of glycols and menthols, and synthesis of peptides and other chemicals. An increasing number of lipases with suitable properties are becoming available and efforts are underway to commercialize biotransformation and syntheses based on lipases 16. Enzyme sales for use in washing powders still remain the single biggest market for industrial enzymes. The major commercial application for hydrolytic lipases is their use in laundry detergents. Detergent enzymes make up nearly 32% of the total lipase sales. Lipase for use in detergents needs to be thermostable and remain active in the alkaline environment of a typical machine wash. An estimated 1000 tons of lipases are added to approximately 13 billion tons of detergents produced each year Because of their ability to hydrolyzes fats, lipases find a major use as additives in industrial laundry and household detergents. Detergent Upases are especially selected to meet the following requirements: (1) a low substrate specificity, i.e., an ability to hydrolyze fats of various compositions; (2) ability to withstand relatively harsh washing conditions (pH 10-11, -60 _C); (3) ability to withstand damaging surfactants and enzymes [e.g., linear alkyl benzene sulfonates (LAS) and proteases], which are important ingredients of many detergent formulations. Lipases with the desired properties are obtained through a combination of continuous screening 17-19 and protein engineering 20 . In 1994, Novo Nordisk introduced the first commercial recombinant lipase 'Lipolase/ which originated from the fungus Thermomyces lanuginosus and was expressed in Aspergillus oryzae. In 1995, two bacterial lipases were introduced — 'Lumafast' from Pseudomonas mendocina and 'Lipomax' from P. alcaligenes — by Genencor International15. According to a report an alkaline lipase, produced by P. alcaligenes M-l, which was well suited to removing fatty stains under conditions of a modern machine wash. The patent literature contains examples of many microbial lipases that are said to be suitable for use in detergents 22. Lipase-producing microorganisms include bacteria, fungi, yeasts, and actinomyces. Bacillus subtilis is a Gram-positive, aerobic, spore-forming bacterium that has generated substantial commercial interest because of its highly efficient protein secretion system Though extracellular lipolytic activity of B.subtlis was observed as early as in 1979 23, molecular research started in 1992 when a lipase gene, lipA, was cloned and sequenced 24. Subsequently the lipase was overexpressed, purified and characterized 25. Later, a second gene, lipB, was found that is 68% identical with lip A at the nucleic acid level 26. This gene has been cloned and the protein overexpressed, purified and characterized. The Bacillus subtilis lipase with a molecular weight of 19,348 Da is one of the smallest lipases known. It is one of the few lipases that do not show the interfacial activation in the presence of oil-water interfaces. LipA is very tolerant to basic pH and has its optimum activity at pHlO. It hydrolyses the sn-1 and sn-3 glycerol esters with both short and long chain fatty acids, showing optimum activity with C8 fatty acid chains. Bacterial lipases are classified into eight families according to their sequence similarities, conserved sequence motifs and biological properties 27. The true lipases are classified in family I which contains six subfamilies. Bacillus lipases have been placed in subfamilies 4 and 5. In these two subfamilies alanine replaces the first glycine residue in the conserved G-X-S-X-G pentapeptide around the active site serine residue. Subfamily 4 consists of only three members, LipA and LipB from B.subtilis and a lipase from Bacillus pumilis, which share 74- 77% sequence identity. These are the smallest lipases known and show very little sequence similarity (~ 15%) with the other, much larger, Bacillus lipases that constitute subfamily 5. The crystal structure of the B.subtilis lipase LipA reveals a globular protein with dimensions of 35 X 36 X 42 28. The structure shows a compact domain that consists of six P- strands in a parallel p- sheet, surrounded by a- helices. There are two a- helices on one side of the P- sheet and three on the other side. The fold of the B.subtilis lipase resembles that of the core of the a/p hydrolase fold enzymes. The B.subtilis lipase lacks the first two strands of the canonical a/p hydrolase fold and the helix ccD is replaced by a small 310 helix. The helix aE is exceptionally small, with only one helical turn, and several a-helices start or terminate with 310 helical turns. Due to these structural features, its small size and absence of a lid domain, the B.subtilis lipase is considered a minimal a/p hydrolase fold enzyme. Description of the invention: In accordance with the present invention a methods of directed evolution were applied to the lipase gene ( Gene Seq ID # V) to isolate protein variants of the original sequence which possess increased thermostable properties. The methodology relies initially on the ability to create random variations in the original gene sequence and express the corresponding proteins in the bacteria, E.coli. The produced variants of the original sequence would have altered sequence, hence altered properties. The variants, at a proteins level, would be tested for their thermostability and those sequences which demonstrate improved thermostability would be subjected to the next round of random mutagenesis and screening. Thus by sequential accumulation of the mutants and subsequent pooling of the mutations the thermostability of lipase was improved by 200-fold at high temperature. High temperature range includes temperature ranges from 50-90 C. The method according to the present invention includes four steps in generating the variant Upases and their characterization to obtain thermostable Upases. In the first of the methods variation in the primary sequence of the lipase gene were generated by error prone PCR methods. The adapted protocols are similar to several published protocols. In addition many of the different protocols such as random priming, ITCHY etc could also be applied for generation of variance in the gene sequence 29-30. In the second step of the invention, the mutant sequences are cloned into an expression vector and the protein expressed in the culture lysates. The expressed proteins are screened for their ability to withstand higher temperature was tested in a large population using medium-throughput methods. In the third step of the invention the promising variants were pooled by a family shuffling procedures according to the method of Stemmer 12 and further tested for thermostability. In addition to the shuffling procedures selective changes in the primary sequence were also incorporated by standard molecular biology procedures. In the fourth step of the invention the positive sequences were over-expressed in large cultures and the proteins are purified by the published procedures according to Colson et al24. The purified proteins were tested for their thermostability. The screening procedures of the lipase variants for increased thermostability involve ability to hydrolyse chromogenic substrate esters based on p-nitrophenyl group. Natural substrates of lipase are triglycerides, which are not convenient to design a simple medium trough put assays wherein the source of enzyme is over-expressed lipase in a cell lysate 31. P-nitrophenyl esters of fatty acids are, convenient and the activity of lipase represents the activity of lipase on triglycerides. Long chain esters of p-nitrophenyl, especially p-nitrophenyl oleate, are well suited for this purpose. Detergent solubilized PNPO demonstrates negligible back ground hydrolysis and well suited for lipase present in cell lysate. The strong yellow color of very high extinction coefficient of hydrolysis product p-nitrophenyl can be estimated conveniently in a 96-well plates. Along with p-nitrophenyl esters, many other fluorogenic or chromogenic esters of fatty acids could be used for this purpose. As employed herein the term thermostability refers to the property of the enzymes, which retain their activity subsequent to exposure to higher temperatures. Enzymes lose their tertiary conformation on exposure to higher temperature due to the increased movement of the structural elements, which perturbs the functional structure of the protein. Typically proteins lose their activity at higher temperatures with time. The rate of this loss in activity, reflects in half life i.e., time required to lose half of the initial activity, is a convenient parameter to compare the thermostability of the protein 4. Activity, as defined here, corresponds to the catalytic acitivity represented by the term kcat/Km, where kcat is the rate of the product formation and Km is the apparent affinity constant of the substrate to the enzyme. Retaining the functional structure at elevated temperatures resides in the ability to form interactions within the protein that withstand high temperatures. The range of the temperature that is relevant for the present invention ranges from 35 to 90 C. The naturally occurring lipase from Bacillus lipase has the amino acid sequence of 1-181 as given in the sequence ID # . The corresponding nucleic acid sequence expressing the protein (ID # )was presented in ID # . It was discovered that the amino acid substitutions at positions 68, 71,114,120,132,144,147 and 166 were found to be important for the thermostability of the lipase. In accordance with the present investigation, it was further discovered that the substitutions at positions 114, 132 and 166 are suited for increasing the stability of the proteins. Any of the innumerable combinations of substitutions possible at each of these positions with the other 19 amino acids would be favourable for the thermostability. The specific substitutions of relevance for thermostability in lipase are given below. From To Position N V 166 A D 132 A V 68 L P 114 R S 147 V A 144 N D 120 Examples: Example 1: Purification of lipase from Bacillus subtilis Purification of the lipase was performed from E.coli cells expressing the lipase in an appropriate vector. The purification essentially involves passing the cell lysate in phenyl-sepharose column followed by a Mono-S column. Lipase is an aggregated prone protein, care especially keeping protein concentration below 5mg/ml, was taken to avoid aggregation of the protein. The purification of the lipase was carried out essentially as described earlier32 with minor modifications. Lipase from culture filterates of Bacillus strain or from the E.coli lysates was processed similarly. For purification of the wild type and mutant proteins from E.coli the lip A gene or the mutant genes are cloned into pET 21b. ). For this, the gene corresponding to the full length, mature protein was amplified with primers PrNde I (forward primer) (5'- CCATGATTACGCATATGGCTGAACACAA-3') and JOF. The forward primer had an engineered Nde I site. The forward primer also introduced a start codon at the start of the lipase gene in the form of the ATG sequence that is part of the Nde I recognition sequence. This would introduce a methionine in the Nterminus of the mature protein, expressed in E.coli, just before the N-terminal alanine that occurs in the protein purified from the culture supernatant of B.subtilis. The wild type protein as well as the mutants were amplified, digested with Nde I and BamH I and ligated with pET-21b digested with Nde I and BamH I. The ligation mix was transformed into E.coli DH5a and the positives were selected by plasmid minipreps and restriction digestions (Fig.l). Protein was purified from E.coli BL21 (DE3) cells. Cells containing the appropriate plasmid were grown till mid-log phase before inducing with 0.5 mM IPTG. Cells were harvested 2.5 hours after induction by centrifuging at 15,000 rpm at 4 °C for 20 min. The pellet was washed with STE and resuspended in IX TE containing 0.3 mg/ml lysozyme. The suspension was incubated on ice for 30 min before lysing the cells by sonication. Sonication was carried out by keeping the cells on ice. Short pulses of half-minute duration were applied and 1 min cooling time was allowed between pulses. The sonicated cells were centrifuged at 20,000 rpm at 4 °C. The supernatant was loaded on a phenyl sepharose column. The remaining steps were done as described in chapter 2. The purified proteins were stored in -70 °C till further use. B.subtilis BCL1051 was grown aerobically for 16-18 hrs at 37 °C in 21 Erlenmeyer flasks, each containing 500 ml of medium of the following composition: 2.4 % yeast extract, 1.2 % tryptone, 0.4 % gum Arabic, 0.4 % glycerol, 0.017 M KH2PO4, 0.072 M K2HPO4, 50 mg/ml kanamycin sulfate. The culture medium was inoculated at 1 % from 10 ml precultures. After harvesting the cells by centrifugation at 6000 rpm for 30 min, the culture supernatant was pumped at a flow rate of 30 ml/hr onto a Phenyl Sepharose Fast Flow High sub column (Pharmacia) (20 ml column volume per 11 culture) equilibrated with 100 mM potassium phosphate, pH 8.0. The column was washed at a flow rate of 50 ml/hr first with 10 mM potassium phosphate, pH 8.0 and then with 30% ethylene glycol in 10 mM potassium phosphate, pH 8.0. Elution was performed at a flow rate of 50 ml/hr with 80 % ethylene glycol in 10 mM potassium phosphate, pH 8.0. 2 ml fractions were collected and the fractions containing protein (detected by absorbance at 280 nm) were checked for enzyme activity. The active fractions were pooled and dialyzed against 2 mM glycine-NaOH, pH 10.0. The dialyzed protein was diluted 1:1 with 50 mM Bicine-NaOH, pH 8.5 (buffer A) and loaded onto a MonoS HR5/5 (Pharmacia) column, pre-equilibrated with buffer A, using a Superloop (Pharmacia) on a FPLC (Pharmacia) system. The protein-boundcolumn was washed thoroughly with the buffer A to remove unbound proteins. The protein was eluted using a linear gradient with buffer A to buffer B (50 mM Bicine-NaOH, pH 8.5,1 M NaCl). The enzyme eluted around 300 mM NaCl as a single peak. The active fractions eluted from the MonoS column were dialyzed overnight against 2 mM glycine, pH 10.0 and concentrated using an Amicon concentrator fitted with a YMIO membrane (10 kD cutoff). Purity of the protein was checked on a 12% SDS-PAGE gel containing 5 M urea (Lessuisse et al, 1993). The protein was 95 % pure on a Coomassie stained gel (Fig.2). Example 2: Assay of lipase: Lipase belongs to a class of enzymes known as interfacially active enzymes. These enzymes have very little activity on the substrate monomers but their activity increases dramatically on insoluble substrate such as emulsified triglycerides, monolayers etc. This property makes lipases dissimilar to other enzymes which act on soluble substrate monomers. Triglycerides, natural substrates of lipase are not very convenient to set up simple chromogenic assays (Fig.3). Activity of pure lipases, sometimes, can be monitored by detecting the pH changes using pH-sensitive dyes. However, such assays yield complications when the enzyme source is a lysate and when there are other processes that may alter the pH. P-nitrophenyl esters are most convenient to monitor the activity. Short chain ester, p-nitrophenyl acetate and long chain ester, p-nitro phenyl oleate (PNPO), were synthesized by routine synthetic methods (given below). PNPO is a insoluble ester, was used in our assays using triton X-100 as a solubilizing agent. Triton X-100: PNPO co-micelles showed low back ground hydrolysis and were also stable at elevated temperatures. 96-well plate assays for screening the variants of lipase, though very useful to screen large number of samples, quantitates the activity approximately. All positives obtained in 96-well screens were confirmed in a tube assays, where the number of samples are less and more accurate specific activity calculations could be made. Synthesis of chromogenic substrates for lipase assays The following chromogenic substrates were synthesized for lipase assays: 1) p-nitrophenyl oleate 2) p-nitrophenyl stearate 3) p-nitrophenyl caprylate The fatty acid, N, N'- methyl tetrayl biscyclohexamine (dicyclohexylcarbodiimide, DCC), N, N'- dimethylamino pyridine (DMAP), and p-nitrophenol were taken in mole ratios of 1:1:1:2. The fatty acid was taken in a round bottom flask containing 20 ml of dry DCM and a few ml of chloroform. The mixture was stirred for two minutes followed by addition of DCC. A white precipitate was formed. This was followed by the addition of DMAP. Subsequent addition of p-nitrophenol led to the formation of a yellow precipitate. The reaction vessel was flushed with nitrogen and stirred for 5 hours. The progress of the reaction was monitored by thin layer chromatography. After completion of the reaction, the DCM was evaporated to dryness and the ester was purified by column chromatography (silica gel column, elution with petroleum etheracetone). The purity and identity of the product was confirmed by JR-NMR spectroscopy. Example 3 Lipase assay in 96-well microtitre plates): The colonies obtained from the cloning of the PCR product generated by error-prone PCR were patched on another similar plate and simultaneously inoculated in separate wells of a microtitre plate containing 200 ^il 2XYT containing 25 ng/ml chloramphenicol and 0.2 % glucose. The cells were grown for 24 hours in the microtitre plate with continuous shaking at 200 rpm. After 24 hrs, 5 j^l culture from each well was taken and added to the corresponding well another microtitre plate containing 200 ul 2XYT supplemented with 25 ng/ml chloramphenicol. After 3 hours of growth the cultures were induced with 1 mM IPTG. After 3 more hours 25 ul of culture was taken from every well into the corresponding wells of two fresh microtitre plates containing 25 ul phosphate buffer pH 7.0. One of the plates was exposed to high temperature for 20 min, cooled on ice for 15 min and then allowed to come to room temperature. The other plate was kept at room temperature. 25 ul of the PNPO-Triton X-100 substrate solution prepared as described above was added to each well. The plates were incubated at 37 °C and absorbance at 405 nm was recorded in an ELISA reader at definite time intervals. The clones showing less than 20 % of the activity of the wild-type protein (or the parent from which it is generated) were removed from further consideration. The residual activity for each clone after exposure to high temperature was calculated. The clones showing highest residual activity were chosen for the next level of screening. Lipase assays in tubes:: The colonies that showed highest residual activity in the microtire plate level screen were grown for 12 hours in 5 ml 2XYT medium 25 ug/ml chloramphenicol and 0.2 % glucose. 10 ml of 2XYT containing 25 ng/ml chloramphenicol and 0.2 % glucose was inoculated with 100 \il of the overnight grown culture. After 2.5 hours growth, the cultures were induced with 1.5 mM IPTG and were harvested after another 2.5 hours. The cell pellet was washed with STE and resuspended in 1 ml 0.05 M potassium phosphate buffer pH 7.2. The cell suspension was sonicated with a Branson sonicator with four pulses of 30 sec and 1 min cooling time in between the pulses. The tubes were kept on ice during sonication and cooling of the samples. The sonicated samples were centrifuged at 15,000 rpm for 45 min and the supernatant was used for the assays. The supernatant was divided into four 250 ul aliquots. Three of the aliquots were exposed to higher temperatures and the fourth was kept on ice. The tubes were exposed to high temperatures for 20 min, chilled on ice, centrifuged at 4 °C at 15,000 rpm and then allowed to come to room temperature before assaying for enzymatic activity. The lipase activity in the cell lysates was determined at room temperature in sodium phosphate buffer pH 7.2 by using pnitrophenyl oleate as substrate. The enzymatic activity was measured by following the change of absorbance at 405 nm with time. Lysates of cells that do not contain the lipase gene but otherwise processed in the same way as mentioned above, were used to determine the background hydrolysis of pnitrophenyl oleate in E.coli cell lysate. The background hydrolysis values were subtracted from the enzymatic activity value. The total protein in the cell lysates was determined by Lowry's method and was used to normalize the activity. Example 4 Half-lives of thermal inactivation Exposing the enzymes to higher temperatures and then assaying the activity at room temperature normally assess thermostability of enzymes. At higher temperature the protein denatures and irreversible unfolds. Thermostable enzymes possess additional stabilizing interactions which would make them less susceptible for heat denaturations. The activity remaining is residual activity, which decrease both with increase in temperature or with increase in time at a given temperature. Heat treatment of the purified proteins was carried out in a programmable thermal cycler (GeneAmp PCR system 9700) in 0.2 ml thin-walled PCR tubes to allow precise temperature control of the samples. The proteins were taken at a concentration of 0.05 mg/ml in 0.05 M sodium phosphate buffer, pH 7.0. 25 ^il of protein samples were taken in each tube. The proteins were heated for the required time, cooled at 4 °C for 20 min, centrifuged and equilibrated at room temperature before assaying for enzymatic activity. 20 ul of the heat-treated protein sample was added to 1 ml 0.05 M sodium phosphate, pH 7.2 containing 2 mM p-nitrophenyl acetate. Enzymatic activity was measured at 25 °C by monitoring the rate of increase in absorbance at 405 nm. Typically, inactivation was followed until > 80 % of the activity was lost. Plots of log(residual activity) versus time were linear. Inactivation rate constants (kinact) were obtained from the slope and half-lives were calculated as ti/2 = Iog2/kmact. The half lives of various mutants obtained were presented in figure (Fig.4) where the residual activities were measured using PNPA as substrate. The enzyme mutants were exposed to 55 C. In fig.5 data obtained with residual activities with three mutants using olive oil as a substrate was presented. The activities were measured using pH stat equipment. This data demonstrates that the enhancement seen with mutants was independent of the substrate and nature of the assay. Preparation of substrate stocks: Appropriate amounts of the insoluble p-nitrophenyl ester and Triton X-100 were weighed out in a glass vial and mixed with a magnetic stirrer till the ester completely dissolved in Triton X-100. Buffer was added slowly while stirring to prepare a 2X stock solution containing 0.4 mM p-nitrophenyl ester and 40 mM Triton X-100. Substrate solutions prepared in this way were optically clear. 100X substrate stocks of the water-soluble p-nitrophenyl acetate were made in acetone and 2 mM p-nitrophenyl acetate was used for each reaction. The reactions were carried out in absence of Triton X-100 and all the measurements to determine kinetic parameters were done with this reaction system. Example 5 Assay with olive oil Assay with the olive oil is performed pH stat equipment. All lipases subsequent to thir activity reduce th pH off the reaction medium by releasing a proton. The decrease in pH could be neutralized by addition of known amounts of alkali. The rate of additon of alkali would represent the activity of the lipase. We have prepared the lipase substrate by mixing gum Arabic (0.5%), olive oil ( ) and CaC12( ). The mixture was sonicated in a bath till we obtain a uniform emulsion. We have used 10 ml of the substrate for each assay.At the beginning of the asay the pH of the substrate was brought to 8.4 by addition of alkali. The reaction was started with the addition of 10 microlitres of Img/ml enzyme solution. The rate of reaction was calculated from the slopes of amount of alkali vs. time curves. ).l N NaOH was sued as alkali. Example 6 Methods of generation of variations in the Lipase genes: The sequence ot Lip A, whose product is lipase gene of interest in this invention, from Bacillus subtilis was published. In Bacillus LipA gene product is secreted into the culture medium owing to the presence of a signal sequence at the Nterminal of the sequence, which aids in its transport out of the cell. Molecular biology of Bacillus species has been well studied and it is a Gram-positive strain. For routine molecular biology techniques such as transformation, cloning, expression etc. Bacillus sp. is less suited compared to E.coli 33'34. The main difficulty is in transforming the Bacillus sp with the plasmids. The efficiency is lower by several orders of magnitude compared to E.coli. Further, the observed efficiencies are only detectable with electroporation, which is a harsher method. In E. coli the transformation efficiency is higher and reproducible and the choice of plasmids is wide. To perform various gene manipulations, E.coli was used. The clone pLipA containing the complete lipA gene in pBR322 plasmid was a kind gift from Dr Frens Pierce (Fig.6). The lipase gene along with the region coding for the signal sequence was amplified with primers Forl (forward primer) (5'-GGAGGATCATATGAAATTTGTAAAAA-3') and Revl (reverse primer) (5'- CCCGGGATCCATTGTCCGTTACC-3'). The primers contained engineered Ndel and Bam HI sites respectively. The ATG of the Ndel site in Forl coincided with the natural start codon of the lipase and the BamHl site was beyond the natural stop codon. The amplified product was digested with Nde 1 and Bam HI and cloned into the Nde 1-BamHl sites of the plasmid pET-21b yielding the plasmid pET-lipwt (Fig. 7) . The lipase gene coding for the mature protein was amplified from pET-lipwt by using primers PREcoR I (forward primer) (5'- CGTCAGCGAATTCCGCTGAACACAT-3') and PRBamH I (reverse primer) (5'- GCGGGAAGGATCCGAATTCGAGCT-3'). The primers had an engineered EcoR I and BamH I site respectively. The amplified product was cleaved by EcoR I and BamH I and cloned into the EcoRl-BamHl sites of the plasmid pJO290. This construct (pJ02901ip) was used for screening thermostable mutants (Fig.8). The E.coli strain JM109 was used for all the screening steps and all media contained 0.2 % glucose unless otherwise mentioned. This system was chosen because it allows low-level, controlled and inducible expression of the gene product in E.coli, which is necessary to prevent the reported toxicity of the protein to E.coli and to prevent complications from in vivo insolubility of this highly hydrophobic and aggregation-prone protein. Methods of random mutagenesis: The critical step in the invention is in the ability to create variations in the gene. The variation generated should be "sufficient' to yield functional variants. Enzymes have evolved over millions of years of evolution and in the process the enzymes may have tested and avoided deleterious mutations and also tested and incorporated beneficial mutations. It is also believed that most of the gene mutations would be silent i.e., they do not bring about a change in amino acid sequence. In random mutagenesis protocols, it is essential to obtain variations in the gene sequence that result in non-silent mutations and excess of variations, wherein the gene product would be non-functional or may not form. Error-prone PCR based mutagenesis protocols need to be optimized to obtain sufficient variation in the activity of the lipase. The success of the directed evolution protocols strongly depends on the control of this variable. The protocols used in the present example were modifications of the published procedures. The lipase gene was mutagenised by error-prone PCR (Cadwell and Joyce, 1992). Primers JOF (5'-CGCCAGGGTTTTCCCAGTCACGAC-3') and JOR (5'- TGACACAGGAAACAGCTATGAC-3') flank the gene beyond the Eco Rl and Bam HI sites present on the plasmid. Error-prone PCR was carried out in a 100 ul reaction volume containing 20 femtomoles of the plasmid pJO290-lip, 50 pmoles each of primers JOF and JOR, 100 mM Tris.Cl (pH 8.3 at 25 °C), 500 mM KC1, 0.1 % gelatin (w/v), 7 mM MgCb, 0.25 mM MnCl2,1 mM each of dTTP and dCTP, 0.2 mM each of dATP and dCTP and 5 units Taq DNA polymerase. After an initial denaturation of 3 min at 94 °C, the following steps were repeated for 30 cycles in a thermal cycler: 1 min at 94 °C, 1 min at 45 °C and 1 min at 72 °C. The amplified product was precipitated with ethanol, eluted from a 1 % agarose gel and digested with EcoR I and BamH I. The digested product was again eluted from a 1 % agarose gel and ligated with pJO290 digested with EcoR I and BamH I. The ligation mix was transformed into E.coli JM109 and selection was done on LB-agar supplemented with 25 l^g/ml chloramphenicol and 0.2 % glucose. Site-directed mutagenesis Site directed mutagenesis was carried out on the lipase gene cloned in pET-21b by a modified PCR technique (Chen and Arnold, 1991). For each substitution an oligonucleotide containing the desired mutation was used as the primer (mismatch primer) to initiate chain extension between the 5' and 3' PCR primers. In the first PCR, the mismatch primer and the 3' primer were used to generate a DNA fragment containing the new base substitution. The fragment was separated from the template and primers by agarose gel electrophoresis, purified and used as the new 3' primer in a second PCR with the 5' primer to generate full length product, which was cloned into pET-21b for expression of the mutant protein. Example 7 Recombination of the clones obtained in generation 2 The mutant 3B1 was created from the clone 2-8G10 and wt by using the unique restriction site Hae II at position 910 of the lipase gene. The genes coding for the two proteins were amplified by PCR using the T7 promoter and terminator primers. The PCR products were purified by gel extraction and digested with Hae II and Nde I. The upper and lower bands correspond to the C-terminal and N-terminal regions of the protein, respectively. The upper band from clone 2- 8G10 and the lower one from the wild-type protein were eluted. The higher molecular weight fragment was digested with BamH I and purified. A three point ligation containing the Nde I- Hae II fragment (from the wt), the Hae IIBamH I fragment (from 2-8G10) and pET-21b cut with Nde I and Hae II was set up, the ligation mix transformed into DH5a and the positives selected (Fig.9). The sequence of the gene was confirmed by DNA sequencing. The mutant 4B1 (triple mutant) was created by site-directed mutagenesis on the 3B1 template using the mutagenic primer PROLF: 5'- GGC AAG GCG CCT CCG GGA ACA GAT- 3' to incorporate a codon change CTT -> CCT that led to L114P change in the amino acid sequence. The sequences of all the genes were confirmed by automated DNA sequencing. Example 8 Enzyme kinetics All kinetic measurements were made using a thermostatted spectrophotometer using the water-soluble substrate p-nitrophenyl acetate. Initial rates of hydrolysis of p-nitrophenyl acetate at various concentrations were determined at 25 °C in sodium phosphate buffer pH 7.2. The values for KM and kcat were derived from the corresponding Lineweaver-Burke plots. The kinetic parameters obtained with wild type and the mutants was presented in fig. 10. Example 9 Activity of lipase and its mutants in the presence of organic solvents The activity of the lipasea nd its mutants was checked in the presence of various solvents. The organic solvents tested were acetonitrile, isopropanol, dimethyl sulfoxide and dimethyl formamide. The activity assay was performed using PNPA as a substrate. The substrate (2 mM) was dissolved in various percents (v/v) of the organic solvent in buffer (50 mM pH 8.0) and the reaction was started with the addition of lipase at a concentration of 0.246 mg/ml. The activity was monitored as n increase in absorption at 410 nm and the specific activity was calculated using the initial slopes of the curve. In fig 11 the data obtained with acetonitrile is presented. The preceding examples demonstrate the usefulness of the present invention in generating, identifying and isolating lipases which have improved stability and/or ester hydrolysis activity at higher temperature in organic media relative to the natural enzyme. Having thus described exemplary embodiments of the present invention, it should be noted by those skilled in the art that the disclosures herein are exemplary only and that various other alternations, adaptations and modifications may be made within the scope of the present invention. Accordingly, the present invention is not limited to the specific embodiments as illustrated herein. Description of figures Fig.l : Subcloning of lipA without the signal sequence into pET 21b Fig.2. SDS-PAGE profiles of the purified proteins. All the lipases were purified by the procedures given in examples. Lane 1: Low Molecular weight marker, 2: wild-type lipase, 3:1-1E5,4: 2-8G10, 5: 2-3H5, 6: 3B1, 7: 4B1 Fig.3 Hydrolysis of a triglyceride catalyzed by a lipase Fig.4: Residual activity of various mutants and the wild type at a various times on exposure to a temperature of 55 C. The substrate used is PNPA. Fig. 5 Residual activity of various mutants and the wild type at various times on exposure to temperature of 50 C. The substrate used is olive oil. Fig.6 The lipA gene pBR 322 Fig.7 Subcloning lipA along with the suignal sequence into pET 21b Fig.8 Subcloning of lip A without signal sequence into pJO290 Fig. 9 Recombination of the clones obtained in generation II Fig.10 Kinetic parameters and half life of stability at 55 C of wild type and mutant lipase. Fig.ll Activity of lipase and its mutants in the presence of acetonitrile at various concentrations in water. References: 1. Karsherkoff, A. and Ladenstein, R. TIBS (2001) 26, 550-556 2 Kumar,S, Tsai,C-J and Nussinov,R. (2000) Protein Engg. 13:179-191. 3 Lehmann,B. et.al. (2000) Protein Engg. 13: 49-57. 4 Jaenicke, R. and Bohm, G. (1998) Curr. Opin. Struct. Bio. 8, 738-748. 5 Vogt, G., Woell, S. and Argos, P. (1997a) J. Mol. Biol. 269, 631-643. 6 Vogt, G., Woell, S. and Argos, P. (1997b) Folding Design, 1, S40-S46. 7 Ladenstein, R. and Antranikian, G. (1998) Adv. Biochem. Eng. Biotechnol. 61, 37-85. 8. Reetz MT, Zonta A, Simpelkamp J. (1995) Angew Chem, Int Ed Engl 34, 301-303. 9 Plou, F.J. and BallesterosA. TIBS (1999) 17, 304-306 10 Argos, P., Rossmann, M. G., Grau, U., Zuber, H., Frank, G., and Tratschin, J. D. (1979) Biochemistry, 18, 5698-5703. 11 Arnold, F. (1999) Engineering and Science No. 1/2,41-50. 12 Stemmer, W. P. C. (1994) Nature (London) 370: 389-39 13 Ostermeier M, Shim JH, Benkovic SJ. (1999) Nat Biotechnol.,17,1205-9. 14 Woolley,P and Petersen,S.B. (Ed.) Lipase: their structure, biochemistry and application (1994) Cambridge University Press. 15 Jaeger, K-E., Dijkstra, B.W. and Reetz, M.T. (1999) Annu. Rev. Microbiol. 53:315-351. 16 Schmid,R.D. and Verger, R. (1998) Angew. Chem.. Int. Ed. Engl. 37, 1608- 1633. 17 Jaeger KE, Reetz TM. (1998) Trends Biotechnol. 16,396-403. 18 Wang, L.J., Kong, X. D., Zhang, H.Y., Wang, X.P, and Zhang J. Biochem Biophys Res Commun Sep 276:346-349. 19 Rubin,B. and Dennis,E.A. (1997) Lipases PartA: Biotechnology, Methods Enzymol. 286, pp563, Academic press, San diego. 20 Kazlauskas RJ, Bornscheuer UT. Biotransformations with Upases. In: Rehm HJ, Pihler G, Stadler A, Kelly PJW, editors. Biotechnology, vol. 8. New York: VCH, 1998. pp. 37-192. 21 Gerritse G, Hommes RW, and Quax WJ. (1998) J Appl Environ Microbiol, 64,2644-51. 22 Bycroft, A.L. and Byng, G.S. (1992) European patent application 0468102A1 23 Kennedy, M.B. and Lennarz, W.J. (1979) J. Biol Chem. 254,1080-1089. 24 Dartois, V., Baulard, A., Schanck, K. and Colson, C. (1992) Biochim. Biophys. Acta 1131: 253-260. 25 Lesuisse, E., Schanck, K. and Colson, C. (1993) Eur. J. Biochem. 216,155-160. 26 Eggert, T., Pencreac'h, G., Douchet, I., Verger, R. and Jaeger, K.-E. (2000) Eur. J. Biochem. 267, 6459-6469. 27 Arpigny, J.L and Jaeger, K.-E. (1999) Biochem J. 343,177-183. 28 Pouderoyen, G., Eggert, T., Jaeger, K.E. and Djikstra, B.W. (2001) JMolBiol. 309:215-226. 29 Lutz,S. and benkovic, S.J. (2000) Cur.Opi. Biotech. 11,319-324. 30 Shao Z, Zhao H, Giver L, and Arnold FH (1998) Nucleic Acids Res. 26,681- 683. 31 Beisson,F., Alim Tiss, Riviere, C. and Verger, R. (2000) Eur.J.lipid Sci. Tech 133-153 32 SambrookJ., Fritsch,E.F. and Maniatis, T. (1989) Molecular cloning: a laboratory manual (2nd Edition) Cold spring Harbor laboratory Press 33 Hoch, J.A., Sonenshein, A.L. and Losick, R. (1993) Bacillus subtislis and other gram positive bacteria, ASM. We Claim: 1. Novel thermostable, organic solvent resistant and high pH tolerant lipase gene variants having SEQ ID No. 2 of molecular wt 19443, SEQ ID No. 3 of molecular wt 19515 SEQ ID No. 4 of molecular wt 19456.9, SEQ ID No. 5 of molecular wt. 19487and SEQ ID No. 6 of molecular wt. 19470.9 2. Novel gene variants as claimed in claim 1, wherein said gene variants are thermostable in the temperature range of about 45 to 95 C. 3. Novel gene variants as claimed in claim 1, wherein said gene variants are resistant to organic solvents selected from group consisting of acetonitrile, isopropanol, dimethyl sulfoxide and dimethyl formide. 4. Novel gene variants as claimed in claim 1, wherein residual activity of the gene variants is in the range of 25 to 100 % in presence of acetonitrile. 5. Novel gene variants as claimed in claim 1, wherein residual activity of the gene variants is in the range of 28.7 to 85.5% in presence of acetonitrile. 6. Novel gene variants as claimed in claim 1, wherein the gene variants have inherent ability to withstand high pH in the range of 9 to 13; ability to withstand damaging surfactants and enzymes comprising groups of linear alkyl benzene sulfonates, proteases and compounds thereof. 7. An expression system for novel thermostable, organic solvent resistant and high pH tolerant lipase gene variants as claimed in claim 1-6, wherein said expression system comprising of having SEQ ID No. 2 of molecular wt 19443, SEQ ID No. 3 of molecular wt 19515, SEQ ID No. 4 of molecular wt 19456.9, SEQ ID No. 5 of molecular wt. 19487 and SEQ ID No. 6 of molecular wt 19470.9 present in the vector pJ0290. 8. A method of preparing novel thermostable, organic solvent resistant and high pH tolerant lipase gene variants as claimed in claim 1-6, having SEQ ID No. 2 of molecular wt 19443, SEQ ID No. 3 of molecular wt 19515, SEQ ID No. 4 of molecular wt 19456. 9, SEQ ID No. 5 of molecular wt. 19487 and SEQ ID No. 6 of molecular wt 19470.9 said method comprising the steps of: (a) isolating and purifying lipase gene from Bacillus subtilis, (b) cloning lipase gene isolated in step (a) in vector pJ0290, (c) generating gene variants from lipase gene isolated in step (a) by random mutagenesis and site-directed mutagenesis using forward primer JOF having SEQ ID No. 13 and reverse primer JOR having SEQ ID No. 14, (d) cloning the gene variants obtained in step (c) in plasmid vector pJ0290, and (e) ligating the cloned gene variants of step (d) in E.coli JM109 to further obtain expression of the protein.

Full Text

Field of Invention:
The present invention relates to creation, optimization and production of a
lipase. More specifically, the invention relates to the generation and production
of lipase variants that are thermostable and also stable in organic solvents. The
invention also relates to methods of selecting lipase variants for high
temperatures and for their purification.
BACKGROUND OF THE INVENTION
Enzymes are the workhorses of a cell that affect virtually every biological process
that characterizes a living organism. They catalyze chemical reactions with
remarkable specificity and rate enhancements. The awesome catalytic power and
versatility of enzymes has long been recognized and enzymes have proved to be
very useful outside the living system as well. Enzymes today have widespread
application in industry and are seen as environment friendly alternatives to
chemical reagents because enzymatic reactions require milder conditions and
tend to be cleaner with lesser byproduct and waste generation. Enzymes are
being used in numerous new applications in the food, feed, agriculture, paper,
leather, and textiles industries, resulting in significant cost reductions and
environment-friendly operations.
Enzymes have evolved to function best under the physiological conditions of the
parent organism. In vitro applications often call upon enzymes to work under
non-physiological conditions or to perform functions they have not evolved for.
For example, enzymes may have to catalyze reactions involving novel substrates;
they may have to work under extreme conditions of salt, temperature, pH etc., or
in the presence of potentially inhibiting or denaturing chemicals. Such
applications have brought to light the severe disabilities of enzymes to function
as industrial catalysts. In order to extract optimum performance from enzymes in
the test-tube and in industrial reactors, these biocatalysts need to be tailored to
suit specific applications.
The commercial success of these enzymes can be attributed to their ease of use. In
addition to functioning at high temperatures, thermostable enzymes generally
posses an increased shelf life which markedly improves handling conditions. If
enzymes are to play a significant role in large scale processing of chemicals, they
must be able to endure the harsh conditions associated with these processes.
Thermostable enzymes are easier to handle, last longer, and given the proper
immobilization support should be reusable for multiple applications
In obtaining thermostable enzymes the conventional approach is to screen the
microbial collections collected from extremephillic environments1. The promising
candidate enzymes are further investigated for suitability for a specific process.
For example, for applications requiring thermostable or salt stable enzymes,
enzymes from thermophilic or halophilic organisms were used, respectively.
However, such an approach severely restricted the use of enzymes because
enzymes for all applications cannot be found in nature. There may not be a
natural enzyme for many kinds of transformations. Moreover, enzyme usage is
often restricted by undesirable properties of enzymes like product inhibition, low
stability etc. Very often an enzyme is required to have a combination of several
properties that may be impossible to find in a natural enzyme.
Another approach to obtain thermostable enzymes is based on the current
knowledge on the protein structures (crystal structures) of homologous enzymes
from mesophiles and thermophiles 2-3. Such comparisons yielded information on
the probable interactions that enhance thermostability. Using such information
efforts were made to incorporate these changes in mesophillic enzymes to
improve their thermostability. Such approaches have not been very successful
since interactions that improve thermostability in a protein are many and each
protein acquires, over evolutionary times, those interactions that are best suited
for its sequence and the mileu in which it functions. Though structural
determinants of protein stability have been objects of numerous studies on model
proteins, no universal stabilization mechanism has yet been found 4. The most
obvious conclusion that can be drawn from the literature is that different
proteins have adapted to different thermal environments by a variety of
evolutionary devices. The lack of understanding of the structural features
leading to protein thermostability has been partly due to a scarcity of data
because experimental studies comparing homologous proteins from
psychrophilic, mesophilic and thermophilic organisms have been limited to only
a few proteins. Moreover, inability to form definite rules for improving protein
thermostability is due to the large number and complexity of possible
contributing factors 4'7. Based on comparisons between mesophiles and
thermophiles, the main mechanisms responsible for increased thermostability
have been identified as increase in the number of hydrogen bonds and salt
bridges, increased core hydrophobicity, better packing efficiency, a-helix and
loop stabilization and resistance to covalent destruction. Often it becomes
difficult to delineate protein interactions that contribute to thermostability from
other selection pressures such as salt, pH etc.
Other strategies adapted to increase the thermostability was based on the
observation that immobilized enzymes acquire thermostability to some extent8.
Hence, several solid supports were tried to immobilize proteins. And also recent
observations made with enzymes in organic solvents indicated that in organic
solvents enzymes acquire thermostability 9. The advent of recombinant DNA
techniques has greatly facilitated protein engineering by allowing facile
mutagenesis and production of proteins.
The term protein thermostability refers to the preservation of the unique
chemical and spatial structure of a polypeptide chain under extremes of
temperature conditions 4. In general, the higher the temperature to which the
enzyme is exposed, the shorter the half-life of the enzyme (i.e., the shorter the
enzyme retains its activity). Similarly, the greater levels of organic solvent to
which said enzymes are exposed, the shorter the half-life of the enzyme. The
phrase "catalytic activity" or simply "activity," means an increase in the k.sub.cat
or a decrease in the K.sub.M for a given substrate, reflected in an increase in the
k.sub.catt /K.sub.M ratio.The structural basis of protein thermostability has been
an actively pursued area of research for at least two decades 10 . However,
enzymes lifted out of the context of living organisms do not always function as
well as they do in their natural milieu. For example, they have optimum activity
at the physiological temperature of the organism and tend to denature at higher
temperatures leading to drop in activity. Thermostable enzymes are important as
they can be used at high temperatures and harsher conditions required in
industrial contexts. Also they generally have higher storage stabilities and bring
down costs by obliviating the need for low temperature storage and decreasing
the loss due to denaturation on storage and handling. Moreover reactions carried
out at higher temperatures generally proceed at higher rates further bringing
down operation times.
In view of the environmental safety reasons, there is a constant pressure to
reduce the use of environmentally polluting processes in industry. Enzymes are
increasingly used to replace chemical processes in leather, food, and
pharmaceutical industries. Comparison of protein structures from
extremeophiles demonstrated that protein structural plasticity is enormous and
is resident in the primary structure. This lent considerable support to strategies
that alter the primary structure of the proteins at the genetic level and screen for
the variants with special properties such as thermostability. The tremendous
success in handling the genes and developing protocols to alter it at will, has
allowed to evolve proteins with special functions. The strategy relies in
generating variation in gene sequences by molecular biology methods and
screening the variants by expressing them and screening the mutant population
n-13. The screening protocols are based on the property of interest, e.g., activity at
high temperature or activity in the presence of organic solvents. The present
invention encompasses methods for generating variation in gene sequences,
protocols for screening the enzymes with higher thermostability and also
protocols for sequencing and expression.
Lipases (triacylglycerol acylhydrolases, E.G. 3.1.1.3) are water-soluble enzymes
that catalyze the hydrolysis of ester bonds in triacylglycerols and often also
exhibit phospholipase, cutinase and amidase activities 14. They are used for the
production of detergents, pharmaceuticals, perfumes, flavour enhancers and
texturising agents in consmetic products. Lipases are crucial for the production
of a wide variety of foods, especially for products from milk, fat and oil. Lipases
are ubiquitous enzymes of considerable physiological significance and industrial
potential. Lipases catalyze the hydrolysis of triacylglycerols to glycerol and free
fatty acids. In contrast to esterases, lipases are activated only when adsorbed to
an oil-water interface and do not hydrolyze dissolved substrates in the bulk
fluid. A true lipase will split emulsified esters of glycerine and long-chain fatty
acids such as triolein and tripalmitin. Lipases are serine hydrolases.
Commercially useful lipases are usually obtained from microorganisms that
produce a wide variety of extracellular lipases 15 . Many lipases are active in
organic solvents where they catalyze a number of useful reactions including
esterification transesterification, regioselective acylation of glycols and menthols,
and synthesis of peptides and other chemicals. An increasing number of lipases
with suitable properties are becoming available and efforts are underway to
commercialize biotransformation and syntheses based on lipases 16. Enzyme
sales for use in washing powders still remain the single biggest market for
industrial enzymes. The major commercial application for hydrolytic lipases is
their use in laundry detergents. Detergent enzymes make up nearly 32% of the
total lipase sales. Lipase for use in detergents needs to be thermostable and
remain active in the alkaline environment of a typical machine wash. An
estimated 1000 tons of lipases are added to approximately 13 billion tons of
detergents produced each year Because of their ability to hydrolyzes fats, lipases
find a major use as additives in industrial laundry and household detergents.
Detergent Upases are especially selected to meet the following requirements: (1) a
low substrate specificity, i.e., an ability to hydrolyze fats of various compositions;
(2) ability to withstand relatively harsh washing conditions (pH 10-11, -60 _C);
(3) ability to withstand damaging surfactants and enzymes [e.g., linear alkyl
benzene sulfonates (LAS) and proteases], which are important ingredients of
many detergent formulations. Lipases with the desired properties are obtained
through a combination of continuous screening 17-19 and protein engineering 20 .
In 1994, Novo Nordisk introduced the first commercial recombinant lipase
'Lipolase/ which originated from the fungus Thermomyces lanuginosus and was
expressed in Aspergillus oryzae. In 1995, two bacterial lipases were introduced —
'Lumafast' from Pseudomonas mendocina and 'Lipomax' from P. alcaligenes —
by Genencor International15. According to a report an alkaline lipase, produced
by P. alcaligenes M-l, which was well suited to removing fatty stains under
conditions of a modern machine wash. The patent literature contains examples of
many microbial lipases that are said to be suitable for use in detergents 22.
Lipase-producing microorganisms include bacteria, fungi, yeasts, and
actinomyces. Bacillus subtilis is a Gram-positive, aerobic, spore-forming
bacterium that has generated substantial commercial interest because of its
highly efficient protein secretion system Though extracellular lipolytic activity of
B.subtlis was observed as early as in 1979 23, molecular research started in 1992
when a lipase gene, lipA, was cloned and sequenced 24. Subsequently the lipase
was overexpressed, purified and characterized 25. Later, a second gene, lipB, was
found that is 68% identical with lip A at the nucleic acid level 26. This gene has
been cloned and the protein overexpressed, purified and characterized.
The Bacillus subtilis lipase with a molecular weight of 19,348 Da is one of the
smallest lipases known. It is one of the few lipases that do not show the
interfacial activation in the presence of oil-water interfaces. LipA is very tolerant
to basic pH and has its optimum activity at pHlO. It hydrolyses the sn-1 and sn-3
glycerol esters with both short and long chain fatty acids, showing optimum
activity with C8 fatty acid chains.
Bacterial lipases are classified into eight families according to their sequence
similarities, conserved sequence motifs and biological properties 27. The true
lipases are classified in family I which contains six subfamilies. Bacillus lipases
have been placed in subfamilies 4 and 5. In these two subfamilies alanine
replaces the first glycine residue in the conserved G-X-S-X-G pentapeptide
around the active site serine residue. Subfamily 4 consists of only three members,
LipA and LipB from B.subtilis and a lipase from Bacillus pumilis, which share 74-
77% sequence identity. These are the smallest lipases known and show very little
sequence similarity (~ 15%) with the other, much larger, Bacillus lipases that
constitute subfamily 5.
The crystal structure of the B.subtilis lipase LipA reveals a globular protein with
dimensions of 35 X 36 X 42 28. The structure shows a compact domain that
consists of six P- strands in a parallel p- sheet, surrounded by a- helices. There
are two a- helices on one side of the P- sheet and three on the other side. The fold
of the B.subtilis lipase resembles that of the core of the a/p hydrolase fold
enzymes. The B.subtilis lipase lacks the first two strands of the canonical a/p
hydrolase fold and the helix ccD is replaced by a small 310 helix. The helix aE is
exceptionally small, with only one helical turn, and several a-helices start or
terminate with 310 helical turns. Due to these structural features, its small size
and absence of a lid domain, the B.subtilis lipase is considered a minimal a/p
hydrolase fold enzyme.
Description of the invention:
In accordance with the present invention a methods of directed evolution were
applied to the lipase gene ( Gene Seq ID # V) to isolate protein variants of the
original sequence which possess increased thermostable properties. The
methodology relies initially on the ability to create random variations in the
original gene sequence and express the corresponding proteins in the bacteria,
E.coli. The produced variants of the original sequence would have altered
sequence, hence altered properties. The variants, at a proteins level, would be
tested for their thermostability and those sequences which demonstrate
improved thermostability would be subjected to the next round of random
mutagenesis and screening. Thus by sequential accumulation of the mutants and
subsequent pooling of the mutations the thermostability of lipase was improved
by 200-fold at high temperature. High temperature range includes temperature
ranges from 50-90 C.
The method according to the present invention includes four steps in generating
the variant Upases and their characterization to obtain thermostable Upases. In
the first of the methods variation in the primary sequence of the lipase gene were
generated by error prone PCR methods. The adapted protocols are similar to
several published protocols. In addition many of the different protocols such as
random priming, ITCHY etc could also be applied for generation of variance in
the gene sequence 29-30. In the second step of the invention, the mutant sequences
are cloned into an expression vector and the protein expressed in the culture
lysates. The expressed proteins are screened for their ability to withstand higher
temperature was tested in a large population using medium-throughput
methods. In the third step of the invention the promising variants were pooled
by a family shuffling procedures according to the method of Stemmer 12 and
further tested for thermostability. In addition to the shuffling procedures
selective changes in the primary sequence were also incorporated by standard
molecular biology procedures. In the fourth step of the invention the positive
sequences were over-expressed in large cultures and the proteins are purified by
the published procedures according to Colson et al24. The purified proteins were
tested for their thermostability.
The screening procedures of the lipase variants for increased thermostability
involve ability to hydrolyse chromogenic substrate esters based on p-nitrophenyl
group. Natural substrates of lipase are triglycerides, which are not convenient to
design a simple medium trough put assays wherein the source of enzyme is
over-expressed lipase in a cell lysate 31. P-nitrophenyl esters of fatty acids are,
convenient and the activity of lipase represents the activity of lipase on
triglycerides. Long chain esters of p-nitrophenyl, especially p-nitrophenyl oleate,
are well suited for this purpose. Detergent solubilized PNPO demonstrates
negligible back ground hydrolysis and well suited for lipase present in cell
lysate. The strong yellow color of very high extinction coefficient of hydrolysis
product p-nitrophenyl can be estimated conveniently in a 96-well plates. Along
with p-nitrophenyl esters, many other fluorogenic or chromogenic esters of fatty
acids could be used for this purpose.
As employed herein the term thermostability refers to the property of the
enzymes, which retain their activity subsequent to exposure to higher
temperatures. Enzymes lose their tertiary conformation on exposure to higher
temperature due to the increased movement of the structural elements, which
perturbs the functional structure of the protein. Typically proteins lose their
activity at higher temperatures with time. The rate of this loss in activity, reflects
in half life i.e., time required to lose half of the initial activity, is a convenient
parameter to compare the thermostability of the protein 4. Activity, as defined
here, corresponds to the catalytic acitivity represented by the term kcat/Km,
where kcat is the rate of the product formation and Km is the apparent affinity
constant of the substrate to the enzyme. Retaining the functional structure at
elevated temperatures resides in the ability to form interactions within the
protein that withstand high temperatures. The range of the temperature that is
relevant for the present invention ranges from 35 to 90 C.
The naturally occurring lipase from Bacillus lipase has the amino acid sequence
of 1-181 as given in the sequence ID # . The corresponding nucleic acid sequence
expressing the protein (ID # )was presented in ID # . It was discovered that the
amino acid substitutions at positions 68, 71,114,120,132,144,147 and 166 were
found to be important for the thermostability of the lipase. In accordance with
the present investigation, it was further discovered that the substitutions at
positions 114, 132 and 166 are suited for increasing the stability of the proteins.
Any of the innumerable combinations of substitutions possible at each of these
positions with the other 19 amino acids would be favourable for the
thermostability.
The specific substitutions of relevance for thermostability in lipase are given
below.
From To Position
N V 166
A D 132
A V 68
L P 114
R S 147
V A 144
N D 120
Examples:
Example 1:
Purification of lipase from Bacillus subtilis
Purification of the lipase was performed from E.coli cells expressing the lipase in
an appropriate vector. The purification essentially involves passing the cell lysate
in phenyl-sepharose column followed by a Mono-S column. Lipase is an
aggregated prone protein, care especially keeping protein concentration below
5mg/ml, was taken to avoid aggregation of the protein. The purification of the
lipase was carried out essentially as described earlier32 with minor modifications.
Lipase from culture filterates of Bacillus strain or from the E.coli lysates was
processed similarly. For purification of the wild type and mutant proteins from
E.coli the lip A gene or the mutant genes are cloned into pET 21b. ). For this, the
gene corresponding to the full length, mature protein was amplified with
primers PrNde I (forward primer) (5'-
CCATGATTACGCATATGGCTGAACACAA-3') and JOF. The forward primer
had an engineered Nde I site. The forward primer also introduced a start codon
at the start of the lipase gene in the form of the ATG sequence that is part of the
Nde I recognition sequence. This would introduce a methionine in the Nterminus
of the mature protein, expressed in E.coli, just before the N-terminal
alanine that occurs in the protein purified from the culture supernatant of
B.subtilis. The wild type protein as well as the mutants were amplified, digested
with Nde I and BamH I and ligated with pET-21b digested with Nde I and BamH
I. The ligation mix was transformed into E.coli DH5a and the positives were
selected by plasmid minipreps and restriction digestions (Fig.l).
Protein was purified from E.coli BL21 (DE3) cells. Cells containing the
appropriate plasmid were grown till mid-log phase before inducing with 0.5 mM
IPTG. Cells were harvested 2.5 hours after induction by centrifuging at 15,000
rpm at 4 °C for 20 min. The pellet was washed with STE and resuspended in IX
TE containing 0.3 mg/ml lysozyme. The suspension was incubated on ice for 30
min before lysing the cells by sonication. Sonication was carried out by keeping
the cells on ice. Short pulses of half-minute duration were applied and 1 min
cooling time was allowed between pulses. The sonicated cells were centrifuged
at 20,000 rpm at 4 °C. The supernatant was loaded on a phenyl sepharose
column. The remaining steps were done as described in chapter 2. The purified
proteins were stored in -70 °C till further use.
B.subtilis BCL1051 was grown aerobically for 16-18 hrs at 37 °C in 21 Erlenmeyer
flasks, each containing 500 ml of medium of the following composition: 2.4 %
yeast extract, 1.2 % tryptone, 0.4 % gum Arabic, 0.4 % glycerol, 0.017 M KH2PO4,
0.072 M K2HPO4, 50 mg/ml kanamycin sulfate. The culture medium was
inoculated at 1 % from 10 ml precultures. After harvesting the cells by
centrifugation at 6000 rpm for 30 min, the culture supernatant was pumped at a
flow rate of 30 ml/hr onto a Phenyl Sepharose Fast Flow High sub column
(Pharmacia) (20 ml column volume per 11 culture) equilibrated with 100 mM
potassium phosphate, pH 8.0. The column was washed at a flow rate of 50 ml/hr
first with 10 mM potassium phosphate, pH 8.0 and then with 30% ethylene
glycol in 10 mM potassium phosphate, pH 8.0. Elution was performed at a flow
rate of 50 ml/hr with 80 % ethylene glycol in 10 mM potassium phosphate, pH
8.0. 2 ml fractions were collected and the fractions containing protein (detected
by absorbance at 280 nm) were checked for enzyme activity. The active fractions
were pooled and dialyzed against 2 mM glycine-NaOH, pH 10.0. The dialyzed
protein was diluted 1:1 with 50 mM Bicine-NaOH, pH 8.5 (buffer A) and loaded
onto a MonoS HR5/5 (Pharmacia) column, pre-equilibrated with buffer A, using
a Superloop (Pharmacia) on a FPLC (Pharmacia) system. The protein-boundcolumn
was washed thoroughly with the buffer A to remove unbound proteins.
The protein was eluted using a linear gradient with buffer A to buffer B (50 mM
Bicine-NaOH, pH 8.5,1 M NaCl). The enzyme eluted around 300 mM NaCl as a
single peak. The active fractions eluted from the MonoS column were dialyzed
overnight against 2 mM glycine, pH 10.0 and concentrated using an Amicon
concentrator fitted with a YMIO membrane (10 kD cutoff). Purity of the protein
was checked on a 12% SDS-PAGE gel containing 5 M urea (Lessuisse et al, 1993).
The protein was 95 % pure on a Coomassie stained gel (Fig.2).
Example 2:
Assay of lipase:
Lipase belongs to a class of enzymes known as interfacially active enzymes.
These enzymes have very little activity on the substrate monomers but their
activity increases dramatically on insoluble substrate such as emulsified
triglycerides, monolayers etc. This property makes lipases dissimilar to other
enzymes which act on soluble substrate monomers. Triglycerides, natural
substrates of lipase are not very convenient to set up simple chromogenic assays
(Fig.3). Activity of pure lipases, sometimes, can be monitored by detecting the
pH changes using pH-sensitive dyes. However, such assays yield complications
when the enzyme source is a lysate and when there are other processes that may
alter the pH. P-nitrophenyl esters are most convenient to monitor the activity.
Short chain ester, p-nitrophenyl acetate and long chain ester, p-nitro phenyl
oleate (PNPO), were synthesized by routine synthetic methods (given below).
PNPO is a insoluble ester, was used in our assays using triton X-100 as a
solubilizing agent. Triton X-100: PNPO co-micelles showed low back ground
hydrolysis and were also stable at elevated temperatures. 96-well plate assays for
screening the variants of lipase, though very useful to screen large number of
samples, quantitates the activity approximately. All positives obtained in 96-well
screens were confirmed in a tube assays, where the number of samples are less
and more accurate specific activity calculations could be made.
Synthesis of chromogenic substrates for lipase assays
The following chromogenic substrates were synthesized for lipase assays:
1) p-nitrophenyl oleate
2) p-nitrophenyl stearate
3) p-nitrophenyl caprylate
The fatty acid, N, N'- methyl tetrayl biscyclohexamine
(dicyclohexylcarbodiimide, DCC), N, N'- dimethylamino pyridine (DMAP), and
p-nitrophenol were taken in mole ratios of 1:1:1:2. The fatty acid was taken in a
round bottom flask containing 20 ml of dry DCM and a few ml of chloroform.
The mixture was stirred for two minutes followed by addition of DCC. A white
precipitate was formed. This was followed by the addition of DMAP. Subsequent
addition of p-nitrophenol led to the formation of a yellow precipitate. The
reaction vessel was flushed with nitrogen and stirred for 5 hours. The progress of
the reaction was monitored by thin layer chromatography. After completion of
the reaction, the DCM was evaporated to dryness and the ester was purified by
column chromatography (silica gel column, elution with petroleum etheracetone).
The purity and identity of the product was confirmed by JR-NMR
spectroscopy.
Example 3
Lipase assay in 96-well microtitre plates): The colonies obtained from the cloning
of the PCR product generated by error-prone PCR were patched on another
similar plate and simultaneously inoculated in separate wells of a microtitre
plate containing 200 ^il 2XYT containing 25 ng/ml chloramphenicol and 0.2 %
glucose. The cells were grown for 24 hours in the microtitre plate with
continuous shaking at 200 rpm. After 24 hrs, 5 j^l culture from each well was
taken and added to the corresponding well another microtitre plate containing
200 ul 2XYT supplemented with 25 ng/ml chloramphenicol. After 3 hours of
growth the cultures were induced with 1 mM IPTG. After 3 more hours 25 ul of
culture was taken from every well into the corresponding wells of two fresh
microtitre plates containing 25 ul phosphate buffer pH 7.0. One of the plates was
exposed to high temperature for 20 min, cooled on ice for 15 min and then
allowed to come to room temperature. The other plate was kept at room
temperature. 25 ul of the PNPO-Triton X-100 substrate solution prepared as
described above was added to each well. The plates were incubated at 37 °C and
absorbance at 405 nm was recorded in an ELISA reader at definite time intervals.
The clones showing less than 20 % of the activity of the wild-type protein (or the
parent from which it is generated) were removed from further consideration. The
residual activity for each clone after exposure to high temperature was
calculated. The clones showing highest residual activity were chosen for the next
level of screening.
Lipase assays in tubes:: The colonies that showed highest residual activity in the
microtire plate level screen were grown for 12 hours in 5 ml 2XYT medium 25
ug/ml chloramphenicol and 0.2 % glucose. 10 ml of 2XYT containing 25 ng/ml
chloramphenicol and 0.2 % glucose was inoculated with 100 \il of the overnight
grown culture. After 2.5 hours growth, the cultures were induced with 1.5 mM
IPTG and were harvested after another 2.5 hours. The cell pellet was washed
with STE and resuspended in 1 ml 0.05 M potassium phosphate buffer pH 7.2.
The cell suspension was sonicated with a Branson sonicator with four pulses of
30 sec and 1 min cooling time in between the pulses. The tubes were kept on ice
during sonication and cooling of the samples. The sonicated samples were
centrifuged at 15,000 rpm for 45 min and the supernatant was used for the
assays. The supernatant was divided into four 250 ul aliquots. Three of the
aliquots were exposed to higher temperatures and the fourth was kept on ice.
The tubes were exposed to high temperatures for 20 min, chilled on ice,
centrifuged at 4 °C at 15,000 rpm and then allowed to come to room temperature
before assaying for enzymatic activity. The lipase activity in the cell lysates was
determined at room temperature in sodium phosphate buffer pH 7.2 by using pnitrophenyl
oleate as substrate. The enzymatic activity was measured by
following the change of absorbance at 405 nm with time. Lysates of cells that do
not contain the lipase gene but otherwise processed in the same way as
mentioned above, were used to determine the background hydrolysis of pnitrophenyl
oleate in E.coli cell lysate. The background hydrolysis values were
subtracted from the enzymatic activity value. The total protein in the cell lysates
was determined by Lowry's method and was used to normalize the activity.
Example 4
Half-lives of thermal inactivation
Exposing the enzymes to higher temperatures and then assaying the activity at
room temperature normally assess thermostability of enzymes. At higher
temperature the protein denatures and irreversible unfolds. Thermostable
enzymes possess additional stabilizing interactions which would make them less
susceptible for heat denaturations. The activity remaining is residual activity,
which decrease both with increase in temperature or with increase in time at a
given temperature. Heat treatment of the purified proteins was carried out in a
programmable thermal cycler (GeneAmp PCR system 9700) in 0.2 ml thin-walled
PCR tubes to allow precise temperature control of the samples. The proteins
were taken at a concentration of 0.05 mg/ml in 0.05 M sodium phosphate buffer,
pH 7.0. 25 ^il of protein samples were taken in each tube. The proteins were
heated for the required time, cooled at 4 °C for 20 min, centrifuged and
equilibrated at room temperature before assaying for enzymatic activity. 20 ul of
the heat-treated protein sample was added to 1 ml 0.05 M sodium phosphate, pH
7.2 containing 2 mM p-nitrophenyl acetate. Enzymatic activity was measured at
25 °C by monitoring the rate of increase in absorbance at 405 nm. Typically,
inactivation was followed until > 80 % of the activity was lost. Plots of
log(residual activity) versus time were linear. Inactivation rate constants (kinact)
were obtained from the slope and half-lives were calculated as ti/2 = Iog2/kmact.
The half lives of various mutants obtained were presented in figure (Fig.4) where
the residual activities were measured using PNPA as substrate. The enzyme
mutants were exposed to 55 C. In fig.5 data obtained with residual activities with
three mutants using olive oil as a substrate was presented. The activities were
measured using pH stat equipment. This data demonstrates that the
enhancement seen with mutants was independent of the substrate and nature of
the assay.
Preparation of substrate stocks:
Appropriate amounts of the insoluble p-nitrophenyl ester and Triton X-100 were
weighed out in a glass vial and mixed with a magnetic stirrer till the ester
completely dissolved in Triton X-100. Buffer was added slowly while stirring to
prepare a 2X stock solution containing 0.4 mM p-nitrophenyl ester and 40 mM
Triton X-100. Substrate solutions prepared in this way were optically clear. 100X
substrate stocks of the water-soluble p-nitrophenyl acetate were made in acetone
and 2 mM p-nitrophenyl acetate was used for each reaction. The reactions were
carried out in absence of Triton X-100 and all the measurements to determine
kinetic parameters were done with this reaction system.
Example 5
Assay with olive oil
Assay with the olive oil is performed pH stat equipment. All lipases subsequent
to thir activity reduce th pH off the reaction medium by releasing a proton. The
decrease in pH could be neutralized by addition of known amounts of alkali. The
rate of additon of alkali would represent the activity of the lipase. We have
prepared the lipase substrate by mixing gum Arabic (0.5%), olive oil ( ) and
CaC12( ). The mixture was sonicated in a bath till we obtain a uniform emulsion.
We have used 10 ml of the substrate for each assay.At the beginning of the asay
the pH of the substrate was brought to 8.4 by addition of alkali. The reaction was
started with the addition of 10 microlitres of Img/ml enzyme solution. The rate
of reaction was calculated from the slopes of amount of alkali vs. time curves. ).l
N NaOH was sued as alkali.
Example 6
Methods of generation of variations in the Lipase genes:
The sequence ot Lip A, whose product is lipase gene of interest in this invention,
from Bacillus subtilis was published. In Bacillus LipA gene product is secreted
into the culture medium owing to the presence of a signal sequence at the Nterminal
of the sequence, which aids in its transport out of the cell. Molecular
biology of Bacillus species has been well studied and it is a Gram-positive strain.
For routine molecular biology techniques such as transformation, cloning,
expression etc. Bacillus sp. is less suited compared to E.coli 33'34. The main
difficulty is in transforming the Bacillus sp with the plasmids. The efficiency is
lower by several orders of magnitude compared to E.coli. Further, the observed
efficiencies are only detectable with electroporation, which is a harsher method.
In E. coli the transformation efficiency is higher and reproducible and the choice
of plasmids is wide. To perform various gene manipulations, E.coli was used.
The clone pLipA containing the complete lipA gene in pBR322 plasmid was a
kind gift from Dr Frens Pierce (Fig.6). The lipase gene along with the region
coding for the signal sequence was amplified with primers Forl (forward primer)
(5'-GGAGGATCATATGAAATTTGTAAAAA-3') and Revl (reverse primer) (5'-
CCCGGGATCCATTGTCCGTTACC-3'). The primers contained engineered
Ndel and Bam HI sites respectively. The ATG of the Ndel site in Forl coincided
with the natural start codon of the lipase and the BamHl site was beyond the
natural stop codon. The amplified product was digested with Nde 1 and Bam HI
and cloned into the Nde 1-BamHl sites of the plasmid pET-21b yielding the
plasmid pET-lipwt (Fig. 7) . The lipase gene coding for the mature protein was
amplified from pET-lipwt by using primers PREcoR I (forward primer) (5'-
CGTCAGCGAATTCCGCTGAACACAT-3') and PRBamH I (reverse primer) (5'-
GCGGGAAGGATCCGAATTCGAGCT-3'). The primers had an engineered EcoR
I and BamH I site respectively. The amplified product was cleaved by EcoR I and
BamH I and cloned into the EcoRl-BamHl sites of the plasmid pJO290. This
construct (pJ02901ip) was used for screening thermostable mutants (Fig.8). The
E.coli strain JM109 was used for all the screening steps and all media contained
0.2 % glucose unless otherwise mentioned. This system was chosen because it
allows low-level, controlled and inducible expression of the gene product in
E.coli, which is necessary to prevent the reported toxicity of the protein to E.coli
and to prevent complications from in vivo insolubility of this highly hydrophobic
and aggregation-prone protein.
Methods of random mutagenesis:
The critical step in the invention is in the ability to create variations in the gene.
The variation generated should be "sufficient' to yield functional variants.
Enzymes have evolved over millions of years of evolution and in the process the
enzymes may have tested and avoided deleterious mutations and also tested and
incorporated beneficial mutations. It is also believed that most of the gene
mutations would be silent i.e., they do not bring about a change in amino acid
sequence. In random mutagenesis protocols, it is essential to obtain variations in
the gene sequence that result in non-silent mutations and excess of variations,
wherein the gene product would be non-functional or may not form. Error-prone
PCR based mutagenesis protocols need to be optimized to obtain sufficient
variation in the activity of the lipase. The success of the directed evolution
protocols strongly depends on the control of this variable. The protocols used in
the present example were modifications of the published procedures.
The lipase gene was mutagenised by error-prone PCR (Cadwell and Joyce, 1992).
Primers JOF (5'-CGCCAGGGTTTTCCCAGTCACGAC-3') and JOR (5'-
TGACACAGGAAACAGCTATGAC-3') flank the gene beyond the Eco Rl and
Bam HI sites present on the plasmid. Error-prone PCR was carried out in a 100
ul reaction volume containing 20 femtomoles of the plasmid pJO290-lip, 50
pmoles each of primers JOF and JOR, 100 mM Tris.Cl (pH 8.3 at 25 °C), 500 mM
KC1, 0.1 % gelatin (w/v), 7 mM MgCb, 0.25 mM MnCl2,1 mM each of dTTP and
dCTP, 0.2 mM each of dATP and dCTP and 5 units Taq DNA polymerase. After
an initial denaturation of 3 min at 94 °C, the following steps were repeated for 30
cycles in a thermal cycler: 1 min at 94 °C, 1 min at 45 °C and 1 min at 72 °C. The
amplified product was precipitated with ethanol, eluted from a 1 % agarose gel
and digested with EcoR I and BamH I. The digested product was again eluted
from a 1 % agarose gel and ligated with pJO290 digested with EcoR I and BamH
I. The ligation mix was transformed into E.coli JM109 and selection was done on
LB-agar supplemented with 25 l^g/ml chloramphenicol and 0.2 % glucose.
Site-directed mutagenesis
Site directed mutagenesis was carried out on the lipase gene cloned in pET-21b
by a modified PCR technique (Chen and Arnold, 1991). For each substitution an
oligonucleotide containing the desired mutation was used as the primer
(mismatch primer) to initiate chain extension between the 5' and 3' PCR primers.
In the first PCR, the mismatch primer and the 3' primer were used to generate a
DNA fragment containing the new base substitution. The fragment was
separated from the template and primers by agarose gel electrophoresis, purified
and used as the new 3' primer in a second PCR with the 5' primer to generate full
length product, which was cloned into pET-21b for expression of the mutant
protein.
Example 7
Recombination of the clones obtained in generation 2
The mutant 3B1 was created from the clone 2-8G10 and wt by using the unique
restriction site Hae II at position 910 of the lipase gene. The genes coding for the
two proteins were amplified by PCR using the T7 promoter and terminator
primers. The PCR products were purified by gel extraction and digested with
Hae II and Nde I. The upper and lower bands correspond to the C-terminal and
N-terminal regions of the protein, respectively. The upper band from clone 2-
8G10 and the lower one from the wild-type protein were eluted. The higher
molecular weight fragment was digested with BamH I and purified. A three
point ligation containing the Nde I- Hae II fragment (from the wt), the Hae IIBamH
I fragment (from 2-8G10) and pET-21b cut with Nde I and Hae II was set
up, the ligation mix transformed into DH5a and the positives selected (Fig.9).
The sequence of the gene was confirmed by DNA sequencing.
The mutant 4B1 (triple mutant) was created by site-directed mutagenesis on the
3B1 template using the mutagenic primer PROLF: 5'- GGC AAG GCG CCT CCG
GGA ACA GAT- 3' to incorporate a codon change CTT -> CCT that led to L114P
change in the amino acid sequence. The sequences of all the genes were
confirmed by automated DNA sequencing.
Example 8
Enzyme kinetics
All kinetic measurements were made using a thermostatted spectrophotometer
using the water-soluble substrate p-nitrophenyl acetate. Initial rates of hydrolysis
of p-nitrophenyl acetate at various concentrations were determined at 25 °C in
sodium phosphate buffer pH 7.2. The values for KM and kcat were derived from
the corresponding Lineweaver-Burke plots. The kinetic parameters obtained
with wild type and the mutants was presented in fig. 10.
Example 9
Activity of lipase and its mutants in the presence of organic solvents
The activity of the lipasea nd its mutants was checked in the presence of various
solvents. The organic solvents tested were acetonitrile, isopropanol, dimethyl
sulfoxide and dimethyl formamide. The activity assay was performed using
PNPA as a substrate. The substrate (2 mM) was dissolved in various percents
(v/v) of the organic solvent in buffer (50 mM pH 8.0) and the reaction was
started with the addition of lipase at a concentration of 0.246 mg/ml. The activity
was monitored as n increase in absorption at 410 nm and the specific activity was
calculated using the initial slopes of the curve. In fig 11 the data obtained with
acetonitrile is presented.
The preceding examples demonstrate the usefulness of the present invention in
generating, identifying and isolating lipases which have improved stability
and/or ester hydrolysis activity at higher temperature in organic media relative
to the natural enzyme.
Having thus described exemplary embodiments of the present invention, it
should be noted by those skilled in the art that the disclosures herein are
exemplary only and that various other alternations, adaptations and
modifications may be made within the scope of the present invention.
Accordingly, the present invention is not limited to the specific embodiments as
illustrated herein.
Description of figures
Fig.l : Subcloning of lipA without the signal sequence into pET 21b
Fig.2. SDS-PAGE profiles of the purified proteins. All the lipases were purified
by the procedures given in examples. Lane 1: Low Molecular weight marker, 2:
wild-type lipase, 3:1-1E5,4: 2-8G10, 5: 2-3H5, 6: 3B1, 7: 4B1
Fig.3 Hydrolysis of a triglyceride catalyzed by a lipase
Fig.4: Residual activity of various mutants and the wild type at a various times
on exposure to a temperature of 55 C. The substrate used is PNPA.
Fig. 5 Residual activity of various mutants and the wild type at various times on
exposure to temperature of 50 C. The substrate used is olive oil.
Fig.6 The lipA gene pBR 322
Fig.7 Subcloning lipA along with the suignal sequence into pET 21b
Fig.8 Subcloning of lip A without signal sequence into pJO290
Fig. 9 Recombination of the clones obtained in generation II
Fig.10 Kinetic parameters and half life of stability at 55 C of wild type and
mutant lipase.
Fig.ll Activity of lipase and its mutants in the presence of acetonitrile at various
concentrations in water.
References:
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We Claim:
1. Novel thermostable, organic solvent resistant and high pH tolerant lipase gene variants having SEQ ID No. 2 of molecular wt 19443, SEQ ID No. 3 of molecular wt 19515 SEQ ID No. 4 of molecular wt 19456.9, SEQ ID No. 5 of molecular wt. 19487and SEQ ID No. 6 of molecular wt. 19470.9
2. Novel gene variants as claimed in claim 1, wherein said gene variants are thermostable in the temperature range of about 45 to 95 C.
3. Novel gene variants as claimed in claim 1, wherein said gene variants are resistant to organic solvents selected from group consisting of acetonitrile, isopropanol, dimethyl sulfoxide and dimethyl formide.
4. Novel gene variants as claimed in claim 1, wherein residual activity of the gene variants is in the range of 25 to 100 % in presence of acetonitrile.
5. Novel gene variants as claimed in claim 1, wherein residual activity of the gene variants is in the range of 28.7 to 85.5% in presence of acetonitrile.
6. Novel gene variants as claimed in claim 1, wherein the gene variants have inherent ability to withstand high pH in the range of 9 to 13; ability to withstand damaging surfactants and enzymes comprising groups of linear alkyl benzene sulfonates, proteases and compounds thereof.
7. An expression system for novel thermostable, organic solvent resistant and high pH tolerant lipase gene variants as claimed in claim 1-6, wherein said expression system comprising of having SEQ ID No. 2 of molecular wt 19443, SEQ ID No. 3 of molecular wt 19515, SEQ ID No. 4 of molecular wt 19456.9, SEQ ID No. 5 of molecular wt. 19487 and SEQ ID No. 6 of molecular wt 19470.9 present in the vector pJ0290.
8. A method of preparing novel thermostable, organic solvent resistant and high pH tolerant lipase gene variants as claimed in claim 1-6, having SEQ ID No. 2 of molecular wt 19443, SEQ ID No. 3 of molecular wt 19515, SEQ ID No. 4 of molecular wt 19456. 9, SEQ ID No. 5 of molecular wt. 19487 and SEQ ID No. 6 of molecular wt 19470.9 said method comprising the steps of:

(a) isolating and purifying lipase gene from Bacillus subtilis,
(b) cloning lipase gene isolated in step (a) in vector pJ0290,
(c) generating gene variants from lipase gene isolated in step (a) by random mutagenesis and site-directed mutagenesis using forward primer JOF having SEQ ID
No. 13 and reverse primer JOR having SEQ ID No. 14,
(d) cloning the gene variants obtained in step (c) in plasmid vector pJ0290, and
(e) ligating the cloned gene variants of step (d) in E.coli JM109 to further obtain expression of the protein.

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75-DEL-2003-Correspondence-Others-(30-06-2009).pdf

75-del-2003-correspondence-others.pdf

75-del-2003-correspondence-po.pdf

75-del-2003-description (complete).pdf

75-DEL-2003-Drawings-(25-02-2009).pdf

75-del-2003-drawings.pdf

75-del-2003-form-1.pdf

75-del-2003-form-18.pdf

75-del-2003-form-2.pdf

75-DEL-2003-Form-3-(25-02-2009).pdf

75-del-2003-form-3.pdf

75-DEL-2003-Form-5-(25-02-2009).pdf

75-del-2003-form-5.pdf

75-DEL-2003-Others-Document-(25-02-2009).pdf

75-DEL-2003-Others-Documents-(16-04-2005).pdf

75-DEL-2003-PCT-210-(25-02-2009).pdf

75-DEL-2003-PCT-237-(25-02-2009).pdf

75-DEL-2003-PCT-409-(25-02-2009).pdf

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Patent Number

235486

Indian Patent Application Number

75/DEL/2003

PG Journal Number

31/2009

Publication Date

31-Jul-2009

Grant Date

06-Jul-2009

Date of Filing

30-Jan-2003

Name of Patentee

COUNCIL OF SCIENTIFIC AND INDUSTRIAL RESEARCH

Applicant Address

RAFI MARG, NEW DELHI-110 001, INDIA.

Inventors:

#	Inventor's Name	Inventor's Address
1	NALAM MADHUSUDHANA RAO	CENTREA FOR CELLULAR AND MOLECULAR BIOLOGY, HYDERABAD, INDIA.
2	MS PRIYAMVADA ACHARYA	CENTREAFOR CELLULAR AND MOLECULAR BIOLOGY, HYDERABAD, INDIA.

PCT International Classification Number

C12N 9/00

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA