Abstract:
A biologically pure culture of  Geobacillus  Strain T1 bacteria isolated from Palm Oil Mill Effluent capable of producing thermostable lipase T1 gene. The production of thermostable lipase T1 gene from a novel  Geobacillus  bacterium having a designated as strain T1, was aerobic, gram positive and endospore-forming, rod-shaped. The G+C content of the genomic DNA was 52.6%. On the basic of physiological data, phenotypic traits and molecular analysis, strain T1 represents a novel species within the genus  Geobacillus , The thermostable T1 lipase gene was subcloned into the pGEX-4T1 vector with and without signal peptide in prokaryotic system. The 28 amino acid residues signal peptide alter the conformation of GST moiety and preventing it from bind to affinity glutathione Sepharose column. By expression and simplification of the purification through single step affinity chromatography shows a specific activity of 292.929 U/mg and 72.55% of fusion lipase was recovered from crude cell lysate. T1 mature lipase was successfully purified with a final recovery of 51.49% and specificity activity of 959.033 U/mg.

Description:
CROSS-REFERENCE TO A RELATED APPLICATION  
       [0001]     The subject application claims priority to Malaysian Application No. PI 2004 3110, filed Aug. 2, 2004.  
       FIELD OF INVENTION  
       [0002]     This invention relates to a novel microorganism and a production method. More particularly, to a novel microorganism which produces a thermostable lipase T1.  
       BACKGROUND OF THE INVENTION  
       [0003]     Lipase is a very important metabolic enzyme for ordinary biological body; it can hydrolyze fat to produce free fatty acid. Lipases or acylglycerol hydrolases are enzymes that catalyze the hydrolysis of long chain triglyceride into diacylglyceride, monoglyceride, glycerol and free fatty acids. However, lipases are also capable of catalyzing the reverse reaction of hydrolysis in the formation of esters from alcohols and fatty acids or via transesterification.  
         [0004]     The lipase is extensively used as an enzyme for food processing to flavor dairy products, medicines as digestive, improvements of fats and oils, and the like. The lipase is required to have various characteristics for each use, and a thermostable lipase is applied to a wide various characteristics for each use, and a thermostable lipase is applied to a wide variety of fields and requested to be variously used.  
         [0005]     Microbial extracellular lipases are usually more thermostable than animals or plants lipases. Microbial extracellular lipase has a potential use in industries and diagnostics. A major requirement for commercial enzyme is thermal stability because thermal denaturation is a common cause of enzyme inactivation. In addition, increasing enzyme thermostability would allow enzymatic reactions to be carried out at higher temperatures; this would help to increase conversion rates, substrate solubility and to reduce the possibility of microbial growth and the viscosity of the reaction medium.  
         [0006]     Although thermophiles could be a good candidate in producing thermostable enzyme, but it is often impractical because of low yield and also high temperature fermentation equipment may be needed. To overcome this problem, a molecular approach through genetic engineering becomes a good alternative to achieve high-level expression towards bulk production economically via prokaryotic system. So far, several thermostable lipases have been successfully cloned and expressed in heterologous hosts intracellularly.  
         [0007]     For fundamental studies and for commercial purposes expression of foreign protein in prokaryotic systems is most widely used to achieve high-level expression. Expression vector and host are an important issue for achieving maximal expression of cloned gene, however molecular cloning of foreign gene does not ensure that the gene been expressed successfully.  
         [0008]     At this present invention, rapid cloning of thermostable lipase through Polymerase Chain Reaction (PCR) technique and manipulation of the thermostable lipase T1 gene is express through prokaryotic system using various kinds of promoters.  
       SUMMARY OF THE INVENTION  
       [0009]     The bacterium strain  Geobacillus  sp. Strain T1 (DSMZ Deposit No.  Geobacillus  sp. T1-DSM 17139) of the present invention provides a thermostable T1 lipase gene and a method of producing it. The present invention also provides a transformant having a recombinant host cell expressing the thermostable T1 lipase gene and a method preparing.  
     
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0010]      FIG. 1  is a diagram showing the micrograph of strain T1.  
         [0011]      FIG. 2  is a diagram showing temperature activity of T1 mature lipase. Purified T1 lipase was assayed at different temperatures for 30 minute under shaking rate of 250 rpm with olive oil as substrate.  
         [0012]      FIG. 3  is a diagram showing activity test of T1 lipase that was assayed at various pH or 30 minute.  
         [0013]      FIG. 4  is a diagram showing stability of T1 lipase that was assayed after pre-treatment with various pH from pH 4 to pH 12 for 30 minute.  
         [0014]      FIG. 5  is a diagram showing effect of metal ions on lipase activity.  
         [0015]      FIG. 6  is a diagram showing effect of surfactants on lipase activity.  
         [0016]      FIG. 7  is a diagram showing effect of substrates on lipase activity.  
         [0017]      FIG. 8  is a diagram showing effect of inhibitors on lipase activity. 
     
    
     BRIEF DESCRIPTION OF THE SEQUENCES  
       [0018]     SEQ ID NO:1 is the complete 16S rDNA sequence of 1519 bp (AY166603).  
         [0019]     SEQ ID NO:2 is forward primers pGEX-For.  
         [0020]     SEQ ID NO:3 is reverse primers pGEX-Rev.  
         [0021]     SEQ ID NO:4 is forward primer EH2-F.  
       DETAILED DESCRIPTION OF THE INVENTION  
       [0022]     Screening of  Geobacillus  sp. Strain T1 According to the present invention,  Geobacillus  sp. Strain T1 was isolated and identified based on thermophilic species. The  Geobacillus  sp. Strain T1 culture could be obtained from Enzyme and Microbial Technology Research Department of Biochemistry and Microbiology, Faculty of Science and Environmantal Studies, University Putra Malaysia.  
         [0023]     In the process of screening for lipase producing bacterium from Palm Oil Mill Effluent in Malaysia, isolate T1 which gave positive result on Triolein agar plate was isolated. To verify the systematic position of this lipase-producing bacterium, a study of morphological and physiological characteristics, 16S rRNA analysis, cellular fatty acids analysis, DNA composition, DNA/DNA hybridization and RiboPrint analysis were undertaken.  
         [0000]     Isolation and Identification of  Geobacillus  sp. Strain T1  
         [0024]     Samples were taken from a Palm Oil Mill Effluent in Selangor, Malaysia.  Geobacillus  sp. Strain T1 was isolated from an enriched medium (pH 7.0) containing NaCl 0.2%, MgSO 4 .7H 2 O 0.04%, MgCl 2 .6H 2 O 0.07%, CaCl 2 .2H 2 O 0.05%, KH 2 PO 4  0.03%,K 2 HPO 4 0.03%, (NH 4 ) 2 SO 4  0.05% with the sole carbon source of Olive oil (2%) at 60° C. under shaking condition of 150 rpm. It was plated on Triolein agar plate form screening of lipase producer.  
         [0025]     For morphological studies, pure bacterial strain was streaked on nutrient agar plate and incubated at 60° C. prior to gram staining, the results were then observed under light microscope. Morphological and physiological characteristics were sent to DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen) (German Collection of Microorganism and Cell Cultures). The physiological characteristics study includes catalase and oxidase test, anaerobic growth, Voges-Proskauer test, growth at 30° C., 40° C. and 70° C., growth in medium with pH 5.7, NaCl 2%, 5% and lysozyme broth, fermentation of D-glucose, L-arabinose, D-xylose, D-mannitol and D-glucose, hydrolysis of starch, gelatin, casein and tween 80, decomposition of tyrosine, use of citrate and propionate, nitrate reduction, indol production, phenylalanine deaminase and arginine dihydrolase test were conducted. Fatty acids were extracted and analysed following the instructions of Sherlock microbial identification system.  
         [0026]     16S rDNA was amplified by PCR using two universal primers and the PCR product was purified. A purified PCR product was cloned into TOPO TA PCR 2.1 cloning vector (Invitrogen). The recombinant plasmid was extracted and was then sequenced using an ABI PRISM 377 DNA sequencer (Applied Biosystems).  
         [0027]     A chromosomal DNA was isolated and purified and G+C content was determined by using chromatography conditions. The DNA was hydrolyzed and the resultant nucleotides were analyzed by reverse-phase HPLC. DNA/DNA hybridization was carried out with the using a model 2600 spectrophotometer.  
         [0028]     A standardized, automated ribotyping was performed using the Qualicon™ RiboPrinter system. The RiboPrinter system combines molecular processing steps for ribotyping in a stand-alone, automated instrument. Steps including cell lysis, digestion of chromosomal DNA with restriction enzyme EcoR1, separation of fragments by electrophoresis, transfer of DNA fragments to a nylon membrane, hybridization to a probe generated from the rrnB operon from  E. coli , chemiluminescent detection of the probe to the fragments containing rrn operon sequences, image detection and computerized analysis of RiboPrint patterns.  
         [0029]     The isolated Strain T1 was aerobic, gram positive, endospore forming, rod-shaped bacteria (0.8-1.0 μm width and 2.5-6.0 μm long) ( FIG. 1 ). The DNA base composition of strain T1 was around 52.6% mol G+C. The cylindrical endospores were appeared terminally in swollen sporangia. The growth of strain T1 occurred even at 70° C. but no growth observed at 30° C. and 40° C. It was tolerant with the presence of NaCl up to 2%. Acid was produced from D-fructose, but not from D-glucose and D-mannitol. It showed positive result in citrate and nitrate test. The morphological and physiological characteristics are presented in Table 1 and 2.  
                                                                                             TABLE 1                           Morphological and physiological properties of  Geobacillus  strain T1.                Characteristic     Geobacillus  strain T1                       Cell width (μm)   0.8-1.0           Cell length (μm)   2.5-6.0           Spores oval/cylindrical   +           Swollen sporangium   d           Catalase   +           Oxidase   −           Anaerobic growth   −           VP reaction   −           pH in VP broth   4.9                Growth at   30° C.   −               40° C.   −               70° C.   +                Growth in               Medium pH 5.7   +                NaCl   2%   +               5%   −                Lysozyme broth   −           Production of acid from:           D-glucose   −           L-arabinose   w           D-xylose   w           D-mannitol   −           D-fructose   +           Gas from glucose   −           Lecithinase   n.g.           Hydrolysis of:           Starch   +           Gelatin   −           Casein   −           Tween 80   −           Decomposition of tyrosine   +           Use of:           Citrate   +           Propionate   −           Nitrate reduction   +           Indole production   −           Phenylalanine deaminase   −           Arginine dihydrolase   −                         +—positive;                −—negative;                w—weak reaction;                n.d.—not determined;                n.g.—not growth.             
 
         [0030]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                   
               
               
                 Comparison of biochemical, morphological and physiological properties 
               
               
                 of  Geobacillus  strain T1 and related thermophilic bacilli. 
               
             
          
           
               
                   
                 Characteristic 
                 1 
                 2 
                 3 
               
               
                   
                   
               
               
                   
                 G + C mol % of DNA 
                 52.6 
                 53.7 
                 51.0 
               
             
          
           
               
                   
                 Growth at 
                 30° C. 
                 − 
                 − 
                 − 
               
               
                   
                   
                 70° C. 
                 + 
                 + 
                 + 
               
             
          
           
               
                   
                 Spore 
                 shape 
                 C 
                 O 
                 O/C 
               
               
                   
                   
                 Position 
                 T 
                 T 
                 T 
               
             
          
           
               
                   
                 Catalase 
                 + 
                 + 
                 n.d. 
               
               
                   
                 Oxidase 
                 − 
                 + 
                 − 
               
               
                   
                 Anaerobic growth 
                 − 
                 − 
                 − 
               
               
                   
                 Acid from glucose 
                 − 
                 + 
                 + 
               
             
          
           
               
                   
                 Formation of 
                 acetoin 
                   
                 − 
                 − 
               
               
                   
                   
                 Indole 
                 − 
                 − 
                 − 
               
               
                   
                   
                 H 2 S 
                   
                 n.d. 
                 n.d. 
               
             
          
           
               
                   
                 Citrate utilization 
                 + 
                 − 
                 + 
               
               
                   
                 Hydrolysis of: 
               
               
                   
                 Starch 
                 + 
                 + 
                 + 
               
               
                   
                 Gelatin 
                 − 
                 n.d. 
                 + 
               
               
                   
                 Casein 
                 − 
                 w 
                 − 
               
               
                   
                 Nitrate reduction 
                 + 
                 n.d. 
                 + 
               
               
                   
                   
               
               
                   
                   +—positive;    
               
               
                   
                   −—negative;    
               
               
                   
                   w—weak reaction;    
               
               
                   
                   n.d.—not determined;    
               
               
                   
                   C—cylindrical;    
               
               
                   
                   O—oval;    
               
               
                   
                   T—terminal.    
               
               
                   
                   Taxa are indicated as: 1,  Geobacillus zalihaii ; 2,  Geobacillus thermoleovorans  (Zarilla and Perry, 1986); 3,  Geobacillus kaustophilus  (White et al., 1993).    
               
             
          
         
       
     
         [0031]     A complete 16S rDNA sequence of 1519 bp (AY166603) is determined as below (SEQ ID NO: 1).  
                               1   gagtttgatc ctggctcagg acgaacgctg gcggcgtgcc taatacatgc aagtcgagcg                   61   gaccggatcg gagcttgctc tgatttggtc agcggcggac gggtgagtaa cacgtgggca               121   acctgcccgc aagaccggga taactccggg aaaccggagc taataccgga taacaccgaa               181   gaccgcatgg tctttggttg aaaggcggcc tttggctgtc acttgcggat gggcccgcgg               241   cgcattagct agttggtgag gtaacggctc accaaggcga cgatgcgtag ccggcctgag               301   agggtgaccg gccacactgg gactgagaca cggcccagac tcctacggga ggcagcagta               361   gggaatcttc cgcaatgggc gaaagcctga cggagcgacg ccgcgtgagc gaagaaggcc               421   ttcgggtcgt aaagctctgt tgtgagggac gaaggagcgc cgttcgaaga gggcggcgcg               481   gtgacggtac ctcacgagga agccccggct aactacgtgc cagcagccgc ggtaatacgt               541   agggggcgag cgttgtccgg aattattggg cgtaaagcgc gcgcaggcgg ttccttaagt               601   ctgatgtgaa agcccacggc tcaaccgtgg agggtcattg gaaactgggg gacttgagtg               661   caggagagga gagcggaatt ccacgtgtag cggtgaaatg cgtagagatg tggaggaaca               721   ccagtggcga aggcggctct ctggcctgca actgacgctg aggcgcgaaa gcgtggggag               781   caaacaggat tagataccot ggtagtccac gccgtaaacg atgagtgcta agtgttagag               841   gggtcacacc ctttagtgct gcagctaacg cgataagcac tccgccgggg gagtacggcc               901   gcaaggctga aactcaaagg aattgacggg ggcccgcaca agcggtggag catgtggttt               961   aattcgaagc aacgcgaaga accttaccag gtcttgacat cccctgacaa cccaagagat               1021   tgggcgttcc cccttcgggg ggacagggtg acaggtggtg catggttgtc gtcagctcgt               1081   gtcgtgagat gttgggttaa gtcccgcaac gagcgcaacc ctcgcctcta gttgccagca               1141   cgaaggtggg cactctagag ggactgccgg cgacaagtcg gaggaaggtg gggatgacgt               1201   caaatcatca tgccccttat gacctgggct acacacgtgc tacaatgggc ggtacaaagg               1261   gctgcgaacc cgcgaggggg agcgaatccc aaaaagccgc tctcagttcg gattgcaggc               1321   tgcaactcgc ctgcatgaag ccggaatcgc tagtaatcgc ggatcagcat gccgcggtga               1381   atacgttccc gggccttgta cacaccgccc gtcacaccac gagagcttgc aacacccgaa               1441   gtcggtgagg caacccgcaa gggagccagc cgccgaaggt ggggcaagtg attggggtga               1501   agtcgtaaca aggtagccg          
 
         [0032]     The major amount of cellular fatty acids of strain T1 was iso-fatty acids (Table 3). Among them, iso-branched pentadecanoic acid (iso-C15), hexadecanoic acid (iso-C16) and heptadecanoic acid (iso-C17) making up 78.33% of the total fatty acids especially iso-C15 and iso-C17 were in abundant. The fatty acid profile distinguishes the genus  Geobacillus  clearly from other mesophiles and thermophiles of the genera  Bacillus Alicyclobacillus, Brevibacillus, Aneurinibacillus, Sulfobacillus  and  Thermobacillus. Although the strain T 1 and  Geobacillus thermoleovorans  DSM 5366 T  shared typical fatty acid profile of  Bacillus  rRNA group 5 but it could be differentiated by % composition of iso-C 16 as indicated in Table 3.  
                                           TABLE 3                           Cellular fatty acids composition of  Geobacillus  strain T1 and       phylogenitical neighbor.                  Geobacillus  sp.     Geobacillus thermoleovorans         Fatty acid   strain T1   (DSM 5366 T )                    10.0       2.7       14.0 ISO       1.0       14:0   7.22   1.4       15:0 ISO   32.42   22.6       15:0 ANTEISO   1.01   1.3       15:0   0.82   2.1       16:0 ISO   6.14   21.0       16:0   4.98   11.2       17:0 ISO   39.77   18.5       17:0 ANTEISO   4.97   4.6       17:0   0.53   1.3       18:1 ISO H   0.38       18:0 ISO   0.36   0.9       18:0   0.47   3.4       18.1       1.2       19:0 ISO   0.91       unsaturated C16       6.6       other       0.2                  
 
         [0033]     DNA/DNA hybridization study was carried out to verify the taxonomic relationship between strain T1 and phylogenetical neighbors. The genomic DNA/DNA relatedness between strain T1 and type strains  Geobacillus kaustophilus  DSM 7263 T  and  Geobacillus thermoleovorans  DSM 5366 T  were 64.9 and 68.8, respectively (Table 4).  
                         TABLE 4                           Levels of DNA/DNA reassociation (%) among  Geobacillus  strain       T1 and thermophilic bacilli.            Organisms     Geobacillus  strain T1                 Geobacillus kaustophilus  DSM 7263 T     66.9; 62.9 (average = 64.9)         Geobacillus thermoleovorans  DSM 5366 T     70.0; 67.6 (average = 68.8)                  
 
         [0034]     The DNA/DNA reassociation values were fall below the threshold value of 70% DNA/DNA similarity for definition of species. The RiboPrint analysis was carried out for the decision on the affiliation of strain T1. However, the RiboPrint pattern of strain T1 was not identified by the Dupont identification library to give rise to the identification at the species level (&gt;0.85). Its RiboPrint pattern showed the highest similarity to  Geobacillus kaustophilus  DSM 7263 T  (0.69). The similarity to the pattern of  Geobacillus therleovorans  DSM 5366T was somewhat lower (0.57). The patterns of  Geobacillus kaustophilus  DSM 7263 T  and  Geobacillus thermoleovorans  DSM 5366 T  show a binary similarity of 0.64.  
         [0035]     As a consequence, the strain T1 merits recognition as a member of a novel species through morphology and physiological studies, cellular fatty acids composition, DNA composition, DNA/DNA hybridization and RiboPrint analysis. Therefore, we proposed the creation of a novel species,  Geobacillus zalihae  sp. nov., for strain T1 T . (za.li.&#39;hae N.L. gen. n. zalihae of zaliha, in honour to the scientist from Universiti Putra Malaysia, who has contributed significantly to extremophiles). Cells are rod-shaped, 0.8-1.0 width and 2.5-6.0 length, gram positive bacteria. The terminal spores are cylindrical and swollen the sporangium. The DNA base composition of strain T1 was around 52.6% mol G+C. It contained major amount of iso-fatty acids with iso-C15, and iso-C17 were in abundant (77.19%). Growth is aerobic and still growth at 70° C. and tolerant up to 2% NaCl. It can not perform anaerobic growth. Catalase and nitrate test are positive. No acid from D-glucose and D-mannitol. It can utilize citrate. Starch is hydrolysed but not gelatin and casein.  
         [0000]     Cloning and Sequencing of Thermostable Lipase Gene from  Geobacillus  sp. Strain T1  
         [0000]     Bacterial Strains and Plasmids  
         [0036]     Recombinant plasmid pBAD/T1 (Leow et al., 2004) carrying thermostable T1 lipase gene was used as source for subcloning.  E. coli  strains were grown in LB medium at 37° C. pRSET C (Invitrogen), pET22b(+) (Novagen) and pGEX-4T1 (Amersham Bioscience; United Kingdom, England) was used for subcloning and expression.  
         [0000]     DNA Manipulation  
         [0037]     The plasmid DNA was isolated with a QIAGEN miniprep spin kit (QIAGEN; Hilden, Germany) according to the manufacturer&#39;s instructions. The PCR product was purified with a GeneClean Kit (Qbiogene; Carlsbad, USA) as described by the supplier. Competent cells of  E. coli  were prepared by using a conventional CaCl 2 .  
         [0000]     Subcloning and Expression of the Thermostable T1 Lipase Gene.  
         [0038]     Subcloning of the T1 lipase gene was done by designing a set of primers which incorporated restriction enzyme sites BamH1/EcoR1 which involved primers pGEX-For: 5′-GAA GGG ATC CGT GAT GAA ATG CTG TCG GAT TAT G-3(SEQ ID NO:2) and pGEX-Rev: 5′-AAT AGA ATT CTT AAG GCT GCA AGC TCG CCA A-3′ (SEQ ID NO:3) for subcloning of open reading frame of T1 lipase and EH2-F: 5′-GAC GGG ATC CGC ATC CCT ACG CGC CAA TGA T-3′ (SEQ ID NO:4) and pGEX-Rev: 5′-AAT AGA ATT CTT AAG GCT GCA AGC TCG CCA A-3′ (SEQ ID NO:3) for subcloning of T1 mature lipase gene. The ligated plasmid was used to transform  E. coli  strains and screened with tributyrin LB agar plates with appropriate antibiotics.  E. coli  BL21(De3)plysS harboring recombinant plasmids were grown in 1 L blue cap bottles containing 200 ml of LB medium supplemented with 100 μg/ml ampicillin and 35 μg/ml chloramphenicol on a rotary shaker (200 rpm) at 37° C.  
         [0039]     The recombinant clones with and without signal peptide were induced with 1 mM of isopropyl-β-D-thiogalactopyranoside (IPTG) at OD 600  nm ˜0.5 for different induction periods (0, 4, 8, 12, 20, 28, 36 and 44 h). It was further optimized by varying the concentration of IPTG (w/o, 0.025, 0.05, 0.1, 0.5, 1.0, 1.5 and 2.0 mM) and induction OD 600 nm  (0.25, 0.50, 0.75 and 1.00). Cultures (10 ml) were harvested by centrifugation and resuspended with 2 ml of 50 mM of potassium phosphate buffer (pH 7.0) before sonication (Branson 250 sonifier: output 2, duty cycle 30 and min 2) and cleared by centrifugation (12,000 rpm, 20 min). The clear crude lysate was used for lipase assay.  
         [0000]     Electrophoresis  
         [0040]     SDS-PAGE was done on 12% of gel was used to analyse the protein. A broad range of protein standard (MBI Fermentas; St. Leon-Rot, Germany) was used as a molecular mass marker.  
         [0000]     Purification of T1 Lipase  
         [0041]     Four hundred mL of recombinant culture was harvested by centrifugation and concentrated to 40 mL prior to sonication. The crude cell lysate was loaded on a glutathione-Sepharose HP column (XK 16/20) containing 10 mL column volume equilibrated with PBS (pH 7.3) at a flow rate of 0.2 mL/minute. The column was washed with the same buffer until no protein was detected. The bound lipase was eluted with thrombin cleavage buffer (20 mM Tris-HCl, 100 mL NaCl and 0.33 mM CaCl 2 , pH 8.4 supplemented with 10 mM reduced glutathione. The fusion protein was subjected to thrombin cleavage at 20° C. for 20 h and buffer exchanged with Sephadex G-25 prior to dialysis against PBS (pH 7.3). The GST tag and thrombin enzyme were further removed by using Glutahione-Sepharose HP, HiTrap Glutathione 4FF and HiTrap Benzamidine in series.  
         [0042]     Expression of T1 lipase including signal peptide was achieved through prokaryotic system involving pBAD, pRSET C, pET22b(+) and pGEX-4T1 which under the control of araC, T7, T7 lac and tac promoters. Among them, relatively higher level of expression was achieved with pGEX-4T1 expression system which under the control of tac promoter for inducible high level expression. It was chosen for further study. Simple optimization was carried out to increase the soluble protein of recombinant clone harboring recombinant plasmid pGEX-4T1 with signal peptide. A total of 11,708 U of lipase activity was detected when induced with 0.05 mM of IPTG at OD 600nm  ˜0.5 for 8 hour which corresponded to a specific activity of 30.192 U/mg. (Table 5).  
                                       TABLE 5                           Expression of T1 lipase gene with and without signal peptide.                Total activity from 1L culture (U)            Optimization condition   Signal peptide   Without signal peptide               Induction time (h)    5,627 (8)   23,915 (12)       Inducer concentration (mM)   11,708 (0.050)   31,609 (0.025)       Induction OD 600 nm     —   41,902 (0.750)                 Note:            The host BL21(De3)plysS harboring recombinant plasmid pGEX/T1 and pGEX/T1S were induced with 1 mM of IPTG at OD 600 nm ˜0.5 for different times. The bracket indicates the optimum values of every parameter. The total activity was calculated based on 1 L of culture.             
 
         [0043]     Theoretically, expression of T1 lipase as GST fusion protein in prokaryotic system allows rapid purification of recombinant lipase through affinity chromatography. However, we are failed to purify the soluble fraction of recombinant lipase because it did not bind to glutathione Sepharose 4FF even though a high level of activity was detected. It might be due to high hydrophobic region of GST and signal peptide lead to conformation changes and improperly folded of GST domain. In some cases, GST fusion proteins are totally or partly insoluble. Furthermore, high level expression might be contributing to improper folding of partial and highly insoluble fusion protein and subsequently interfere it from binding to affinity column.  
         [0044]     According to Frangioni and Neel (1993), the insolubility of GST fusion protein was common. So, Sarkosyl lysis method was used to solubilize partially improper folded active T1 fusion lipase. The non-ionic detergent Triton X-100 was used to sequester sarkosyl with the hope to refold back the solubilized fusion protein in correct folding which subsequently allow GST fusion protein to bind with affinity glutathione Sepharose 4FF. As shown in Table 6, a recovery of around 25% and purification fold of 7.3 was achieved through anionic and non-ionic detergents treatment of fusion protein.  
                                                                   TABLE 6                           Purification of GST fusion lipase with signal peptide.                Total   Total   Specific               Purification   activity   protein   activity   Recovery   Purification       steps   (U)   (mg)   (U/mg)   (%)   fold                    Cell lysate   1,824.90   37.90   48.15   100.00   1.00       Glutathione   454.79   1.29   352.55   24.92   7.31       Sepharose                 Note:            The fusion protein was solubilized with 2% anionic detergent Sarkosyl and 1% non-ionic detergent Triton X-100 for binding to affinity chromatography.             
 
         [0045]     Signal peptide control the entry of all virtually all proteins to the secretory pathway, both in eukaryotes and prokaryotes and cleaved off while the protein is translocated through the membrane. However, it does not contribute to the structural gene of thermostable T1 lipase. Thus, we tried to express the T1 lipase by omitting the signal peptide with the aimed to improve the folding and interaction of GST tag and T1 mature lipase without interfering of the affinity binding of GST fusion protein to immobilized glutathione at matrix support.  
         [0046]     Removing of signal peptide rigidify the fusion lipase since a total of 28 amino acid residues limited the movement of T1 lipase in covering the GST moiety. Manipulation of T1 lipase gene significantly improved the expression level by 4.25 and 2.70 fold for induction time and inducer concentration, respectively (Table 1). Further optimization expression encountered an expression level of 41,902 U/L of culture when induced with 0.025 mM of IPTG at OD 600nm  ˜0.75 for 12 hour. It was around 279 fold increased in expression level when compared to wild-type  Geobacillus  sp. Strain T1.  
         [0047]     Removing the signal peptide from the fusion protein not only improved the yield of active T1 lipase but also simplifying the purification through affinity chromatography glutathione Sepharose 4FF column to homogeneity ( FIG. 2 ). As shown in Table 7, the recovery obtained was around 72.55% with the purification fold of 2.87. Purification of fusion lipase increased the specific activity from 103.762 U/mg (crude cell lysate) to 297.929 U/mg (purified fusion lipase).  
         [0048]     T1 mature lipase was obtained through subsequent purification steps (Table 8). The fusion lipase was cleavage with thrombin protease at 20° C. for 20 hour and subjected to Sephadex G-25 to exchange buffer to PBS (pH 7.3) and remove free glutathione prior to dialysis. It was subjected to affinity chromatography glutathione sepharose HP, HiTrap glutathione sepharose 4FF and Benzamidine FF (high sub) in series to remove the GST tag and thrombin enzyme.  
                                                                   TABLE 7                           Purification of GST fusion lipase without signal peptide                Total   Total   Specific               Purification   activity   protein   activity   Recovery   Purification       Steps   (U)   (mg)   (U/mg)   (%)   fold                    Cell lysate   3,732.331   35.970   103.762   100.00   1.00       Glutathione   2,707.880   9.089   297.929   72.55   2.87       Sepharose                 Notes:            Direct purification of fusion protein was conducted with affinity Glutathione Sepharose 4FF column without any detergent.             
 
         [0049]                                                                        TABLE 8                           Purification of T1 mature lipase                Total   Total   Specific               Purification   activity   protein   activity   Recovery   Purification       Steps   (U)   (mg)   (U/mg)   (%)   fold                    Cell lysate   28,848.120   138.255   208.659   1.00   100.00       Affinity 1   20,557.051   42.038   489.011   71.26   2.34       Affinity 2   14,852.742   15.487   959.033   51.49   4.60                 Sepharose HP, HiTrap glutathione Sepharose 4FF and Hitrap Benzamidine (high sub) attached in series.               
 Characterization of T1 Fusion and Mature Lipase 
 
         [0050]     The T1 lipase was tested at temperatures ranging from 40° C. to 80° C. at 5° C. intervals for 30 min for enzyme activity. The thermostability test was conducted at various temperatures for different hours. Recombinant T1 lipase was also kept at a wide range of pH values ranging from pH 4-12 for pH activity and stability determination. The buffer systems used were 50 mM acetate buffer (pH 4-6), potassium phosphate buffer (pH 6-8), Tris-HCl buffer (pH 8-9), glycine-NaOH buffer (pH 9-11), and Na 2 HPO 3 /NaOH buffer (pH 11-12). The effect of effectors (metal ions, surfactants) and inhibitors were studied at a concentration of 1 mM and 5 mM, respectively at 50° C. for 30 min with an exceptional for pepstatin (1.0 mM).  
         [0051]     The effect of temperature activity and stability was tested at various temperatures for 30 min. It was most active in the temperature range 60° C. to 80° C., with maximal activity at 70° C. ( FIG. 2 ). The effect of pH on activity and stability on lipase activity was conducted at various pHs for 30 min under shaking condition. As shown in  FIG. 3 , it was active over wide pH range especially pH 6 to 10 with an optimum pH of 9 with olive oil as substrate. pH treatment showed that T1 mature lipase was relatively stable at alkaline pH ( FIG. 4 ).  
         [0052]     The effect of metal ion at final concentration of 1 mM was tested by treatment at 50° C. for 30 min. As shown in  FIG. 5 , Na + , Mn2 +  and K +  slightly enhanced the lipase activity after 15 min of treatment. Mg 2+ , Mn 2+ , Ca 2+ , K +  and Zn 2+  showed stabilizing effect even after 30 min of treatment especially Ca 2+  without significant lost of enzyme activity. However, Fe 2 + and Cu 2+  strongly inhibited the lipase activity by 60% and 52% after 30 min. The effect of surfactants on lipase activity on lipase activity was studied at concentration of 0.1% ( FIG. 6 ). Addition of 0.1% Tween, obvious enhancement was seen especially Tween 80 which increased the lipase activity by 63% and 88% after 0 and 30 min of treatment, respectively.  
         [0053]     The effect of substrates on lipase activity of various triglycerides and natural oils were used to study the substrate specificity.  FIG. 7  showed that the T1 lipase was preferred natural oils as substrates compared to triglycerides. It more selective to long carbon chain natural oils such as olive oil, corn oil, sunflower oil and palm. This opened the good opportunity for T1 lipase as catalyst in industrial application.  
         [0054]     The effect of inhibitors on lipase activity on several metal chelating agent, reducing agents, serine and aspartate inhibitors were used to study the inhibition effect to T1 mature lipase. It was not a metalloenzyme since EDTA showed slightly effect on lipase activity even at 5 mM. The reducing agents such as β-mercaptoethanol and DTT gave little inhibition effect on T1 lipase. The lipase was strongly inhibited by the addition of 5 mM PMSF and 1 mM of Pepstatin, showing that serine and aspartate residues play an important key role in the catalytic mechanism ( FIG. 8 ). The P1 lipase from  Bacillus stearothermophilus  showed 77% inhibition with the presence of 10 mM of PMSF (Sunchaikul et al., 2001).  
         [0055]     The properties of purified T1 fusion and mature lipase were compared to study the effect of GST tag on lipase activity. Both lipases was purified and their physiochemical properties was studied (Table 9). The fusion partner slightly decreased the optimum temperature and pH to 65° C. and pH 8, respectively. There are no significant different was observed with metal ions and substrates. However, Tween 20-80 gave enhancement effect to T1 mature lipase but only stable with the presence of fusion partner. Both of them were inhibited by serine and aspartate inhibitor at tested concentration.  
                                 TABLE 9                           A comparison of thermostable T1 mature lipase and fusion lipase                Properties   Fusion lipase   mature lipase                       Optimum   65° C.   70° C.           temperature           Temperature   T 1/2  = 2.5 h at 65° C.   T1 /2  = 12 h at 60° C.           stability           Optimum pH   8   9           pH stability   8-10   8-11           Metal ions   Mg 2+ , Ca 2+ , Mn 2+ ,   Mg 2+ , Ca 2+ , Mn 2+ ,               K +  and Na +     K +  and Zn 2+             Surfactants   Stable with Tween   Stable with Tween               and Triton X-100   and Triton X-100           Substrates   natural oils   natural oils           Inhibitors   PMSF and Pepstatin   PMSF and Pepstatin                      
 
         [0056]     Since the GST tag was rigidly fused to T1 mature lipase without signal peptide, so it only conferred a minimal effect on tertiary structure and biological activity without significantly changing of physicochemical properties. There are no significant difference between T1 fusion and mature lipases except the optimum pH and temperature in which only one unit pH and 5° C. higher was encountered for T1 mature lipase. The behavior of T1 fusion and mature lipase to serine and aspartate inhibitors were the same. So, we proposed the T1 lipase fused to GST tag for higher production economically to simplify fusion protein purification for industrial application especially in detergent formulation for warm wash laundry detergent. In addition, the precipitate formed by fusion protein because of aggregation at high concentration of fusion lipase was easily solubilized by resolving it in buffer pH 9 that is far away from the pI of fusion lipase.