KR100509178B1 - Discovery of Bacillus megaterium 20-1[KCTC 10435BP] producing a novle esterase and development of its efficient production method using Escherichia coli BL21(DE3)/pMLE[KCTC10434BP] - Google Patents

Abstract

The present invention relates to a bacterium Bacillus megaterium 20-1, a novel esterase produced therefrom, and a method for mass production thereof, and more specifically, a new high esterase activity from soil. The strain Bacillus megaterium 20-1 was isolated, and the esterase produced by the strain was isolated to examine the characteristics of the enzyme, and it was confirmed that it is a novel enzyme whose activity is greatly increased by a nonionic surfactant. The present invention relates to a method for mass production of an esterase using the recombinant expression vector containing the first gene and introducing the vector to develop a transformed bacterium.

Description

Discovery of Bacillus megaterium 20-1 [KCTC 10435BP] producing a novle esterase and development of its efficient production method using Escherichia coli BL21 (DE3) / pMLE [KCTC10434BP]}

The present invention relates to a bacterium Bacillus megaterium 20-1, a novel esterase produced therefrom, and a method for mass production thereof, and more specifically, a new high esterase activity from soil. The strain Bacillus megaterium 20-1 was isolated, and the esterase produced by the strain was isolated to examine the characteristics of the enzyme, and it was confirmed that it is a novel enzyme whose activity is greatly increased by a nonionic surfactant. The present invention relates to a method for mass production of an esterase using the recombinant expression vector containing the first gene and introducing the vector to develop a transformed bacterium.

Esterase enzymes have been widely studied as intracellular enzyme catalysts for promoting various metabolic reactions in vivo, and are used in various bioconversion reactions as industrial enzymes. The reason why this is important industrially is that the chiral material widely used in the pharmaceutical industry contains a lot of ester bonds. When the ester compound is synthesized by asymmetric chemical synthesis, the reaction proceeds at a high pressure of up to 700 psi and a high temperature of 250 ° C. or higher, which consumes a lot of energy and causes various side reactions that adversely affect the quality. Low purity has difficulty in producing high purity chiral compounds. On the other hand, the biotransformation process using esterases can efficiently produce high-purity chiral materials because it has unique site-specificity, steric specificity and various substrate specificity. In addition, with the recent development of enzymatic reaction systems of ideal, non-aqueous, non-aqueous and reversed phases using enzymes, esterase / lipase enzyme catalysts are increasingly used for the preparation of high value chiral materials.

Esterases / lipases are produced from a variety of animals, plants and microorganisms, among which microbial enzymes are the most biochemically diverse enzymes, many of which have industrially useful special functions. Esterases have their own special functions depending on their origin of production, so the biochemical properties of enzymes should be carefully considered in industrial applications.

Recently, the search and development of esterases, which do not lose their activity in a long time enzymatic reaction process and show great activity even in poor reaction environments such as low temperature, high temperature, alkali, organic solvent, surfactant treatment, etc., are actively progressing. In particular, esterases having properties such as low temperature and surfactant resistance are required for the synthesis of materials used as detergent additives or unstable at high temperatures. However, in most esterases developed to date, the activity of the enzyme is very weak at low temperatures, and the activity of the enzyme is largely inactivated by the nonionic surfactant.

Thus, the present inventors to search for the producing strain having excellent activated by a surface active agent, the activity of the enzyme even at a low temperature novel S. atmospheres sought results, the novel esterase novel strain Bacillus MEGATHERIUM 20-1 (Bacillus megaterium to produce 20-1) was isolated, and cloned genes encoding esterases produced from this strain were analyzed for sequencing. As a result, new homology with less than 55.8% homology with esterases produced by previously reported microorganisms was observed. It was confirmed that the gene, the present invention was completed by producing a transformed bacterium to which the plasmid for mass production was introduced for industrial use of the new esterase.

Accordingly, an object of the present invention is to provide a Bacillus megaterium 20-1 [KCTC 10435BP] producing a new esterase.

It is another object of the present invention to provide a novel esterase produced from the strain and a method for mass production thereof.

The present invention is characterized by Bacillus megaterium 20-1 [KCTC 10435BP], which produces an esterase.

Also included are esterases produced from the strains and genes encoding them.

In addition, the recombinant expression vector containing the gene, the bacteria transformed thereby and a mass production method of the esterase using the same is another feature.

Referring to the present invention in more detail as follows.

The present invention isolates the bacterium Bacillus megaterium 20-1 having high esterase activity from soil, isolates the esterase enzyme produced by the strain, and examines the properties of the enzyme, which is activated by a nonionic surfactant. It was confirmed that this new enzyme is greatly increased, and a recombinant expression vector containing an esterase gene is completed, and the vector is introduced to a method for mass production of esterase using the transformed bacteria.

First, the bacterium Bacillus megaterium 20-1, which produces a stable and activated esterase that is surfactant-activated, was isolated from the soil. Treatment of the esterase produced from this strain with Tween 80 increased the hydrolytic activity on the substrate, especially 14-fold increased activity on the PNPC (C6) substrate. A chromosome of about 1.6 kb containing the esterase gene was obtained from the Bacillus megaterium 20-1, and the sequencing analysis confirmed that a protease composed of 310 amino acids was produced. It was confirmed that the enzyme is a novel enzyme showing a homology of 55.8% or less. In addition, a recombinant expression vector containing the esterase gene was prepared and transformed to perform high expression experiments of the esterase in the transformed bacteria, which confirmed that it produced up to 3,600 units per liter, thereby transforming. Effective separation and purification of esterases produced in isolated bacteria has been established. The esterase purified from the transformed bacteria was 124 U / mg inactive and stable for most nonionic surfactants, rather it was confirmed that the activity of the enzyme was increased by these surfactants.

Therefore, the present invention was able to produce 3,600 units of esterase per liter using a mass expression system through genetic manipulation as described above, in the case of esterases and lipases, which are enzyme catalysts related to ester bonds, Considering the fact that it is difficult to use except for some high value-added substance conversion, it is possible to secure price competitiveness by mass-producing enzymes through high expression of esterase using this mass expression system. It is expected to be.

Hereinafter, the present invention will be described in more detail based on the following examples, but the present invention is not limited thereto.

Example 1 Microbial Separation

1 g of soil sample was completely suspended in 10 ml of sterile water, and then diluted 10 ⁶ fold. 0.1 ml of the diluted solution was plated on a tributyrin-LB flat medium and incubated at 37 ° C. for 24 hours, and the bacteria forming a clear ring around colonies were selected. The bacteria were shaken for 24 hours at 37 ° C. in LB liquid medium, and finally, strains having excellent esterase activity were selected.

Example 2: Microbial Identification

 The isolates were motile and Gram-positive bacillus, and analyzed using API 50 CHB, a microbial identification kit, to be most similar to Bacillus megaterium [Table 1]. Analysis of the nucleotide sequence of the bacterium 16S rRNA [FIG. 1] showed 99.4% homology with Bacillus megaterium, so the isolate was named Bacillus megaterium 20-1. It was. The strain was deposited on February 27, 2003 with the Korea Biotechnology Research Institute Gene Bank, and the accession number was assigned to KCTC 10435BP.

GLY + GLU + MDG - SAC + LYX - ERY - FRU + NAG - TRE + TAG - DARA - MNE - AMY + INU - DFUC - LARA + SBE - ARB + MLZ - LFUC - RIB + RHA - ESC + RAF + DARL - DXYL + DUL - SAL - AMD - LARL - LXYL - INO - CEL + GLYG + GNT - ADO - MAN + MAL + XLT - 2KG - MDX - SOR - LAC - GEN - 5KG - GAL - MDM - MEL + TUR +

Example 3: Cloning and Amino Acid Sequencing of Esterase Genes

The chromosome DNA of Bacillus megaterium 20-1 was completely cut using the restriction enzyme HindIII, and then transformed into E. coli XL1 blue by ligation with the plasmid pUC19 cut with the same restriction enzyme. One colony showing activity in a medium containing intributyrin was obtained. When the plasmid was isolated from this colony, 1.6 kb of foreign DNA was inserted into the 2.7 kb vector DNA. The recombinant plasmid having this 1.6 kb of foreign DNA was named pL20-1 [FIG. 2].

The nucleotide sequence of the recombinant plasmid pL20-1 into which the esterase DNA fragment was inserted was analyzed, and an ORF of 933 bp was identified through the 'ORF search' program of 'DNAstar' (Fig. 3). The ORF consists of 310 amino acids (SEQ ID NO: 3) and encodes an esterase with a molecular weight of about 34,638. Comparing amino acid sequences with existing proteins in the 'Swissprot' database using NCBI's 'BLAST' program, Esterase 20-1 was Alicyclobacillus acidocaldarius [SEQ ID NO: 4] and new enzymes were found to have 56%, 38%, 34% similarity with esterases produced by Archaeoglobus fulgidus and Bacillus halodurans . 4].

Example 4 Preparation of Recombinant Expression Vector for Esterase Mass Production and High Expression of Esterase in E. Coli

Esterase 20-1 has the advantage of performing bioconversion reaction under various reaction conditions because the activity of the enzyme is greatly increased by the nonionic surfactant and shows the optimum activity in a wide temperature range of 20 ~ 40 ℃. It is also very advantageous because the enzyme is not inactivated by the surfactant during the process. However, the yield of the enzyme expressed in the transformed Escherichia coli XL1 blue / pL20-1 is low, so that a high expression experiment was performed in Escherichia coli BL21 (DE3) using pET22-b with a strong T7 promoter to mass produce the enzyme. It was.

1) Production of high expression vector

Two primers were prepared from the DNA nucleotide sequence shown in FIG. 2 for amplification of the esterase gene, followed by PCR to amplify the 0.9 kb esterase gene. Recombinant expression vectors were constructed by cleaving both ends of the amplified genes with NcoI and BamHI restriction enzymes and inserting them with T4DNA ligase into the pET22-b expression vector digested with the same restriction enzymes and named pMLE. pET22-b contains a T7 promoter and was purchased from Novagen.

5'-primer: 5'-T TCC ATG GCT ATG CCG TTA GAT CCG CAT-3 '(SEQ ID NO: 5)

3'-primer: 5'-T TGG ATC CGG AAT AGA ATC AAA TAC TTG-3 '(SEQ ID NO: 6)

2) High expression of esterase using T7 promoter

As the host cell, the expression vector pMLE prepared above was introduced using E. coli BL21 (DE3) having a T7 RNA polymerase. The transformed Escherichia coli was cultured at 37 ° C. using an LB medium containing 100 μg / ml of ampicillin, and when the absorbance reached 0.5 at OD _{600 nm} , IPTG was added to a final concentration of 1 mM. After inducing the first expression and incubating for 4 hours, the cells were centrifuged. Cells were pulverized by ultrasonication and centrifuged, followed by 12% SDS-PAGE of water-soluble proteins. As a result, the esterase content in the total protein was measured to be about 20% [FIG. 5]. The transformed E. coli BL21 (DE3) / pMLE showed a maximum activity of 3,600 U / L of esterase activity after 4 hours. The transformed Escherichia coli BL21 (DE3) / pMLE was deposited with the Korea Biotechnology Research Institute Gene Bank on February 27, 2003. The accession number was assigned 10434BP.

Example 5: Esterase Protein Isolation from Transformed Escherichia Coli BL21 (DE3) / pMLE

Esterase produced by the transformed E. coli BL21 (DE3) / pMLE has a His-tag at the C-terminal, it was purely isolated as follows. The cells obtained by incubation for 4 hours after 1 mM IPTG treatment were crushed by ultrasonic grinding, and the supernatant was obtained by centrifugation, and dialyzed with 20 mM imidazole, 300 mM NaCl, and 50 mM phosphate buffer (pH 7.0). . After the protein solution was added to the top of the Ni-NTA column, the column was washed with the same buffer. Esterase 20-1 was isolated purely by eluting the bound protein by raising the imidazole concentration of the buffer to 200 mM [Fig. 5].

Example 6: Temperature and pH Characterization of Novel Esterase 20-1

The purely isolated esterase 20-1 enzyme was examined for hydrolytic activity on the PNP nitrophenyl caproate substrate. 10 μl of 10 mM PNP caproate substrate, 40 μl of ethanol, 940 μl of 50 mM Tris-HCl buffer, and 10 μl of enzyme solution were mixed and reacted for 5 minutes, and the absorbance increase at 405 nm was measured. In the case of measuring the stability of pH and temperature of the enzyme, after remaining for 30 minutes under each condition, the residual activity was measured.

As a result of investigating the effect of pH on the esterase activity by the above method, it showed the maximum activity at pH 8.0, and when the pH stability of the enzyme was examined, it showed stable enzyme activity in the pH 6-9 range. As a result of investigating the effect of temperature on the enzyme activity, the optimum activity was shown in a wide range of 20-40 ℃, it was found to be stable in the temperature range up to 45 ℃ [Fig. 6].

Example 7: Effect of Esterase 20-1 on Surfactants

In order to examine the effect of the surfactant on the enzyme activity, various surfactants were added to the enzyme solution so as to be 1% (v / v, w / v) and left at room temperature for 30 minutes to measure the activity of the enzyme. As a result of examining the effect of the surfactant, the activity of the esterase 20-1 was increased by 2 to 4 times by the nonionic surfactant, and anionic surfactant such as SDS decreased the activity of the enzyme. And bile acids deoxycholate (deoxycholate) and taurocholate (taurocholate) did not affect the activity of the esterase (Fig. 7).

Example 8 Kinetic Parameter Conversion Study of Esterase 20-1 by Tween 80 Treatment

As a result of treatment of Esterase protein with different concentrations of Tween 80, the enzyme activity increased as the Tween 80 concentration increased, and the enzyme activity increased to a constant level at the concentration of 0.05% (v / v) or more. In addition, by treating 0.1% Tween 80 to the esterase protein and measuring the enzyme activity by time, it was confirmed that the increase in the enzyme activity was made almost simultaneously with the Tween 80 treatment [FIG. 7].

As a result of measuring the kinetic parameters of the enzyme for the PNP ester, the longer the length of fatty acids, the higher the kcat / Km value, and showed the highest activity against PNP caproate [Table 2]. In the case of Tween 80 treated enzymes, the kcat / Km value increased by 14 times compared to untreated enzymes. This is due to the increased affinity of the enzyme for the substrate.

temperament Dynamics parameters 20-1 enzyme Tween80 Treated 20-1 Enzyme PNPacetate K _m (mM) 1.43 1.15 k _cat (s ^-1 ) 108 89.7 k _cat / K _m 75.5 78.0 PNP propionate K _m (mM) 1.57 1.39 k _cat (s ^-1 ) 322 529 k _cat / K _m 205 381 PNPbutyrate K _m (mM) 0.14 0.45 k _cat (s ^-1 ) 46.7 255 k _cat / K _m 334 567 PNP caproate K _m (mM) 0.67 0.037 k _cat (s ^-1 ) 352 274 k _cat / K _m 525 7410

Example 9 Secondary Structure Analysis of Enzymes via CD Spectrum

After the concentration of the purely isolated esterase 20-1 protein was adjusted to 0.2 mg / ml, the secondary structure of the enzyme was analyzed using a Jasco CD spectrometer. The spectrum as shown in FIG. 8 was obtained by measuring the rotation angle of polarized light while irradiating polarized light from 250 to 200 nm.

As a result of analyzing the CD spectrum, it was confirmed that even when 0.1% Tween 80 was treated with the esterase 20-1 protein, no significant change in the secondary structure of the protein occurred.

As described above, the present invention provides a novel bacterium Bacillus megaterium 20-1 producing esterase that is activated to a new surfactant, a gene encoding an esterase, and the application of a highly expressed esterase in Escherichia coli. The new esterase is found to increase the activity of the enzyme 14 times by a nonionic surfactant and has been found to be excellent in the activity of the enzyme in a wide temperature range of 20 to 40 ℃ containing unstable compounds and unsaturated fatty acids at high temperatures It may be usefully used in bioprocesses for producing expensive unsaturated fatty acids by hydrolyzing natural fats or synthesizing esters and peptides.

Figure 1 shows the nucleotide sequence of 16S rRNA of Bacillus megaterium 20-1, and compares homology with Bacillus megaterium (CON).

Figure 2 shows the cloning of the esterase 20-1 and the preparation of pMLE, a mass expression vector.

Figure 3 shows the nucleic acid sequence and amino acid sequence of the esterase 20-1 gene.

Figure 4 shows the result of comparing the amino acid sequence of esterase 20-1 and esterase derived from Alicyclobacillus acidocaldarius (EST2).

Figure 5 shows that the mass expression of the esterase from E. coli transformed with pMLE and purely separated through the Ni-NTA column.

Figure 6 shows the effect of temperature and pH on the activity and stability of esterases.

7 shows that the activity of the esterase is increased by various surfactants and that the activity of the esterase is changed depending on the concentration of Tween 80 and the treatment time.

8 shows CD spectra of esterase proteins before and after Tween80 treatment.

<110> KOREA RESEARCH INSTITUTE OF BIOSCIENCE AND BIOTECHNOLOGY <120> Discovery of Bacillus megaterium 20-1 [KCTC 10435BP] producing a novle esterase and development of its efficient production method using Escherichia coli BL21 (DE3) / pMLE [KCTC10434BP] <160> 6 <170> KopatentIn 1.71 <210> 1 <211> 1533 <212> DNA <213> Bacillus megaterium 20-1 <400> 1 tggctcagga tgaacgctgg cggcgtgcct aatacatgca agtcgagcga actgattaga 60 agcttgcttc tatgacgtta gcggcggacg ggtgagtaac acgtgggcga cctgcctgta 120 agactgggat aacttcggga aaccgaagct aataccggat aggatcttct ccttcatggg 180 agatgattga aagatggttt cggctatcac ttacagatgg gcccgcggtg cattagctag 240 ttggtgaggt aacggctcac caaggcaacg atgcatagcc gacctgagag ggtgatcggc 300 cacactggga ctgagacacg gcccagactc ctacgggagg cagcagtagg gaatcttccg 360 caatggacga aagtctgacg gagcaacgcc gcgtgagtga tgaaggcttt cgggtcgtaa 420 aactctgttg ttagggagga acaagtacaa gagtaactgc ttgtaccttg acggtaccta 480 accagaaagc cacggctaac tacgtgccag cagccgcggt aatacgtagg tggcaagcgt 540 tatccggaat tattgggcgt aaagcgcgcg caggcggttt cttaagtctg atgtgaaagc 600 ccacggctca accgtggagg gtcattggaa actggggaac ttgagtgcag aagagaaaag 660 cggaattcca cgtgtagcgg tgaaatgcgt agagatgtgg aggaacacca gtggcgaagg 720 cggctttttg gtctgtaact gacgctgagg cgcgaaagcg tggggagcaa acaggattag 780 ataccctggt agtccacgcc gtaaacgatg agtgctaagt gttagagggt ttccgccctt 840 tagtgctgca gctaacgcat taagcactcc gcctggggag tacggtcgca agactgaaac 900 tcaaaggaat tgacgggggc ccgcacaagc ggtggagcat gtggtttaat tcgaagcaac 960 gcgaagaacc ttaccaggtc ttgacatcct ctgacaactc tagagataga gcgttcccct 1020 tcgggggaca gagtgacagg tggtgcatgg ttgtcgtcag ctcgtgtcgt gagatgttgg 1080 gttaagtccc gcaacgagcg caacccttga tcttagttgc cagcatttag ttgggcactc 1140 taaggtgact gccggtgaca aaccggagga aggtggggat gacgtcaaat catcatgccc 1200 cttatgacct gggctacaca cgtgctacaa tggatggtac aaagggctgc aagaccgcga 1260 ggtcaagcca atcccataaa accattctca gttcggattg taggctgcaa ctcgcctaca 1320 tgaagctgga atcgctagta atcgcggatc agcatgccgc ggtgaatacg ttcccgggcc 1380 ttgtacacac cgcccgtcac accacgagag tttgtaacac ccgaagtcgg tggagtaccg 1440 taaggagcta gccgcctaag gtgggacaga tgattggggt gaagtcgtaa caaggtagcc 1500 gtatcggaag gtgcggctgg atcactcctt tct 1533 <210> 2 <211> 933 <212> DNA <213> Bacillus megaterium 20-1 <400> 2 atgccgttag atccgcatat tcaaatattt ctaaatcaat ataatgaaat gccccgtcct 60 tctttagagg acgttacacc cccacagctg agggaaatgg aaaagatgtc cttaactcct 120 tctaaagaag cagttaaaaa agtatataat gaagaaattg aattaaatga acgtacgctc 180 actctacgag tgtatgaacc tgaaggaaca gggccatttc ccgctcttgt ttattatcac 240 ggaggaggat gggtattagg cagcctggat actcatgact ccatatgcag gtcgtatgca 300 aatgaaacaa actgtattgt agtgtctgtt gattaccgtc ttgctcctga gagtaaattt 360 cccgctgcag taaacgatgc ctatgacgcc ttggattgga tttcagctca tgcgtctcaa 420 ttaaatatcg attcaaacaa aattgccgtc ggtggcgaca gtgccggtgg taaccttgct 480 gcggttgtaa gcattttagc aaaacaaaga caaggtccat ccattgttca tcagctgctt 540 atttatccgt ctgtaggatt taaaaatcaa caccctgcct ctatgaaaga aaacgccgaa 600 ggatatcttc tttcaaaaga tctcatggat tggtttcgcc ttcagtactt aaataataaa 660 gaagaagaac agcaccccta taacgctcca gtattactag aagatttatc gagtctaccg 720 agcgctacca ttattacagc acaatatgat cctttaagag atagcggaaa agactacgcg 780 gacgcattaa aaaatcacgg tgtccccgtc acctatgaaa attatgaaac aatgattcac 840 gggtttttag ggtttcatga attcgtccca ctcgctcagc aggcgatcaa taaaagcgca 900 gctcaactgc gtcaagtatt tgattctatt taa 933 <210> 3 <211> 310 <212> PRT <213> Bacillus megaterium 20-1 <400> 3 Met Pro Leu Asp Pro His Ile Gln Ile Phe Leu Asn Gln Tyr Asn Glu 1 5 10 15 Met Pro Arg Pro Ser Leu Glu Asp Val Thr Pro Pro Gln Leu Arg Glu 20 25 30 Met Glu Lys Met Ser Leu Thr Pro Ser Lys Glu Ala Val Lys Lys Val 35 40 45 Tyr Asn Glu Glu Ile Glu Leu Asn Glu Arg Thr Leu Thr Leu Arg Val 50 55 60 Tyr Glu Pro Glu Gly Thr Gly Pro Phe Pro Ala Leu Val Tyr Tyr His 65 70 75 80 Gly Gly Gly Trp Val Leu Gly Ser Leu Asp Thr His Asp Ser Ile Cys 85 90 95 Arg Ser Tyr Ala Asn Glu Thr Asn Cys Ile Val Val Ser Val Asp Tyr 100 105 110 Arg Leu Ala Pro Glu Ser Lys Phe Pro Ala Ala Val Asn Asp Ala Tyr 115 120 125 Asp Ala Leu Asp Trp Ile Ser Ala His Ala Ser Gln Leu Asn Ile Asp 130 135 140 Ser Asn Lys Ile Ala Val Gly Gly Asp Ser Ala Gly Gly Asn Leu Ala 145 150 155 160 Ala Val Val Ser Ile Leu Ala Lys Gln Arg Gln Gly Pro Ser Ile Val 165 170 175 His Gln Leu Leu Ile Tyr Pro Ser Val Gly Phe Lys Asn Gln His Pro 180 185 190 Ala Ser Met Lys Glu Asn Ala Glu Gly Tyr Leu Leu Ser Lys Asp Leu 195 200 205 Met Asp Trp Phe Arg Leu Gln Tyr Leu Asn Asn Lys Glu Glu Glu Gln 210 215 220 His Pro Tyr Asn Ala Pro Val Leu Leu Glu Asp Leu Ser Ser Leu Pro 225 230 235 240 Ser Ala Thr Ile Ile Thr Ala Gln Tyr Asp Pro Leu Arg Asp Ser Gly 245 250 255 Lys Asp Tyr Ala Asp Ala Leu Lys Asn His Gly Val Pro Val Thr Tyr 260 265 270 Glu Asn Tyr Glu Thr Met Ile His Gly Phe Leu Gly Phe His Glu Phe 275 280 285 Val Pro Leu Ala Gln Gln Ala Ile Asn Lys Ser Ala Ala Gln Leu Arg 290 295 300 Gln Val Phe Asp Ser Ile 305 310 <210> 4 <211> 310 <212> PRT <213> Alicyclobacillus acidocaldarius <400> 4 Met Pro Leu Asp Pro Val Ile Gln Gln Val Leu Asp Gln Leu Asn Arg 1 5 10 15 Met Pro Ala Pro Asp Tyr Lys His Leu Ser Ala Gln Gln Phe Arg Ser 20 25 30 Gln Gln Ser Leu Phe Pro Pro Val Lys Lys Glu Pro Val Ala Glu Val 35 40 45 Arg Glu Phe Asp Met Asp Leu Pro Gly Arg Thr Leu Lys Val Arg Met 50 55 60 Tyr Arg Pro Glu Gly Val Glu Pro Pro Tyr Pro Ala Leu Val Tyr Tyr 65 70 75 80 His Gly Gly Gly Trp Val Val Gly Asp Leu Glu Thr His Asp Pro Val 85 90 95 Cys Arg Val Leu Ala Lys Asp Gly Arg Ala Val Val Phe Ser Val Asp 100 105 110 Tyr Arg Leu Ala Pro Glu His Lys Phe Pro Ala Ala Val Glu Asp Ala 115 120 125 Tyr Asp Ala Leu Gln Trp Ile Ala Glu Arg Ala Ala Asp Phe His Leu 130 135 140 Asp Pro Ala Arg Ile Ala Val Gly Gly Asp Ser Ala Gly Gly Asn Leu 145 150 155 160 Ala Ala Val Thr Ser Ile Leu Ala Lys Glu Arg Gly Gly Pro Ala Leu 165 170 175 Ala Phe Gln Leu Leu Ile Tyr Pro Ser Thr Gly Tyr Asp Pro Ala His 180 185 190 Pro Pro Ala Ser Ile Glu Glu Asn Ala Glu Gly Tyr Leu Leu Thr Gly 195 200 205 Gly Met Met Leu Trp Phe Arg Asp Gln Tyr Leu Asn Ser Leu Glu Glu 210 215 220 Leu Thr His Pro Trp Phe Ser Pro Val Leu Tyr Pro Asp Leu Ser Gly 225 230 235 240 Leu Pro Pro Ala Tyr Ile Ala Thr Ala Gln Tyr Asp Pro Leu Arg Asp 245 250 255 Val Gly Lys Leu Tyr Ala Glu Ala Leu Asn Lys Ala Gly Val Lys Val 260 265 270 Glu Ile Glu Asn Phe Glu Asp Leu Ile His Gly Phe Ala Gln Phe Tyr 275 280 285 Ser Leu Ser Pro Gly Ala Thr Lys Ala Leu Val Arg Ile Ala Glu Lys 290 295 300 Leu Arg Asp Ala Leu Ala 305 310 <210> 5 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> 5'-PRIMER <400> 5 ttccatggct atgccgttag atccgcat 28 <210> 6 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> 3'-PRIMER <400> 6 ttggatccgg aatagaatca aatacttg 28

Claims (9)

Bacillus megaterium 20-1 [KCTC 10435BP] to produce esterases. A gene which is represented by the nucleotide sequence of SEQ ID NO: 2 and which coats an esterase whose hydrolytic activity increases by nonionic surfactant. 3. The gene of claim 2, wherein the gene is derived from Bacillus megaterium 20-1 [KCTC 10435BP]. Esterase represented by the amino acid sequence of SEQ ID NO: 3, the hydrolytic activity is increased by the nonionic surfactant. The esterase according to claim 4, wherein the esterase is derived from Bacillus megaterium 20-1 [KCTC 10435BP]. A recombinant expression vector comprising the gene of claim 2 or 3. Bacteria transformed with the expression vector of claim 6. 8. The transformed bacterium of claim 7, wherein the transformant is Escherichia coli. Method for mass production of esterases, characterized in that using the transformed bacteria of claim 7 or 8.