CN102286441A

CN102286441A - Low-temperature esterase and coding gene and use thereof

Info

Publication number: CN102286441A
Application number: CN2011102082110A
Authority: CN
Inventors: 许学伟; 吴敏; 江夏薇; 王春生; 杨俊毅; 刘镇盛
Original assignee: Second Institute of Oceanography SOA
Current assignee: Second Institute of Oceanography SOA
Priority date: 2011-07-24
Filing date: 2011-07-24
Publication date: 2011-12-21
Anticipated expiration: 2031-07-24
Also published as: CN102286441B

Abstract

The invention discloses low-temperature esterase and a coding gene and use thereof. In the invention, a new esterase gene is screened out of a metagenomic library of abyssal deposit from Pacific Ocean, and the protein coded by the gene is found to have excellent enzymatic characteristics; and the catalytic hydrolysis temperature of the protein ranges from 10 to 50 DEG C, and the hydrolysis pH value is 6.0 to 9.0. Under a condition of a temperature of 0 to 20 DEG C, the zymoprotein can keep good stability; and under a condition of adding bivalent ions such as Mg<2+> and Mn<2+> or organic solvent Tween-20 and Tween-80, the enzymatic activity is improved. The obtained esterase gene can be cloned into a proper host to realize heterologous expression, and thus, the industrial production of esterase can be realized. The esterase gene can be used in the production process for synthesizing an ester product by a catalytic ester hydrolysis and enzyme method and demonstrates high economic and social values in process such as preparation of detergent, printing and dyeing aid and biodiesel.

Description

A kind of low temperature esterase and encoding gene and application

Technical field

The present invention relates to the genetically engineered field, specifically, relate to a kind of low temperature esterase, its encoding gene and application thereof.

Background technology

Esterase (esterase, EC 3.1) is a kind of lytic enzyme, can via hydrolytic action, the ester class be cut into acids and alcohols in the presence of water molecules.Esterase is present in animal, plant and the microorganism cells.According to aminoacid sequence difference, the bacterium esterase can be divided into 8 families (I-VIII) at present.In recent years, development along with genetic engineering technique, some new esterases are found successively, for example: screen the EstA and the EstF of the LipG to the Korea S marine site, southern China sea water body and settling environment, the EstGK1 of the EstD2 of plant root soil and sheep cud and EstZ3 etc.

Grand genome claims first genome again, comes from the direct extractive technique of the soil microbe genome DNA seventies in 20th century, is proposed first in 1998 and perfect this noun by people such as Handelsman.Grand genome is meant whole tiny organism genetic material summations in the habitat.Grand genome-based technologies can directly be extracted microbe genome DNA from environmental sample, clone in different carriers, recombinant vectors is transferred to suitable host to set up grand genomic library, in conjunction with different triage techniqueses, screening obtains new gene or new biologically active substance from gene library again.Use this new technology of exempting to cultivate, can walk around microbial strains separation and Culture technological difficulties, directly research on gene level, development and utilization cultured microorganism resource not are for industry, agricultural, medicine and environment sustainable development provide the microbial gene resource.Chinese patent 200710143545.8 provides the gene of planting encoding beta-glucosidase, it by make up soil not the activity of beta-glucosidase of the macro genome DNA library of culturing micro-organisms and library clone detect sieve method and obtain.

The bacterial strain that present bacterium esterase of authorizing and gene patent majority thereof are directed to separation and Culture.Chinese patent 98811110.1 provides a kind of aminoacid sequence, coding gene sequence of heat resisting basic phosphatase, and the method for producing recombinase.Chinese patent 200410091990.0 provides a kind of glycerophosphate phosphatase gene of pseudomonas and cloning process thereof.Chinese patent 200810222671.7 provides a kind of esterase and encoding gene and application, and the esterase of invention is applicable to chemical industry, food, bio-transformation, medicine industry and other related industries.Chinese patent 200910144213.0 provides thermostable carboxylesterase gene, proteins encoded and the application thereof of obtaining in a kind of therefrom warm streptomycete.Yet bacterium esterase and gene patent thereof also have by culture technique not screens in grand gene library.Chinese patent 200810226942.6 provides a kind of novel gene of esterase Est p1 and recombinant expression system thereof, and this gene clone is from the grand genomic library of 100m deep-sea, South China Sea seabed ocean floor mud.Chinese patent 200810222671.7 provides a kind of esterase and encoding gene and application, this gene clone is from the grand genomic library of South China Sea 778.5m abyssal sediment, and the esterase of invention is applicable to chemical industry, food, bio-transformation, medicine industry and other related industries.

Summary of the invention

The purpose of this invention is to provide a kind of new low temperature esterase, encoding gene and preparation method thereof, this esterase is used for the biocatalysis and the conversion of lipid degradation and other oil compounds.

The present invention is therefrom in the grand genomic library of nero deep settling (depth of water 5886m), obtain a kind of new esterase gene Est6 by specific substrate (tributyrin) screening, comprise this expression of gene reorganization bacterium and be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, the address is No. 3 Institute of Microorganism, Academia Sinica in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City (100101), deposit number is CGMCC No.5084, preservation date is on July 18th, 2011, Latin classification called after Escherichia coli.Through the PCR cloning and sequencing, the nucleotide sequence of esterase gene Est6 is shown in SEQ ID No.1.The Est6 size is 909bp, and based composition is: 148A (16.28%), 329C (36.19%), 292G (32.12%) and 140T (15.40%), and the proteins encoded size is 302 amino-acid residues, its aminoacid sequence is shown in SEQ ID No.2.This gene order is carried out homology search in GenBank, find one similarly property be up to 61% dietary protein origin in the strain esterase of culturing bacterium (its number of registration in the GenBank database is ACL67845) not.Structure prediction shows that esterase Est6 has the catalyst structure domain (amino acid position is 142 to 146) that glycine, aspartic acid, Serine, L-Ala and glycine are formed, and constitutes esterase catalyzed center; Also has the oxygen anion hole (amino acid position is 74 to 77) that Histidine, glycine, glycine and glycine are formed.In sum, Est6 should be newcomer of esterase family.

Do not influencing under the Est6 protein-active prerequisite, can carry out various replacements, interpolation and/or lack the derived protein that one or several amino acid obtains to have the Est6 esterase activity the aminoacid sequence shown in the SEQ ID NO.2.According to the common practise of art technology, proteinic biologic activity is closely-related with its functional domain.In general, the site mutation that only occurs in functional domain may exert an influence to proteinic 2 and 3 dimensional organization, thereby influences its biologic activity.And for the amino acid sites that occurs in away from functional domain, because this zone does not participate in the protein function conformation, thereby amino acid whose indivedual point mutation can not produce substantial effect to proteinic biologic activity, thereby can keep the biological function of crude protein substantially.

Thereby, be on the basis of active structure domain of esterase in known 74-77 and 142-146 amino acids, according to common practise and reasoning from logic, can obtain Est6 albumen 1-73 by rational experimental establishment, 78-141 or 147-302 amino acids sequence are carried out various replacements, learn active protein mutant thereby add and/or lack the basic Est6 of reservation of one or several amino acid acquisition esterase biological, preferably to Est6 albumen 1-73, esterase biological is learned active protein mutant thereby 78-141 or 147-302 amino acids are carried out the basic Est6 of reservation of point mutation acquisition.

Correspondingly, consider the degeneracy of codon, for example can be in its coding region, under the condition that does not change aminoacid sequence, or at its non-coding region under the condition that does not influence Est6 genetic expression, can make amendment to the above-mentioned proteic gene order of encoding.Therefore, thus the present invention also protects replacement, the interpolation that the nucleotide sequence shown in the SEQ ID NO.1 is carried out and/or lacks one or several Nucleotide and obtains the dna molecular that coding can keep Est6 esterase protein biologic activity substantially.For example thereby Est6 gene order 1-219,234-423 or 441-906 position nucleotide sequence are carried out various replacements, interpolation and/or lack one or several Nucleotide obtaining the dna molecular that coding can keep Est6 esterase protein biologic activity substantially, thereby preferably Est6 gene order 1-219,234-423 or 441-906 position nucleotide sequence are carried out the dna molecular that point mutation acquisition coding can keep Est6 esterase protein biologic activity substantially.

Utilize gene clone technology, the esterase gene of above-mentioned acquisition can be connected on the suitable carrier, and conversion or transfection are to protokaryon or eukaryote host expresses preparation reorganization esterase.Suitable prokaryotic organism host comprises various bacteriums such as E.coli etc., and suitable eukaryote host comprises yeast (as methanol yeast) and mammalian cell (as Chinese hamster ovary cell) etc., preferably adopts prokaryotic expression system E.coli.

A preferred example is that the esterase gene Est6 that the present invention screens is connected on the coli expression carrier pET28b (Novagen), and is transformed among the intestinal bacteria Rosetta, goes out highly active reorganization esterase through abduction delivering.

The present invention also provides described Est6 application in catalytic hydrolysis ester, transesterify or synthetic and albumen transportation preservation.Show that by esterase activity mensuration Est6 esterase or the above-mentioned host bacterium that can express the Est6 esterase can be used for hydrolysis short chain fatty acid fat.Preferred short chain fatty acid fat is the p-nitrophenyl phenolic ester with C2-C10 short carbon chain, for example paranitrophenylacetic acid ester, p-nitrophenyl butyric ester, p-nitrophenyl capronate, p-nitrophenyl octanoate and p-nitrophenyl decylate etc., catalytic activity was the highest when wherein substrate was p-NP butyric ester (C4).

The Est6 esterase is a kind of low temperature esterase, and the catalytic hydrolysis temperature range is 10～50 ℃, is preferably 20～30 ℃; The pH value of described hydrolysis is 6.0～9.0, is preferably 7.5～8.0.Under 0～20 ℃ of condition, zymoprotein can keep satisfactory stability, is adding divalent ion Mg ²⁺, Mn ²⁺Or under organic solvent polysorbas20 and 80 conditions, the enzyme activity increases.

The present invention therefrom screens the new esterase gene of acquisition in the grand genomic library of nero deep settling, has found that this gene coded protein has good enzymatic property, can be applicable to catalysis and separates in ester and the synthetic ester process of producing product of enzyme process.The esterase gene that obtains can be cloned into and be realized heterogenous expression in the appropriate host, realizes the suitability for industrialized production esterase, for follow-up industrial application provides esterase parent material with low cost.The production of this enzyme will demonstrate important economy and social value in technologies such as washing composition, dyeing and printing auxiliary and biodiesel manufacture.

Description of drawings

Fig. 1 is the policapram gel electrophoresis analysis figure of purifying esterase Est6.

Fig. 2 is the substrate specificity figure of esterase Est6.C2: paranitrophenylacetic acid ester; C4: p-nitrophenyl butyric ester, C6: p-nitrophenyl capronate; C8: p-nitrophenyl octanoate; C10: p-nitrophenyl decylate; C12: p-nitrophenyl laurate; C14: p-nitrophenyl myristinate; C16: p-nitrophenyl Palmitate; Measured value was 100% when the definition substrate was C4.

Fig. 3 is esterase Est6 optimal reactive temperature figure.

Fig. 4 is esterase Est6 optimal reaction pH figure.

Fig. 5 is esterase Est6 thermostability figure.

Fig. 6 is that divalent cation is to esterase Est6 activity influence figure.

Fig. 7 is that organic solvent and washing agent are to esterase Est6 activity influence figure.

Embodiment

Embodiment 1: esterase gene Est6 obtains

The pelagic deposit matter sample adopts the multitube sampler to gather from Middle Pacific depth of water 5886m in August, 2008.Grand genomic library construction adopts CopyControlTM HTP fosmid library production kit (Epicentre, the U.S.), cloning vector is pCC2 FOS fosmid vector (Epicentre, the U.S.), the host is EPI300-T1R E.coli (Epicentre, the U.S.).Through the pulsed field electrophoresis detection, the insertion clip size is 36～48kb.

Adopt the screening of paraxin esterase dull and stereotyped [1% (w/v) tributyrin, 12.5 μ g ml ^-1Paraxin, LB substratum (10g sodium-chlor, pH 7.0 for 10g Tryptones, 5g yeast extract)] screening has clone's of esterase gene.Get 10 μ l library bacterium liquid and be diluted to 100 μ l, it is dull and stereotyped to coat the esterase screening, cultivates 2 days for 30 ℃, and periphery of bacterial colonies obvious transparent circle occurs and is positive colony.The positive colony that screens is chosen the repeated authentication of on paraxin esterase screening flat board, ruling.Obtain esterase positive colony E6 through screening and repeated authentication.

Insert 3ml LB liquid nutrient medium through the esterase positive colony E6 of repeated authentication and (contain 12.5 μ g ml ^-1Paraxin and 6 μ l, 500 * CopyControl Fosmid Autoinduction Solution), 18h is cultivated in 37 ℃ of concussions, and concussion speed is 250rpm.Adopt plasmid extraction test kit (Axygen, the U.S.) extracting fosmid plasmid.The fosmid plasmid that carries esterase gene adopts restriction enzyme Sau3AI to carry out incomplete enzyme and cuts, the dna fragmentation of 1.5～4kb size is reclaimed in rubber tapping, be cloned into the pUC19 plasmid, Transformed E .coli DH5 α bacterial strain again, be coated with the screening of penbritin esterase dull and stereotyped [1% (w/v) tributyrin, 100 μ g ml at last ^-1Penbritin, LB substratum] screen positive subclone.Cultivate for 30 ℃ and select the subclone that produces transparent circle after 2 days, after the line repeated authentication, check order, obtain to insert the fragment dna sequence dna.Obtain 1748bp through positive subclone order-checking and insert fragment.

At inserting fragments sequence, ORF based on NCBI/ORF Finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html) analyzes, obtain to insert open reading frame sequence information in the fragment, by the homology of Blastx (http://blast.ncbi.nlm.nih.gov/) aligned sequences and known esterase gene sequence.Obtain the Est6 gene through the database compare of analysis, size is 909bp, and based composition is: 148A (16.28%), 329C (36.19%), 292G (32.12%) and 140T (15.40%), its nucleotide sequence is shown in SEQ ID No.1.The proteins encoded size is 302 amino-acid residues, and its aminoacid sequence is shown in SEQ ID No.2.This gene order is carried out homology search in GenBank, find one similarly property be up to 61% dietary protein origin in the strain esterase of culturing bacterium not, its number of registration in the GenBank database is ACL67845.

The structure prediction result shows that esterase Est6 has the catalyst structure domain (amino acid position is 142 to 146) that glycine, aspartic acid, Serine, L-Ala and glycine are formed, and constitutes esterase catalyzed center; Also have the oxygen anion hole (amino acid position is 74 to 77) that Histidine, glycine, glycine and glycine are formed, it can provide hydrogen bond by electronegative Sauerstoffatom in transition state in catalytic process.Phylogenetic Analysis result shows that esterase Est6 belongs to the IV family in the esterase family.

In sum, Est6 should be a newcomer in the esterase family.

Embodiment 2: the recombinant expression plasmid of esterase gene Est6 and the structure of recombinant bacterial strain

The esterase gene Est6 that the present invention obtains is cloned on the expression vector, makes up recombinant strains.Analyze the open reading frame sequence of the esterase gene that obtains, upstream primer Est6F (the 5 '-CGCA of the full gene of design amplification esterase based on the ORF of NCBI/ORF Finder CATATGGCCAGTCCACAGCTCC-3 ' is NdeI) with downstream primer Est6R (5 '-AATT AAGCTTCTAGCGTGCGGCGGCG-3 ', HindIII), pcr amplification is confirmed the full length gene sequence.Adopt the method construction expression plasmid of enzyme cutting clone, promptly use NdeI and HindIII double digestion PCR product, fragment after rubber tapping recovery enzyme is cut is connected with the plasmid pET28b through NdeI and HindIII double digestion, adopt the CaCl2 conversion method to be converted in the bacillus coli DH 5 alpha kalamycin resistance screening positive clone.Adopt plasmid extraction test kit (Axygen, the U.S.) to extract the plasmid of positive colony, identify, obtain the dna fragmentation of 909bp, be accredited as esterase gene Est6 through order-checking through NdeI and HindIII double digestion.Show successfully to have made up expression plasmid, this recombinant expression plasmid is transformed in the intestinal bacteria Rosetta expression strain, made up the express recombinant bacterial strain.Comprise this expression of gene reorganization bacterium and be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, the address is No. 3 Institute of Microorganism, Academia Sinica in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City (100101), deposit number is CGMCC No.5084, preservation date is on July 18th, 2011, Latin classification called after Escherichia coli.

Embodiment 3: utilize recombinant strains express recombinant esterase gene Est6

The 3ml recombinant strains that builds is transferred to 100ml contains in the LB liquid nutrient medium of 20 μ g/ml kantlex and 34 μ g/ml paraxin, 37 ℃ of shaking culture are to OD ₆₀₀Reach 0.6, adding final concentration this moment is that the IPTG of 0.5mM carries out abduction delivering, changes 25 ℃ over to 150r/min shaking culture 7h.Low-temperature centrifugation is collected thalline, is resuspended among the Buffer A (20mM Tris hydrochloric acid, pH 8.0 for 500mM sodium-chlor, 10mM imidazoles), carries out ultrasonic disruption on ice and handles.Low-temperature centrifugation is collected supernatant, carries out NTA-Ni then ²⁺Affinity column chromatography, expressed recombinant protein contain 6 * His tag of N end, and affine being adsorbed onto in layer suction post of energy through behind the imidazoles solution gradient wash-out of different concns, collected elutriant.Detect through SDS-PAGE, obtain electrophoretically pure reorganization esterase protein Est6, molecular weight 32kd, consistent with predictor (Fig. 1).Measure protein concn with the Bradford method, obtain the expression amount of about 2.28mg/100ml fermented liquid.

Embodiment 4: the activity of reorganization esterase gene Est6 detects

Utilize p-nitrophenyl butyric ester method to measure the reorganization esterase Est6 activity of purifying.Concrete operations: in the reaction system of 1ml, comprise 1mM p-nitrophenyl butyric ester, 100mM Tris hydrochloride buffer (pH 7.5) and 0.57 μ g pure enzyme protein (being the diluted purifying enzyme liquid of 10 μ l), adopt ultra-violet and visible spectrophotometer (Beckman DU800 type, the U.S.) METHOD FOR CONTINUOUS DETERMINATION light absorption value A under 20 ℃ of conditions ₄₀₅3min uses the enzyme liquid of inactivation to be used for zeroing in contrast.The esterase activity that records is 104.41U/mg.In addition, the enzyme liquid of purifying is directly dripped on 1% (w/v) tributyrin flat board, can be observed the obvious transparent circle.Show that this recombinant expressed esterase Est6 has the esterase hydrolyzed activity, for follow-up research and application provide material.

Embodiment 5: esterase Est6 substrate specificity is analyzed

System is adopted in the substrate specificity analysis of esterase Est6: 100mM Tris-hydrochloride buffer (pH 7.5), the 1mM substrate adds 0.57 μ g pure enzyme protein, at 20 ℃ of following METHOD FOR CONTINUOUS DETERMINATION light absorption value A ₄₀₅3min.Measuring the substrate that adopts is: p-NP acetic ester (C2), p-NP butyric ester (C4), p-NP capronate (C6), p-NP octanoate (C8), p-NP decylate (C10), p-NP laurate (C12), p-NP myristinate (C14), p-NP Palmitate (C16).Show the ester bond of Est6 energy specificity hydrolysis compound p-NP butyric ester after measured.Esterase Est6 has catalytic activity to the short p-nitrophenyl phenolic ester (C2, C4, C6, C8 and C10) of acyl group carbochain; catalytic activity was the highest when wherein substrate was p-NP butyric ester (C4), and more difficult hydrolysis p-NP myristinate (C14) and p-NP Palmitate (C16) are (Fig. 2).The result shows that esterase Est6 has catalytic activity to the shorter lipid material of acyl group carbochain, is better than long-chain lipid for the hydrolysis vigor of short chain lipid.

Embodiment 6: esterase Est6 optimum reaction conditions is analyzed

Esterase Est6 optimal reactive temperature is measured in 10～50 ℃ of scopes.System is adopted in concrete operations: 100mM Tris-hydrochloride buffer (pH 7.5), 1mM p-NP butyric ester adds 0.57 μ g pure enzyme protein, respectively METHOD FOR CONTINUOUS DETERMINATION light absorption value A under 10,15,20,25,30,35,40,45 and 50 ℃ of conditions ₄₀₅3min.Measurement result shows that Est6 is a cold-adapted enzyme, still has catalytic activity under 10 ℃ of conditions, and optimal reactive temperature is 20 ℃ (Fig. 3).

Esterase Est6 optimal reaction pH measures in 3.0～10.0 scopes.Concrete operations are: add 1mM p-NP butyric ester and 0.57 μ g pure enzyme protein in different pH damping fluids, at 20 ℃ of following METHOD FOR CONTINUOUS DETERMINATION light absorption value A ₃₄₈3min.Measuring the damping fluid that uses is: 100mM citric acid-sodium citrate damping fluid (pH 3.0～6.5), 100mM potassium primary phosphate-sodium hydrate buffer solution (pH 6.5～7.5), 100mM Tris hydrochloride buffer (pH 7.5～9.0) and 50mM 2-cyclohexylamino ethyl sulfonic acid-sodium hydrate buffer solution (pH 9.0～10.0).Measurement result shows that the Est6 optimal pH is pH 7.5, has activity (Fig. 4) in pH 6.0～9.0 scopes.

Embodiment 7: the stability analysis of esterase Est6 zymetology

Esterase Est6 thermal stability analysis is measured in 0～60 ℃ of scope.Concrete operations are: enzyme liquid behind the purifying is incubated respectively at 0,10,20,30,40,50 and 60 ℃ of following 30min, measures enzymic activity then.Surveying the enzyme live body is: 100mM Tris hydrochloride buffer (pH 7.5), 1mM p-NP butyric ester adds 0.57 μ g pure enzyme protein, in 20 ℃ of METHOD FOR CONTINUOUS DETERMINATION light absorption value A ₄₀₅3min.Measurement result shows that Est6 keeps satisfactory stability in the time of 0～20 ℃, loses activity when preserving for 40～60 ℃ (Fig. 5).

Divalent cation to the mensuration concrete operations of esterase Est6 activity influence is: add 10mM Co in reaction system respectively ²⁺, Cu ²⁺, Ca ²⁺, Mg ²⁺, Zn ²⁺, Sr ²⁺, Mn ²⁺, Ni ²⁺, Ba ²⁺And ethylenediamine tetraacetic acid (EDTA), measure enzymic activity.Surveying the enzyme live body is: 100mM Tris hydrochloride buffer (pH 7.5), and 1mM p-NP butyric ester, 0.57 μ g pure enzyme protein is in 20 ℃ of following METHOD FOR CONTINUOUS DETERMINATION light absorption value A ₄₀₅3min.Measurement result shows that esterase Est6 is active can be by Zn ²⁺, Cu ²⁺And Ni ²⁺Suppress, at Co ²⁺, Ca ²⁺, Sr ²⁺And Ba ²⁺Exist down and still can keep stronger activity, at Mg ²⁺And Mn ²⁺There is active down increase (Fig. 6).

Organic solvent and washing agent to the mensuration concrete operations of esterase Est6 activity influence are: add the activity that 15% (v/v) organic solvent (Virahol, acetonitrile, ethanol, methyl alcohol, acetone, dimethyl sulfoxide (DMSO) and dimethyl formamide) and 1% washing agent (w/v or v/v) (sodium lauryl sulphate, polysorbas20, tween 80 and Triton X-100) are measured enzyme then in reaction system respectively.The survey live body is: 100mM Tris hydrochloride buffer (pH 7.5), and 1mM p-NP butyric ester, 0.57 μ g pure enzyme protein is in 20 ℃ of following METHOD FOR CONTINUOUS DETERMINATION light absorption value A ₄₀₅3min.Measurement result shows that esterase Est6 is active can be suppressed by sodium lauryl sulphate, but polysorbas20 and 80 can strengthen its activity (Fig. 7).

Claims

1. Est6 esterase protein is the protein with following (1) or (2) feature:

(1), its aminoacid sequence is consistent with sequence shown in the Seq ID NO.2;

(2), with the amino acid residue sequence of Seq ID NO.2 through replacement, disappearance and/or the interpolation of one or several amino-acid residue and have esterase activity by (1) deutero-protein.

2. Est6 esterase protein according to claim 1 is characterized in that: thus described derived protein is for carrying out various replacements, interpolation and/or lack the basic Est6 of reservation of one or several amino acid acquisition esterase biological learning active protein mutant to Est6 albumen 1-73,78-141 or 147-302 amino acids sequence.

3. according to right 2 described Est6 esterase proteins, it is characterized in that: thus described derived protein obtains the basic Est6 of reservation esterase biological and learns active protein mutant for Est6 albumen 1-73,78-141 or 147-302 amino acids residue being carried out point mutation.

4. the dna molecular of coding claim 1 described Est6 esterase protein, its nucleotide sequence is shown in SEQ ID NO.1; Thereby or for the nucleotide sequence shown in the SEQ ID NO.1 being replaced, is added and/or lacks the dna molecular that one or several Nucleotide acquisition coding can keep Est6 esterase protein biologic activity substantially.

5. dna molecular according to claim 4 is characterized in that: thus it is for carrying out various replacements, interpolation and/or lack one or several Nucleotide obtaining the dna molecular that coding can keep Est6 esterase protein biologic activity substantially to gene order 1-219,234-423 shown in the SEQ ID NO.1 or 441-906 position nucleotide sequence.

6. dna molecular according to claim 5 is characterized in that: thus it is for carrying out the dna molecular that point mutation acquisition coding can keep Est6 esterase protein biologic activity substantially to gene order 1-219,234-423 shown in the SEQ ID NO.1 or 441-906 position nucleotide sequence.

7. carry the carrier of each described dna molecular of claim 4-6.

8. host system, its by the described carrier of claim 7 through transforming or transfection prokaryotic organism or eukaryote host obtain.

9. the application of each described Est6 esterase of claim 1-3 in catalytic hydrolysis ester, transesterify or synthetic and albumen transportation preservation.

10. application according to claim 9 is characterized in that, described Est6 esterase can be used for hydrolysis short chain fatty acid fat.