CN106635941A - Thermophilic esterase derived from aquifex aeolicus strain and functional verification of thermophilic esterase - Google Patents

Abstract

The invention discloses thermophilic esterase derived from an aquifex aeolicus strain and functional verification of the thermophilic esterase, and belongs to the field of bioengineering technology. According to the invention, a novel thermophilic esterase gene is discovered, three expression systems are constructed, and efficient expression and research of enzymatic properties are achieved. Construction of an eukaryotic expression system: preferably, a vector pPIC9K is adopted for expression vector construction, and a pichia pastoris host, preferably GS115, is transformed, so that efficient expression is achieved; construction of a prokaryotic escherichia coli expression system: preferably, a vector MBP3 is adopted for expression vector construction, and escherichia coli hosts, preferably BL21 and Origami2, are transformed, so that efficient expression is achieved; and construction of a prokaryotic bacillus megaterium expression system: preferably, a vector pHIS1525 is adopted for expression vector construction, and a bacillus megaterium host, preferably YYBm1, is transformed, so that efficient expression is achieved. The recombinant enzyme (the thermophilic esterase) has the advantages of esterase activity, ,thermophilic characteristic, thermal stability and the like; and the recombinant enzyme has a great potential in industrial application under a high-temperature condition.

Description

A kind of thermophilic esterase and its functional verification from Aquifex aeolicus bacterial strains

Technical field

The present invention relates to a kind of thermophilic esterase and its functional verification from Aquifex aeolicus bacterial strains, belongs to raw Thing field of engineering technology.

Background technology

Extreme microorganism is the general name of the microorganism for being adapted to live in extreme environment, including thermophilic, thermophilic cold, acidophilus, thermophilic The polytypes such as alkali, piezophilic, thermophilic salt, thermophilic metal ion, radioresistance, resistance to drying and extreme anaerobism.Thermophilic Bacteria (high temperature bacterium, also referred to as Thermophilic microorganism), it is microorganism that a class is lived in 50 DEG C of high temperature above environment.There is temperature in the source of common thermophilic microorganism Spring, volcanic crater, marine sediment, wastewater and waste materials, hot soil etc..Over nearly 30 years, in the boiling point and boiling point temperatures above of water Under the conditions of after the bacterium that can live is found, more promote the research to thermophilic microorganism.In fermentation industry, it is possible to use thermophilic The resistant to elevated temperatures characteristic of hot bacterium, improves reaction temperature, increases reaction speed, reduces the chance of warm type living contaminants in minimizing.Optimal reaction Referred to as Zimadzhunt L 340 of the temperature less than 80 DEG C, referred to as hyperthermophilic enzyme of the optimal reactive temperature higher than 80 DEG C, first of mankind's discovery Zimadzhunt L 340 is the polymerase-Taq enzyme for being applied to modern biotechnology.Hereafter, many Zimadzhunt L 340s are constantly sent out from Thermophilic Bacteria Now and for producing, such as cellulase, protease, amylase, lipase, dextrase etc..

Lipid hydrolyzing enzyme (EC 3.1.1.X) mainly includes lipase and esterase.The difference of lipase and esterase mainly has 3 Point：One is the substrate difference of effect：Esterase is ester-type hydrolysis enzyme of the catalysis containing SCFA, and lipase is catalysis fat containing long-chain The fat (triacylglycerol) of fat acid is hydrolyzed to the enzyme of glycerine and aliphatic acid；Two is the physical state difference of substrate specificity：Esterase Hydrolysis water soluble substrate, reaction system is mostly in oil-water interfaces.Lipase in outphasing system (i.e. oil-water interfaces) or can only have Act in machine phase, hydrolyze fat-soluble substrate；Three is the activation effect at interface：Lipase is higher in water-oil interface catalysis activity, Catalysis activity is low in single solution.All of lipid hydrolyzing enzyme primary structure is all similar, including 1) important area His-Gly-X- Ser-Gly and Gly-X-Ser-X-Gly；2) Ser of active site is covered by alpha-helix, when lipase and interracial contact, α- Spiral is opened.3) generally believe that its avtive spot is made up of 3 catalytic residues：Nucleophilic residues (Ser, Cyc, Asp), catalytic residue (Asp, Glu), His residues.

In recent years, the research with regard to thermostable esterases/lipase is more and more more, many thermophilus strain esterase/lipase by Research.Thermophilic esterase/lipase is microbe-derived mainly prokaryotes, eucaryote and Archimycetes.According to lipid hydrolyzing enzyme Bacterium lipid hydrolyzing enzyme is divided into 8 families by the difference of amino acid sequence and basic biochemistry property, Arpigny and Jaeger et al. Belonging to the 4th family (HSL families) and the 5th family (thermophilic enzyme family) race, wherein Zimadzhunt L 340 more.At present, studied both at home and abroad Mainly have with regard to the thermophilus strain of thermophilic esterase/fat enzyme source：Fervidobacterium belongs to, Thermotoga category, Aureobasidium belongs to, Pseudomonas category, Geobacillus category, Sulfolobus category etc..Esterase is used as a kind of efficient Biocatalyst, because thermophilic esterase has pyroreaction active and good heat endurance, and to organic solvent, detergent With the stronger resistance of denaturant so as to a very wide range of to be applied to food processing, medical industry, leather manufacture, animal feed, cosmetic The industry such as conduct industry and waste water control.

With the progress of science and technology, the scientists gradually micro- life of the isolated hyperthermophilic from some special high-heat environments Thing, also so that people further go deep into the understanding of Zimadzhunt L 340.However, most of Thermophilic Bacteria speeds of growth are slower, bar is cultivated Part is strict, therefore extremely difficult to obtain a large amount of Zimadzhunt L 340s by cultivating wild mushroom.However, sending out with technique for gene engineering Exhibition, people start the genes of interest found in Thermophilic Bacteria to express in some common easy culture hosts, so as to gentle Under the conditions of obtain substantial amounts of Zimadzhunt L 340.At present, existing some thermophilic esterase genes realize the successful expression in Escherichia coli.But In industrialized production application, it is contemplated that the process of enzyme preparation downstream separation and safety problem, the selection of heterogenous expression host is particularly It is important.Pichia pastoris and bacillus megaterium are all food-grade expressive hosts, and can be with exocytosis recombinant protein.

Research to Zimadzhunt L 340 at present is scarcely out of swaddling-clothes, and finds that new Zimadzhunt L 340 is current from hyperthermophilic microorganism The main method of new enzyme preparation.Additionally, in the face of the extensive application of thermophilic esterase, finding new thermophilic esterase and selecting suitable Expressive host is particularly important.

The content of the invention

In order to solve the above problems, present invention finds a kind of new thermophilic ester from Aquifex aeolicus Enzyme.The present invention demonstrates gene function by realizing the heterogenous expression of newfound esterase gene.

The invention solves the problems that first technical problem be define it is a kind of with thermophilic esterase activity novel gene, it is described The amino acid sequence of thermophilic esterase gene is for example 1) or 2) shown：

1) it is amino acid sequence shown in SEQ ID NO.2；Or

2) disappearance of Jing amino acid, replacement, insertion or mutation are formed and had on the basis of the amino acid sequence for 1) limiting Encode the amino acid sequence of activated esterase.

The thermophilic esterase gene source is in Aquifex aeolicus (wind produces liquid bacterium) bacterial strain.Aquifex aeolicus It is a kind of hyperthermophilic eubacteria, in being found in the hot spring of American National Yellowstone, with H₂/CO₂/O₂And other inorganic substances are Somatomedin, maximum growth temperature belongs to several most one of the thermoduric bacterias being currently known at 95 DEG C.

The invention solves the problems that Second Problem be to provide it is a kind of expression thermophilic esterase genetic engineering bacterium or transgenosis 1) or 2) gene of clone, the genetic engineering bacterium or transgenic cell line express amino acid sequence shown in：

1) amino acid sequence is the sequence shown in SEQ ID NO.2；Or

2) amino acid sequence is that the disappearance of Jing amino acid, replacement, insertion or mutation are formed in the sequence basis for 1) limiting And with the sequence of the activated esterase of coding.

In one embodiment of the invention, the nucleotide sequence of the gene is for example 3) or 4) shown：

3) its nucleotide sequence is SEQ ID NO.4 or SEQ ID NO:Sequence shown in 6；

4) disappearance of Jing bases, replacement, insertion or mutation are formed and with volume on the basis of the nucleotide sequence for 3) limiting The nucleotide sequence of the activated esterase of code.

In one embodiment of the invention, the genetic engineering bacterium can be with Pichia pastoris, Escherichia coli or Bacillus megaterium is that host builds what is obtained.

In one embodiment of the invention, the Pichia pastoris may be selected GS115, KM71 or SMD1168 etc..

In one embodiment of the invention, the genetic engineering bacterium is Pichia yeast engineering, first builds restructuring and finishes Red Yeast expression carrier, is expressed by host of Pichia pastoris；The restructured Pichia pastoris in expression carrier can be Build what is obtained on the basis of pPIC9, pPIC3K, pPIC9K, PAO815 or pPICZ α etc..

In one embodiment of the invention, the genetic engineering bacterium is to finish red ferment by vector construction restructuring of pPIC9K Female expression vector, builds what is obtained by host of Pichia pastoris GS115.

In one embodiment of the invention, the Pichia yeast engineering expresses nucleotide sequence such as SEQ ID NO: Gene shown in 6.

In one embodiment of the invention, the Escherichia coli may be selected BL21, Rosctta 2, Origami 2, Rosctta-gami 2 or Origami B etc..

In one embodiment of the invention, the genetic engineering bacterium is colibacillus engineering；First build restructuring big Enterobacteria expression vector, is expressed by host of Escherichia coli；The expression of recombinant e. coli carrier can be Build what is obtained on the basis of pET28T, MBP3, pBR series, pUC series or pAT153 carriers etc..

In one embodiment of the invention, the genetic engineering bacterium is with MBP3 as vector construction recombination bacillus coli Expression vector, builds what is obtained by host of BL21 or Origami 2.

In one embodiment of the invention, the nucleotides sequence of the colibacillus engineering expression is listed in SEQ ID NO:Slight modification on the basis of 6, does not contain and histidine-tagged (eliminates SEQ ID NO:The 678th 's to the 705th on 6 Base).

In one embodiment of the invention, the bacillus megaterium optional WH320, MS941, YYBm1 etc..

In one embodiment of the invention, the genetic engineering bacterium is bacillus megaterium engineering bacteria；First build weight Group bacillus megaterium expression vector, is expressed by host of bacillus megaterium；The restructuring bacillus megaterium expression Carrier can be the structure on the basis of pHIS1525, pHIS1522, pSTREP1525, pSTREPHIS1525, pSTOP 1622 etc. Build what is obtained.

In one embodiment of the invention, the genetic engineering bacterium is huge as vector construction restructuring with pHIS1525 Bacillus expression vector, builds what is obtained by host of bacillus megaterium YYBm1.

In one embodiment of the invention, the bacillus megaterium engineering bacterium expression nucleotides sequence is classified as SEQ ID NO:6.The invention solves the problems that the 3rd problem be to provide a kind of recombinant vector of expression thermophilic esterase, the recombinant vector contains 1) or 2) there is gene of the amino acid sequence shown in：

1) amino acid sequence is the sequence shown in SEQ ID NO.2；Or

2) amino acid sequence is that the disappearance of Jing amino acid, replacement, insertion or mutation are formed in the sequence basis for 1) limiting And with the sequence of the activated esterase of coding.

In one embodiment of the invention, the recombinant vector is with pPIC9, pPIC3K, pPIC9K, PAO815 Or build what is obtained on the basis of pPICZ α etc..

In one embodiment of the invention, the recombinant vector is with pET 28T, MBP3, pBR series, pUC systems Build what is obtained on the basis of row or pAT153 carriers etc..

In one embodiment of the invention, the recombinant vector be with pHIS1525, pHIS1522, Build what is obtained on the basis of pSTREP1525, pSTREPHIS1525 or pSTOP 1622 etc..

The invention solves the problems that the 4th problem be to provide a kind of method for producing thermophilic esterase, it is characterised in that it is described Method is using the recombinant vector for expressing thermophilic esterase of the invention, the genetic engineering bacterium of expression thermophilic esterase or expresses thermophilic The transgenic cell line of esterase.

In one embodiment of the invention, methods described, is that the sequence of SEQ ID NO.6 is connected to into pPIC9K to obtain To recombinant expression carrier pPIC9K-Aaeo2, then recombinant expression carrier electricity is transformed in Pichia pastoris GS115, screening is obtained Positive transformant is recombinant yeast pichia pastoris engineering bacteria；The positive restructuring for obtaining Pichia pastoris GS115/pPIC9K-Aaeo2 is existed 30 DEG C, cultivate to OD under the conditions of 200rpm₆₀₀=2.0-6.0, subsequently centrifugation proceeds to thalline in fresh culture, 28 DEG C, Final concentration of 1.0% methyl alcohol, abduction delivering 30-120h are cultivated and added under the conditions of 200rpm.

The invention solves the problems that the 5th problem be to provide it is described expression thermophilic esterase recombinant vector, expression thermophilic esterase Genetic engineering bacterium or expression thermophilic esterase transgenic cell line application.

In one embodiment of the invention, the application, be applied to food processing, living things catalysis, prepare medicine, The fields such as leather manufacture, animal feed, cosmetics, waste water control.

The invention solves the problems that the 6th problem be to provide a kind of hydrolysis p-nitrophenyl phenols or glycerolipid substrate 1) or 2) method, the gene that methods described is express amino acid sequence shown in, by catalyst of gene expression product water-filling is entered Solution；Wherein gene：

1) amino acid sequence is the sequence shown in SEQ ID NO.2；Or

2) amino acid sequence is that the disappearance of Jing amino acid, replacement, insertion or mutation are formed in the sequence basis for 1) limiting And with the sequence of the activated esterase of coding.

In one embodiment of the invention, the p-nitrophenyl phenols substrate is p-NPC₄、p-NPC₅、p-NPC₈、p- NPC₁₂、p-NPC₁₄Or p-NPC₁₆。

In one embodiment of the invention, the glycerolipid substrate be triacetic acid glyceride, tributyrin, Tricaprylin, three certain herbaceous plants with big flowers acid glycerides, three sour nutmeg glyceride, tripalmitin or olive oil.

Beneficial effects of the present invention：

Present invention finds a kind of new thermophilic esterase, and restructuring expresses the thermophilic esterase.The present invention is obtained to purifying Thermophilic esterase carried out a series of analysis of zymologic property research, including most suitable substrate specificity, most suitable action pH and pH stability, Optimum temperature and temperature stability, organic solvent affect on enzyme stability of recombinating, metal ion is to enzyme stability shadow of recombinating Ring, surfactant affects on enzyme stability of recombinating, inhibitor affects on enzyme stability of recombinating.As a result recombinase Aaeo2 is shown Most suitable substrate is p-NPC₄, optimum temperature and pH are 90 DEG C and 8.0, and with good heat endurance, to metal ion With organic solvent etc. with certain tolerance.

Description of the drawings

Fig. 1 is the chadogram structure figure of the A.aeolicus thermophilic esterase Aaeo2 of the embodiment of the present invention 1；

Fig. 2 is the A.aeolicus thermophilic esterase Aaeo2 signal peptide prediction result figures of the embodiment of the present invention 2；

Fig. 3 is the thermophilic esters of A.aeolicus that the embodiment of the present invention 2 utilizes the prediction of " homologous modeling " method and its software analysis Enzyme Aaeo2 functional structures are illustrated；

Fig. 4 is the pPIC9K-Aaeo2 double digestion qualification figures of the embodiment of the present invention 3；

Fig. 5 is the pPIC9K-Aaeo2 recombinase SDS-PAGE electrophoresis result figures of the embodiment of the present invention 3；M is albumen Marker；Swimming lane 1 is recombinase albumin A aeo2 in fermented supernatant fluid, and swimming lane 2 is purification of Recombinant zymoprotein Aaeo2；

Fig. 6 is the A.aeolicus restructuring thermophilic esterase Aaeo2 substrate specificity analyses of purifying；

Fig. 7 is A.aeolicus restructuring thermophilic esterase Aaeo2 optimum temperatures (Fig. 7 A) and temperature stability of purifying (Fig. 7 B) is analyzed；

Fig. 8 is the Tm values analysis of the A.aeolicus restructuring thermophilic esterase Aaeo2 of purifying；

Fig. 9 is the most suitable action pHs (Fig. 9 A) of A.aeolicus restructuring thermophilic esterase Aaeo2 of purifying and pH stability (figure 9B) analyze.

Specific embodiment

The term for being used in the present invention, unless otherwise specified, typically has those of ordinary skill in the art usual The implication of understanding.

Embodiment and Application Example are prepared with reference to specific, and this is described in further detail with reference to data It is bright.It should be understood that these embodiments are of the invention solely for the purpose of illustration, rather than the scope of the present invention is limited by any way.

Below in an example, the various processes and method not described in detail are conventional methods as known in the art.

Embodiment 1：The acquisition of A.aeolicus restructuring thermophilic esterase genes and gene sequencing

According to conserved sequence " Gly-Xaa-Ser-Xaa-Gly " in esterase primary structure and key enzyme activity site " Ser, The feature of His, Asp ", from two kinds of putative protein sequences of gene group selection of A.aeolicus bacterial strains, in SWISS-PROT data Blast analyses are carried out to it in storehouse, Aaeo1 sequences and a kind of methyl from Nitrosomonas europaea is obtained ester esterase(Reference sequence:Q82SL8.2) there is 27% similitude.Aaeo2 sequences with derive from A kind of mycophenolic acid acyl-glucuronide esterase (Reference of Bos taurus sequence:Q5E9H9.1) there is 25% similitude.Therefore, it can tentatively guess both sequence codified esterase proteins.

5 kinds of thermophilic esterase/lipase studied are selected, with two kinds of thermophilic esterases of A.aeolicus sequence alignment point is carried out Analysis, esterase/lipase known to 5 kinds is respectively esterase (the GenBank NO. in Sulfolobus solfataricus sources: CCQ48704), lipolytic enzyme (the GenBank NO. in S.acidocaldarius sources:AAC67392)、 Carboxylesterase (the GenBank NO. in Archaeoglobusfulgidus sources:WP_010879825)、 Lipase (the GenBank NO. in F.changbaicum sources:EF138832) and Thermotoga maritima source lipase(GenBank NO.:AAD36421).As a result find, there is two kinds of putative proteins of A.aeolicus typical esterase to protect Defending zone domain and key enzyme activity site, in addition, the also presence of other same areas.The key enzyme activity site of Aaeo1 For Ser62, Asp163, His190, sequence Gly60-Gly64 is conservative region.The key enzyme activity site of Aaeo2 be Ser82, Asp185, His212, sequence Gly80-Gly84 is conservative region.

Based on the classification of Arpigny and Jaeger et al. to bacterial origin esterase/lipase, 8 families are classified as；Make With MEGA5.0, using neighbor-joining methods, set bootstrap as 1000, build chadogram (see Fig. 1).By Fig. 1 As can be seen that the recombinase of Aaeo2 sequential codings is classified as Family VIII families.

Wherein, the amino acid sequence of Aaeo1 such as SEQ ID NO:Shown in 1, nucleotide sequence such as SEQ ID NO:Shown in 3.

The amino acid sequence of Aaeo2 such as SEQ ID NO:Shown in 2, nucleotide sequence such as SEQ ID NO:Shown in 4.

Embodiment 2 utilizes " Blast search " method to obtain two kinds of thermophilic esterase three-dimensional prediction structures of A.aeolicus

It is 621bp from a kind of thermophilic esterase Gene A aeo1 length of A.aeolicus, encodes 207 amino acid, point Son amount size is 23.4kDa, and the nucleotide sequence and protein sequence of the albumen are shown in SEQ ID NO:3 and SEQ ID NO:1.It is another kind of Thermophilic esterase Gene A aeo2 length is 678bp, encodes 226 amino acid, and molecular size range is 26.8kDa, the nucleic acid of the albumen Sequence and protein sequence are shown in SEQ ID:4 and SEQ ID NO:2.

Predicted according to SignalP, it is assumed that the possibility that the N-terminal of albumin A aeo2 exists without signal peptide (see Fig. 2).

The online mould of I-TASSER protein is submitted to build clothes respectively the amino acid sequence of two kinds of thermophilic esterases of A.aeolicus Business device (http://zhanglab.ccmb.med.umich.edu/I-TASSER/) homologous modeling is carried out, followed by Pymol Software is analyzed (see Fig. 3) to the homologous modeling structure of two kinds of thermophilic esterases of A.aeolicus.Enzymatic activity position as seen from Figure 3 Point (red-label), negative oxygen ion hole (blue markings) and cap structures (white marking).The template of Aaeo2 albumen homologies modeling For 2wtmA, 2wtmA is derived from the wheat bran esterase of rumen bacteria Butyrivibrio Proteoclasticus.Aaeo2's Sequence homology similitudes of the model (Fig. 3) and template between be 25%, catalytic triads be Ser82, Asp185, His212, structure Into two the residues Leu19 and His83 in negative oxygen ion hole.

The structure of two kinds of thermophilic esterase carrier for expression of eukaryon of embodiment 3A.aeolicus, recombinant expressed and its protein expression

First, the structure of carrier for expression of eukaryon

Two kinds of thermophilic esterase genes of A.aeolicus according to Pichia pastoris codon preference, in the C-terminal of DNA encoding frame 6xHis labels are introduced, and AvrII and NotI restriction enzyme sites are introduced respectively in 5 ' and 3 ' both sides, synthesized by biotech firm Sequence after optimization, i.e. SEQ ID NO:5 (Aaeo1) and SEQ ID NO:6 (Aaeo2), and it is connected to Expression vector pPIC9K On, obtain recombinant expression carrier pPIC9K-Aaeo1 and pPIC9K-Aaeo2.

With AvrII and NotI double digestion recombinant expression carrier plasmid pPIC9K-Aaeo2,37 DEG C of digestion 30min.Double digestion Simply identified, 1% agarose nucleic acid gel as shown in figure 4, positive pPIC9K-Aaeo2 plasmid double digestion figures (AvrII and NotI), M is 15000bp Marker.

2nd, the expression of recombinant vector pPIC9K-Aaeo1 and pPIC9K-Aaeo2 in Pichia pastoris GS115.

1) screening of electricity conversion pichia methanolica and positive transformant

According to pichia yeast expression system operation manual, will the correct recombinant plasmid pPIC9K-Aaeo2 of sequencing with restricted interior After enzyme cutting SacI linearisations, Electroporation Pichia pastoris GS115 to be coated and screen target gene integration on MD flat boards (containing G418) Transformant on recipient bacterium chromosome.Picking transformant, extracts STb gene, with it as template, with 5 ' AOX and 3 ' AOX as primer Enter performing PCR identification, compared with empty carrier transformed bacteria, verifying purpose gene has been integrated in Yeast genome.

2) abduction delivering of the restructuring pPIC9K-Aaeo2 carriers in Pichia pastoris

Recombinant yeast pichia pastoris in 25mL BMGY culture mediums (250mL triangular flasks) 30 DEG C cultivate to OD be 2.0～6.0, from Heart collects thalline, is added to 100mL BMMY culture mediums after 28 DEG C, continues to cultivate under the conditions of 200rpm, adds per 12-24h 1.0% (v/v) methyl alcohol, timing sampling determines cell density and protein concentration, while carrying out the SDS-PAGE and enzyme activity of nutrient solution Determine.Protein concentration adopts Bradford methods.

3) purifying of recombinase and its PAGE gel electrophoretic analysis (see Fig. 5)

Protein purification refer to GE Healthcare guides, SDS-PAGE analysis according to《Molecular Cloning:A Laboratory guide》(the Three editions), the gel resolving gel concentration for using is 12%, and concentration gum concentration is 5%, and applied sample amount is 5-25 μ L.Protein is with examining horse This light blue R-250 is dyeed.In Fig. 5 as can be seen that land2 bands show the single purifying protein Aaeo2 for obtaining, molecular weight is 25kDa or so.

4) thermophilic esterase Enzyme activity assay

Thermophilic esterase enzyme activity determination method with reference to the happy papers of Wang Le (gene cloning of rhizopus chinensis lipase, expression, purify and Zymologic property research, Southern Yangtze University, 2008)：Using p-NPC4 as substrate, pH8.0Tris-HCl buffer solutions, in 70 DEG C of conditions Lower measure enzymatic activity.It is an enzyme that enzyme activity is defined as the enzyme amount for producing 1 μm of ol p-nitrophenol per minute under certain reaction condition International unit living.

Enzyme assay is shown with esterase active.

The avtive spot rite-directed mutagenesis of embodiment 4 further identifies two kinds of hypothesis thermophilic esterases of A.aeolicus

For further two kinds of hypothesis thermophilic esterases of identification A.aeolicus, primer is designed, by full plasmid PCR to key enzyme Avtive spot (Ser-His-Asp) carries out rite-directed mutagenesis, builds mutant strain.For the enzyme active sites of Aaeo1 esterases (Ser62, Asp163, His190), successively using primer g9 and g10, g11 and g12, g13 and g14.The enzymatic activity of Aaeo2 esterases Site (Ser82, Asp185, His212) uses successively primer g15 and g16, g17 and g18, g19 and g20.(mutational site is with slightly Body surface shows)

PCR primer (the sequence such as SEQ ID NO:7～SEQ ID NO:Shown in 18)：

g9：5’-CATGACGTAGTTGGTTGGGCTTTGGGAGGTTCTCTTGC-3’

g10：5’-GCAAGAGAACCTCCCAAAGCCCAACCAACTACGTCATG-3’

g11：5’-GATACACGGCGTCTCCAATCGAATTGTTCCCTAT-3’

g12：5’-ATAGGGAACAATTCGATTGGAGACGCCGTGTATC-3’

g13：5’-CTAATTTTATTAGGTGGCGGAAATTTTCCTGTTCGTAACGAAG-3’

g14：5’-CTTCGTTACGAACAGGAAAATTTCCGCCACCTAATAAAATTAG-3’

g15：5’-GAAATCACTTTGTGTGGATCTGCTCATGGTGGATATATTGCTA-3’

g16：5’-TAGCAATATATCCACCATGAGCAGATCCACACAAAGTGATTTC-3’

g17：5’-CCTGTTGAAATTGTTATTATGCATGGTATTAGAAACGACATTGTTCCTATT-3’

g18：5’-AATAGGAACAATGTCGTTTCTAATACCATGCATAATAACAATTTCAACAGG-3’

g19：

5’-GTTAAAAAGTTTTTGAAAGTTGACGATGATAACCAATTGAATGAATCTTTGCAAA-3’

g20：5’-TTTGCAAAGATTCATTCAATTGGTTATCATCGTCAACTTTCAAAAACTTTTTAAC-3’

The recombinase that mutant strain is expressed is purified, enzymatic activity is determined, it is found that enzymatic activity disappears, illustrated these three positions Importance of the point to recombinase, also further demonstrate that the two putative proteins are esterase.

Embodiment 5：The substrate specificity of the A.aeolicus thermophilic esterases of purifying

Two kinds of restructuring thermophilic esterases are determined respectively to p-nitrophenyl phenols and the substrate specificity of glycerolipid.

Act on the enzymatic activity such as enzyme activity determination of embodiment 3 of p-nitrophenyl phenols substrate.P-NPC is selected respectively₄、p-NPC₅、 p-NPC₈、p-NPC₁₂、p-NPC₁₄、p-NPC₁₆6 kinds of substrates, determine enzymatic activity, select most suitable substrate.

Act on the activity determination method of glyceride type substrate with reference to jade Song into paper (stearothermophilus enzyme molecular clone and Zymologic property research, Jilin University, 2008).From triacetic acid glyceride, tributyrin, tricaprylin, the acid of three certain herbaceous plants with big flowers Glyceride, three sour nutmeg glyceride, tripalmitin and olive oil are substrate, in the phosphoric acid buffer of 50mM pH 8.0 In the buffer solution system of liquid, under the conditions of 70 DEG C esterase activity is determined, determine recombinase to triglycerin esters be substrate when most Suitable substrate.

As a result as shown in fig. 6, Fig. 6 recombinase Aaeo2 tend to hydrolyzing short-chain substrate, centering long-chain substrate has relatively low Relative activity, therefore the most suitable substrates of recombinase Aaeo2 are p-NPC₄, Aaeo2 is to glyceride type almost without enzymatic activity.

Embodiment 6：The A.aeolicus thermophilic esterases optimum temperature of purifying and temperature stability are analyzed

Thermophilic esterase enzyme activity determination method with reference to the happy papers of Wang Le (gene cloning of rhizopus chinensis lipase, expression, purify and Zymologic property research, Southern Yangtze University, 2008)

Optimum temperature and temperature stability analysis have been carried out to the restructuring thermophilic esterase that purifying is obtained.As shown in fig. 7, As a result show, with p-NPC₄For substrate when, the optimum temperature of recombinase Aaeo2 is 90 DEG C, places 2h at 70 and 80 DEG C and still retains 65% residual enzymic activities.In 90 DEG C of half-life 2h.

Experimental result fully shows that restructuring thermophilic esterase Aaeo1 and Aaeo2 are respectively provided with the high temperature being satisfied with industrial production Condition.

Embodiment 7：The T of the A.aeolicus thermophilic esterases of purifying_mValue is determined

Using the method for circular dichroism spectra, the albumen heat endurance of purification of Recombinant enzyme, i.e. T are determined_mValue (melting temperature), dissolving The maximum corresponding temperature of point of the slope of curve is T_mValue, as shown in Figure 8, it can be seen that the melting curve of recombinase Aaeo2 shows Show that curvilinear motion is most fast at 88 DEG C, curve gradually tends to balance at a temperature of higher than 90 DEG C.Therefore T_mIt is worth for 88 DEG C, this Fully further demonstrate the heat endurance experiment of recombinase Aaeo2.

Embodiment 8：The most suitable action pH of A.aeolicus thermophilic esterases of purifying and pH stability analyses

Thermophilic esterase enzyme activity determination method with reference to the happy papers of Wang Le (gene cloning of rhizopus chinensis lipase, expression, purify and Zymologic property research, Southern Yangtze University, 2008)

Most suitable action pH and pH stability analyses have been carried out to the restructuring thermophilic esterase Aaeo2 that purifying is obtained. such as Fig. 9 A institutes Show, in pH8.0, with respect to enzyme activity highest, therefore optimal reaction pH value is 8.0.The pH stability of recombinase Aaeo2 such as Fig. 9 B institutes Show, stability of the recombinase under the conditions of pH7.0-9.0 determined respectively, with the increase of standing time, with respect to enzyme activity drastically under Drop, the half-life is followed successively by 54min, 50min and 46min.

Embodiment 9：The impact of A.aeolicus thermophilic esterase stability of each metal ion species to purifying

Thermophilic esterase enzyme activity determination method with reference to the happy papers of Wang Le (gene cloning of rhizopus chinensis lipase, expression, purify and Zymologic property research, Southern Yangtze University, 2008).

As can be seen from Table 1, for recombinase Aaeo2, EDTA can suppress its enzymatic activity, illustrate its be metal ion according to Bad property enzyme.For Aaeo2, low concentration Fe³⁺、Co²⁺、Mn²⁺Enzymatic activity, low concentration Ca can be improved²⁺Little is affected on enzymatic activity, And high concentration Ca²⁺There is slight inhibitory action to recombinase.

The analysis of A.aeolicus restructuring thermophilic esterase stability of each metal ion species of table 1 to purifying

Embodiment 10：The impact of A.aeolicus thermophilic esterase stability of the various organic solvents to purifying

Thermophilic esterase enzyme activity determination method with reference to the happy papers of Wang Le (gene cloning of rhizopus chinensis lipase, expression, purify and Zymologic property research, Southern Yangtze University, 2008).

The analysis of A.aeolicus restructuring thermophilic esterase stability of the various organic solvents of table 2 to purifying

As can be seen from Table 2, recombinase Aaeo2 has good organic solvent tolerance, and hexamethylene can activate enzyme activity Property, and high concentration n-hexane, n-butanol and isobutanol have stronger inhibitory action to recombinase.The organic solvent pair of low concentration Enzymatic activity affects little.

Embodiment 11：The impact of A.aeolicus thermophilic esterase stability of the various surfactants to purifying

Thermophilic esterase enzyme activity determination method with reference to the happy papers of Wang Le (gene cloning of rhizopus chinensis lipase, expression, purify and Zymologic property research, Southern Yangtze University, 2008).

As can be seen from Table 3, surfactant has certain inhibitory action to recombinase Aaeo2, with surfactant The increase of concentration, inhibitory action strengthens, but low concentration affects less to enzymatic activity.

The analysis of A.aeolicus restructuring thermophilic esterase stability of the various surfactants of table 3 to purifying

Embodiment 12：The impact of A.aeolicus thermophilic esterase stability of the various inhibitor to purifying

Thermophilic esterase enzyme activity determination method with reference to the happy papers of Wang Le (gene cloning of rhizopus chinensis lipase, expression, purify and Zymologic property research, Southern Yangtze University, 2008).

As can be seen from Table 4, for recombinase Aaeo2, PMSF (serine protease and thiol protease inhibitor) and DEPC (His residue trims) has stronger inhibitory action to recombinase.And DTT (is mainly used in disulfide bond in protein Reduction) enzymatic activity can be improved.This shows the importance of Ser, His and Asp to enzymatic activity.

The analysis of A.aeolicus restructuring thermophilic esterase stability of the various inhibitor of table 4 to purifying

Embodiment 13：The structure of A.aeolicus thermophilic esterase coli expression carriers, recombinant expressed and its albumen table Reach

First, the structure of coli expression carrier

1) design of primers：Gene design primer from after optimization, not comprising C-terminal 6xHis labels (sequence such as SEQ ID NO:19～SEQ ID NO:Shown in 22)

g1:5’-GCTCATATGATGCTGAAGAACCCTTTGT-3’

g2:5’-GCTGGATCCTTAATACCCCTTTAAAATAGCTTT-3’

g3:5’-GCCCATATGATGTACTTCTACGATATTAACAA-3’

g4:5’-GGCGGATCCTTACAACAATTCCTCAATATATTTTTG-3’

2) PCR reactions, with carrier pPIC9K-Aaeo1 as template, degenerate temperature is 50 DEG C to g1 and g2；G3 and g4 are with carrier PPIC9K-Aaeo2 is template, and degenerate temperature is 55 DEG C.

Reaction condition：98 DEG C, 3min denaturations；Denaturation, 98 DEG C, 30s, annealing, 50 DEG C (or 55 DEG C), and 1min, renaturation, 72 DEG C, 45s, second step to the 4th step carries out 30 circulations；72℃,10min；10 DEG C of insulations.Electrophoresis detection pcr amplification product, obtains The DNA fragmentation that size is 650 and 700bp or so is obtained, the gel purification kit purifying target DNA provided using omega companies Fragment.

3) PCR primer and plasmid MBP3 of NdeI and BamHI double digestions Aaeo1 and Aaeo2,37 DEG C of digestion 4h.Utilize The gel glue reclaim kit that omega companies provide reclaims target DNA fragment and carrier

4) connect

Take the μ l of T4DNA ligase 1, the μ l of 10x T4DNAligase Buffer 1, genes of interest+carrier (n₁:n₂=1: 3) 8 μ l, are well mixed, and at 16 DEG C 16h is connected.

5) convert

Take 10 μ l connection products to add in competent cell BL21 and Origami 2, be placed in 30min on ice, 42 DEG C of heat shocks It is immediately placed at after 90s on ice, after placing 2min, adds LB culture mediums of the 1ml without antibiotic, 37 DEG C of shaking table cultures 1h to turn The bacterium solution of change is applied on LB (Amp) flat board, 37 DEG C of overnight incubations.Respectively the multiple bacterium colonies of picking are transferred to test tube on flat board, and 37 DEG C culture 8h.

6) plasmid is extracted

Plasmid is extracted with the plasmid extraction kit of omega companies, delivering to biotech firm carries out sequencing identification.

2nd, tables of the recombinant expression carrier MBP3-Aaeo1 and MBP3-Aaeo2 in e. coli bl21 and Origami 2 Reach

1) abduction delivering of the recombinant vector in two kinds of hosts

Picking positive restructuring bacterial strain transformant is inoculated in 5mL LB fluid nutrient mediums on LB flat boards (containing Amp) (containing Amp) In, in 37 DEG C, 200rpm incubated overnights 8h, obtain seed liquor.

Seed liquor is inoculated in 50-100mL LB culture mediums (250mL triangular flasks) with 1% inoculum concentration, 37 DEG C, 200rpm Cultivate to OD600 be 0.6 or so when, add the IPTG of final concentration of 0.2mM, while being cooled to 17 DEG C of cultures, carry out restructuring egg White abduction delivering, culture 12h or so.

2) collection of recombinase and purifying

Thalline is collected by centrifugation, using the resuspended thalline of BufferA, ultrasonication is carried out.Thalline after low-temperature centrifugation is broken is molten Liquid, collecting supernatant carries out protein purification.Purification process refers to GE Healthcare guides.First time protein purification is obtained MBP-Aaeo1 and MBP-Aaeo2 fusion proteins, by ultrafiltration imidazoles is removed, and adds the TEV enzymes for suitably having purified to carry out protease Cut, 16 DEG C of digestion 16h, carry out second protein purification, collection penetrates peak, the recombinant protein A aeo1 and Aaeo2 for as purifying. SDS-PAGE carries out electrophoretic analysis.

Host's extracellular fraction, periplasmic space component, cytoplasm fraction, whole-cell component, cell precipitation component are entered respectively Row electrophoretic analysis, it is seen that recombinant protein is primarily present in cytoplasm fraction.Pichia pastoris protein expression is 0.32mg/mL, E. coli protein expression is 1.2mg/mL.Compared with Pichia pastoris, expression is improved.

3) enzyme activity determination

Bacillus coli expression obtains fusion protein (having TEV protease restriction enzyme site between two albumen), using TEV 16 DEG C place 16h, respectively determine TEV enzyme digestions before and after enzymatic activity, as a result show Origami2/MBP3-Aaeo2 recombinant bacterial strain tables The recombinase for reaching, specific enzyme activity power increases after digestion.

Embodiment 14：The structure of two kinds of thermophilic esterase bacillus megaterium expression vectors of A.aeolicus, it is recombinant expressed and Its protein expression

First, the structure of bacillus megaterium expression vector

1) design of primers：Gene design primer (sequence such as SEQ ID NO from after optimization:23～SEQ ID NO:26 institutes Show)

g5：5’-TAGAGATCTATATGCTGAAGAACCCTTTGTT-3’

g6：5’-TCAGGATCCTTAGTGATGATGATGGTGGT-3’

g7：5’-TAGAGATCTATATGTACTTCTACGACATCAACAA-3’

g8：5’-TGCGGATCCTTAGTGGTGATGATGGTGGTG-3’

2) react according to Standard PCR, with carrier pPIC9K-Aaeo1 as template, degenerate temperature is 51 DEG C to g5 and g6；G7 and With carrier pPIC9K-Aaeo2 as template, degenerate temperature is 57 DEG C to g8.Electrophoresis detection pcr amplification product, acquisition size is 650 Hes The DNA fragmentation of 700bp or so, the gel purification kit purifying target DNA fragment provided using omega companies.

3) PCR primer and plasmid pHIS1525 of BglII and BamHI double digestions Aaeo1 and Aaeo2,37 DEG C of digestion 4h.Profit The gel glue reclaim kit provided with omega companies reclaims target DNA fragment and carrier.

4) conventionally 16 DEG C connection 16h.Thermal shock is converted into DH5 α competence.Plasmid is extracted, biological public affairs are sent to Department's sequencing identification.

2nd, recombinant expression carrier pHIS1525-Aaeo1 and pHIS1525-Aaeo2 is in bacillus megaterium YYBm1 Expression

1) screening of bacillus megaterium and positive transformant is converted

Recombinant vector is transformed in bacillus megaterium YYBm1 protoplasm somatocytes using protoplast transformation, it is former The preparation of raw plastid and protoplast transformation method refer to " Bacillus megaterium Protein Production System " handbooks.The screening on the LB flat boards containing tetracyclin resistance obtains positive transformant, extracts the identification of plasmid double digestion.

2) induction tables of the recombinant vector pHIS1525-Aaeo1 and pHIS1525-Aaeo2 in bacillus megaterium YYBm1 Reach

Picking positive transformant is inoculated in 5mL LB fluid nutrient mediums (containing Tet) from resistant panel, 37 DEG C, 220rpm Shaken overnight culture, obtains seed liquor.

According to 1% inoculum concentration, seed liquor is inoculated in the fresh LB fluid nutrient mediums of 200mL (containing Tet), 37 DEG C, 220rpm shaken cultivations, recombinant bacterium culture grow to OD600 between 0.3-0.4, add 0.5% (w/v) (D)-wood sugar conduct Derivant, still keeps 37 DEG C, 220rpm concussion and cultivates, and a sample is taken per 30-60min, determines OD600nm and protein concentration, 6h terminates culture after induction.

3) purifying of recombinase and SDS-PAGE are analyzed

Centrifuge A sample collects thalline cell and Sample supernatants, 9000r/min is centrifuged 10min.Extracellular protein directly carries out egg White purifying, intracellular protein carries out cracking acquisition using lysis buffer.Purification process refers to GE Healthcare guides.12% The SDS-PAGE of separation gel carries out electrophoretic analysis.

4) enzyme assay

Thermophilic esterase enzyme activity determination method with reference to the happy papers of Wang Le (gene cloning of rhizopus chinensis lipase, expression, purify and Zymologic property research, Southern Yangtze University, 2008)：Using p-NPC4 as substrate, pH8.0Tris-HCl buffer solutions, in 70 DEG C of conditions Lower measure enzymatic activity.It is an enzyme that enzyme activity is defined as the enzyme amount for producing 1 μm of ol p-nitrophenol per minute under certain reaction condition International unit living.

Enzyme assay is shown with esterase active.

These examples show, are esterase protein from two kinds of putative proteins of hyperthermophile Aquifex aeolicus. With the fast development of molecular biology, genetic engineering and enzyme engineering etc., first identified of the present invention simultaneously confirms two kinds of Aquifex The new thermophilic esterase gene in aeolicus sources, and detailed property research has been carried out to esterase protein.These are new thermophilic The industrial applications of hot esterase are laid a good foundation.

Although the present invention is disclosed as above with preferred embodiment, it is not limited to the present invention, any to be familiar with this skill The people of art, without departing from the spirit and scope of the present invention, can do various changes and modification, therefore the protection model of the present invention Enclosing should be by being defined that claims are defined.

<110>Southern Yangtze University

<120>A kind of thermophilic esterase and its functional verification from Aquifex aeolicus bacterial strains

<160> 22

<170> PatentIn version 3.3

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<212> PRT

<213>Artificial sequence

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Met Leu Lys Asn Pro Leu Phe Ile His Gly Trp Ala Phe Ser Ser Lys

1 5 10 15

Val Phe Asn Asp Phe His Gly Ile Lys Tyr Asp Leu Pro Gly His Gly

20 25 30

Lys Asn Lys Asn Pro Tyr Glu Ser Ile Glu Lys Val Val Glu Glu Ile

35 40 45

Gly Lys Ile Ala Thr Ser Lys His Asp Val Val Gly Trp Ser Leu Gly

50 55 60

Gly Ser Leu Ala Leu Leu Phe Ala Tyr Arg Tyr Pro Glu Lys Val Asn

65 70 75 80

Arg Leu Ile Leu Ile Gly Thr Thr Pro His Phe Lys Gly Ala Trp Ser

85 90 95

Glu Lys Asn Ile Arg Ala Met Lys Leu Leu Ile Lys Lys Lys Gly Ile

100 105 110

Lys Ala Phe Arg Glu Leu Ala Tyr Gly Lys Phe Glu Asp Phe Phe Asp

115 120 125

Glu Glu Gln Gly Met Arg Phe Leu Glu Asp Tyr Val Asn Leu Asn Leu

130 135 140

Tyr Thr Val Leu Pro Tyr Ile Lys Lys Glu Val Tyr Leu Ile His Gly

145 150 155 160

Val Ser Asp Arg Ile Val Pro Tyr Ser Glu Ala Tyr Lys Leu His Arg

165 170 175

Ala Leu Lys Cys Ser Lys Leu Ile Leu Leu Gly Gly Gly His Phe Pro

180 185 190

Val Arg Asn Glu Glu His Leu Arg Lys Ala Ile Leu Lys Gly Tyr

195 200 205

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<213>Artificial sequence

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Met Tyr Phe Tyr Asp Ile Asn Asn Glu Lys Arg Leu Trp Leu His Leu

1 5 10 15

His Gly Leu Ala Thr Asn Val Leu Gly Arg Lys Ile Glu Phe Leu Arg

20 25 30

Asn Tyr Phe Lys Glu Lys Lys Leu Tyr Ser Phe Phe Ala Asn Asp Met

35 40 45

Asp Tyr Glu Lys His Thr Thr Thr Asn Thr Leu Asp Phe Leu Glu Val

50 55 60

Leu Val Arg Gly Phe Ser Gln Lys Phe Glu Glu Ile Thr Leu Cys Gly

65 70 75 80

Ser Ser His Gly Gly Tyr Ile Ala Met Asn Tyr Val Arg Lys Arg Pro

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Leu Phe Asn Val Lys Arg Leu Val Leu Leu Ala Pro Ser Tyr Asn Thr

100 105 110

Leu Ser Leu Ile Ile Lys Glu Leu Gly Glu Asp Lys Val Lys Pro Trp

115 120 125

Leu Glu Gly Lys Glu Glu Leu Thr Ile Leu Glu Glu Asp Arg Glu Val

130 135 140

Thr Phe Ile Lys Asp Trp Ala Lys Asp Ile Ile Gln Asn Asp Tyr Glu

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Ile Ile Lys Asp Gly Arg Val Asp Phe Pro Glu Glu Pro Pro Val Glu

165 170 175

Ile Val Ile Met His Gly Ile Arg Asp Asp Ile Val Pro Ile His Tyr

180 185 190

Ser Glu Thr Phe Ala Lys Ser Val Lys Val Lys Lys Phe Leu Lys Val

195 200 205

Asp Asp Asp His Gln Leu Asn Glu Ser Leu Gln Lys Tyr Ile Glu Glu

210 215 220

Leu Leu

225

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<212> DNA

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atgctcaaaa atcccctctt tatccacgga tgggcgtttt cctcaaaggt atttaatgac 60

tttcacggta taaagtacga ccttcccggg cacggaaaaa ataaaaatcc ctacgagagt 120

atagaaaaag tagtggaaga aattggaaaa atagccactt caaaacacga cgttgtaggc 180

tggtcccttg gaggaagtct ggcacttctt ttcgcttaca ggtatccaga aaaggtaaac 240

aggttgatcc ttatagggac cactccccac tttaaaggag cgtggagcga aaagaatata 300

agggctatga aactcctgat aaagaagaag gggataaaag ctttcaggga actagcctac 360

gggaaatttg aggacttctt tgacgaggag cagggcatga gatttcttga ggactacgtg 420

aacctgaact tgtacactgt acttccctat ataaagaagg aggtttacct gatacacgga 480

gtttcggaca gaatagttcc gtattcagaa gcttacaaac tgcatagagc tttaaaatgc 540

tctaaattaa tccttctcgg aggaggacac ttccctgtca gaaatgaaga acaccttagg 600

aaggcaattc tcaagggcta ctga 624

<210> 4

<211> 681

<212> DNA

<213>Artificial sequence

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atgtactttt acgatatcaa caacgagaag agactctggc ttcaccttca cggacttgcg 60

acaaacgtcc tcggaaggaa gatagagttt ttgaggaatt actttaagga aaagaagctt 120

tattcgtttt ttgcgaatga tatggactac gaaaagcaca caactacaaa caccttggat 180

tttttggaag ttctcgtcag aggattttcc cagaagtttg aagaaattac cctttgcggg 240

agttcccacg gcggttacat agcgatgaac tatgtaagga aaaggcctct ctttaatgta 300

aagagactcg tcttactcgc accctcatac aacacactat ccctgataat taaagagctc 360

ggagaagata aggtaaagcc atggcttgag ggaaaggaag aactcaccat acttgaagag 420

gacagggaag tgacctttat aaaggattgg gcaaaggaca taatacagaa cgactacgag 480

attataaaag acggaagggt ggatttcccc gaagagcctc ccgtggaaat agtgataatg 540

cacgggataa gggacgatat agtgccaatc cattattccg agacctttgc aaagagcgta 600

aaggtgaaaa aattcctcaa agtggacgac gaccaccagc taaacgaaag tctgcagaag 660

tatatagaag aactccttta g 681

<210> 5

<211> 651

<212> DNA

<213>Artificial sequence

<400> 5

atgctgaaga accctttgtt catccacggt tgggcatttt catctaaggt tttcaacgac 60

ttccatggta tcaagtacga tttgccagga catggaaaaa acaagaaccc atacgaatct 120

atagagaagg tggtagaaga gatcggtaaa atcgcaacat ctaagcatga cgtagttggt 180

tggagtttgg gaggttctct tgcattgctt tttgcttaca gatacccaga gaaagttaat 240

agactaattc tgataggtac tacccctcat ttcaaaggag cttggtcaga gaagaatatc 300

agagctatga aactgttgat taagaagaaa ggcattaagg ccttcagaga gctggcttat 360

ggtaaatttg aggatttctt tgatgaagaa caagggatga ggtttcttga agactatgtc 420

aatttgaatc tttacacggt gttgccctac attaagaaag aagtctattt gatacacggc 480

gtctccgatc gaattgttcc ctattccgaa gcctacaagc tacacagggc cttgaaatgt 540

tccaaactaa ttttattagg tggcggacat tttcctgttc gtaacgaaga acacttacgt 600

aaagctattt taaaggggta tggatcaagt caccaccatc atcatcacta a 651

<210> 6

<211> 708

<212> DNA

<213>Artificial sequence

<400> 6

atgtacttct acgacatcaa caacgagaag agattgtggt tgcacttgca tggtttggct 60

actaatgttt tgggtagaaa aatcgaattt ttgagaaact acttcaagga gaagaagttg 120

tactctttct tcgctaatga catggattac gagaagcata ctactactaa cactttggat 180

ttcttggagg ttttggttag aggattctct cagaaattcg aagaaatcac tttgtgtgga 240

tcttctcatg gtggatatat tgctatgaac tatgttagaa agagaccatt gtttaatgtt 300

aaaagattgg ttttgttggc tccatcttac aatactttgt ctttgatcat caaggagttg 360

ggagaggata aggttaagcc ttggttggaa ggtaaagaag aattgactat tttggaagaa 420

gatagagaag ttacttttat caaggattgg gctaaagata tcattcagaa cgattatgag 480

attattaagg acggaagagt tgactttcca gaagagccac ctgttgaaat tgttattatg 540

catggtatta gagacgacat tgttcctatt cattattctg agacttttgc taaatctgtt 600

aaagttaaaa agtttttgaa agttgacgat gatcaccaat tgaatgaatc tttgcaaaaa 660

tatattgagg aattgttggg ttcttctcac caccatcatc accactaa 708

<210> 7

<211> 38

<212> DNA

<213>Artificial sequence

<400> 7

catgacgtag ttggttgggc tttgggaggt tctcttgc 38

<210> 8

<211> 38

<212> DNA

<213>Artificial sequence

<400> 8

gcaagagaac ctcccaaagc ccaaccaact acgtcatg 38

<210> 9

<211> 34

<212> DNA

<213>Artificial sequence

<400> 9

gatacacggc gtctccaatc gaattgttcc ctat 34

<210> 10

<211> 34

<212> DNA

<213>Artificial sequence

<400> 10

atagggaaca attcgattgg agacgccgtg tatc 34

<210> 11

<211> 43

<212> DNA

<213>Artificial sequence

<400> 11

ctaattttat taggtggcgg aaattttcct gttcgtaacg aag 43

<210> 12

<211> 43

<212> DNA

<213>Artificial sequence

<400> 12

cttcgttacg aacaggaaaa tttccgccac ctaataaaat tag 43

<210> 13

<211> 43

<212> DNA

<213>Artificial sequence

<400> 13

gaaatcactt tgtgtggatc tgctcatggt ggatatattg cta 43

<210> 14

<211> 43

<212> DNA

<213>Artificial sequence

<400> 14

tagcaatata tccaccatga gcagatccac acaaagtgat ttc 43

<210> 15

<211> 51

<212> DNA

<213>Artificial sequence

<400> 15

cctgttgaaa ttgttattat gcatggtatt agaaacgaca ttgttcctat t 51

<210> 16

<211> 51

<212> DNA

<213>Artificial sequence

<400> 16

aataggaaca atgtcgtttc taataccatg cataataaca atttcaacag g 51

<210> 17

<211> 55

<212> DNA

<213>Artificial sequence

<400> 17

gttaaaaagt ttttgaaagt tgacgatgat aaccaattga atgaatcttt gcaaa 55

<210> 18

<211> 55

<212> DNA

<213>Artificial sequence

<400> 18

tttgcaaaga ttcattcaat tggttatcat cgtcaacttt caaaaacttt ttaac 55

<210> 19

<211> 28

<212> DNA

<213>Artificial sequence

<400> 19

gctcatatga tgctgaagaa ccctttgt 28

<210> 20

<211> 33

<212> DNA

<213>Artificial sequence

<400> 20

gctggatcct taatacccct ttaaaatagc ttt 33

<210> 21

<211> 32

<212> DNA

<213>Artificial sequence

<400> 21

gcccatatga tgtacttcta cgatattaac aa 32

<210> 22

<211> 36

<212> DNA

<213>Artificial sequence

<400> 22

ggcggatcct tacaacaatt cctcaatata tttttg 36

Claims (10)

1. it is a kind of expression thermophilic esterase genetic engineering bacterium or transgenic cell line, it is characterised in that the genetic engineering bacterium 1) or 2) or gene of the transgenic cell line express amino acid sequence shown in：

1) amino acid sequence is the sequence shown in SEQ ID NO.2；Or

2) amino acid sequence is that the disappearance of Jing amino acid, replacement, insertion or mutation are formed and had in the sequence basis for 1) limiting There is the sequence for encoding activated esterase.

2. genetic engineering bacterium according to claim 1, it is characterised in that the genetic engineering bacterium is with Pichia pastoris, big Enterobacteria or bacillus megaterium are that host builds what is obtained.

3. genetic engineering bacterium according to claim 2, it is characterised in that the Pichia pastoris be GS115, KM71 or SMD1168；The Escherichia coli are BL21, Rosctta 2, Origami 2, Rosctta-gami 2 or Origami B；It is described Bacillus megaterium is WH320, MS941 or YYBm1.

4. it is a kind of expression thermophilic esterase recombinant vector, it is characterised in that the recombinant vector contain amino acid sequence for example 1) or 2) gene shown in：

1) amino acid sequence is the sequence shown in SEQ ID NO.2；Or

2) amino acid sequence is that the disappearance of Jing amino acid, replacement, insertion or mutation are formed and had in the sequence basis for 1) limiting There is the sequence for encoding activated esterase.

5. recombinant vector according to claim 4, it is characterised in that the recombinant vector be in pPIC9, pPIC3K, PPIC9K, PAO815, pPICZ α, pET 28T, MBP3, pBR series, pUC series, pAT153 carriers, pHIS1525, Build what is obtained on the basis of pHIS1522, pSTREP1525, pSTREPHIS1525 or pSTOP 1622.

6. a kind of method for producing thermophilic esterase, it is characterised in that methods described is thermophilic using the expression described in claim 4 Expression described in the genetic engineering bacterium or claim 1 of the expression thermophilic esterase described in the recombinant vector of esterase, claim 1 The transgenic cell line of thermophilic esterase.

7. the gene of the recombinant vector for expressing thermophilic esterase described in claim 4, the expression thermophilic esterase described in claim 1 The application of the transgenic cell line of the expression thermophilic esterase described in engineering bacteria or claim 1.

8. application according to claim 7, it is characterised in that the application is to be applied to food processing, living things catalysis, system Standby medicine, leather manufacture, animal feed, cosmetics or field of sewage treatment.

9. it is a kind of hydrolysis p-nitrophenyl phenols or glycerolipid substrate method, it is characterised in that methods described be expression ammonia 1) or 2) gene of the base acid sequence shown in, is hydrolyzed by catalyst of gene expression product；Wherein gene：

1) amino acid sequence is the sequence shown in SEQ ID NO.2；Or

2) amino acid sequence is that the disappearance of Jing amino acid, replacement, insertion or mutation are formed and had in the sequence basis for 1) limiting There is the sequence for encoding activated esterase.

10. method according to claim 9, it is characterised in that the p-nitrophenyl phenols substrate is p-NPC₄、p-NPC₅、 p-NPC₈、p-NPC₁₂、p-NPC₁₄Or p-NPC₁₆；The glycerolipid substrate be triacetic acid glyceride, tributyrin, Tricaprylin, three certain herbaceous plants with big flowers acid glycerides, three sour nutmeg glyceride, tripalmitin or olive oil.