CN106635941B

CN106635941B - A kind of thermophilic esterase and its functional verification from Aquifex aeolicus bacterial strain

Info

Publication number: CN106635941B
Application number: CN201610972926.6A
Authority: CN
Inventors: 喻晓蔚; 徐岩
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2016-11-07
Filing date: 2016-11-07
Publication date: 2019-08-20
Anticipated expiration: 2036-11-07
Also published as: CN106635941A

Abstract

The invention discloses a kind of thermophilic esterase from Aquifex aeolicus bacterial strain and its functional verifications, belong to technical field of bioengineering.Present invention finds a kind of new thermophilic esterase genes, construct 3 kinds of expression systems, realize high efficient expression and study zymologic property.Eukaryotic expression system building: expression vector establishment preferred vector pPIC9K, and Pichia pastoris host, preferably GS115 are converted, realize high efficient expression；The building of protokaryon escherichia expression system: expression vector establishment preferred vector MBP3, and escherichia coli host is converted, preferably BL21 and Origami2 realize high efficient expression；Protokaryon bacillus megaterium expression system construction: expression vector establishment preferred vector pHIS1525, and bacillus megaterium host, preferably YYBm1 are converted, realize high efficient expression.Gained recombinase has many advantages, such as esterase active and thermophilic and thermal stability, has great potentiality in industrial application under the high temperature conditions.

Description

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

Technical field

The present invention relates to a kind of thermophilic esterase from Aquifex aeolicus bacterial strain and its functional verifications, belong to life Object field of engineering technology.

Background technique

Extreme microorganism is to be suitble to live in the general name of the microorganism in extreme environment, including thermophilic, thermophilic cold, acidophilus, thermophilic The multiple types such as alkali, piezophilic, thermophilic salt, thermophilic metal ion, anti-radiation, resistance to drying and extreme anaerobism.Thermophilic Bacteria (high temperature bacterium, also referred to as Thermophilic microorganism), it is a kind of microorganism lived in 50 DEG C or more hot environments.There is temperature in the source of common thermophilic microorganism Spring, volcanic crater, marine sediment, wastewater and waste materials, hot soil etc..In the past 30 years, in the boiling point of water and boiling point temperatures above Under the conditions of after the bacterium that can live is found, more promote the research to thermophilic microorganism.In fermentation industry, it can use thermophilic Hot bacterium characteristic resistant to high temperature 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 lower 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 polymerase-Taq enzyme applied to modern biotechnology.Hereafter, many Zimadzhunt L 340s are constantly sent out from Thermophilic Bacteria It now and is used to produce, 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: first is that the substrate of effect is different: esterase is to be catalyzed the ester-type hydrolysis enzyme containing short chain fatty acids, and lipase is catalysis rouge containing long-chain The fat (triacylglycerol) of fat acid is hydrolyzed to the enzyme of glycerol and fatty acid；Second is that the physical state of substrate specificity is different: esterase Water soluble substrate is hydrolyzed, reaction system is mostly in oil-water interfaces.Lipase in outphasing system (i.e. oil-water interfaces) or can only have It is acted in machine phase, hydrolyzes fat-soluble substrate；Third is that the activation effect at interface: lipase is higher in water-oil interface catalytic activity, Catalytic activity is low in single solution.All lipid hydrolyzing enzyme primary structures are 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 interfacial contact, α- Spiral is opened.3) generally believe that its active site is made of 3 catalytic residues: nucleophilic residues (Ser, Cyc, Asp), catalytic residue (Asp, Glu), His residue.

In recent years, more and more about the research of thermostable esterases/lipase, 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. Race, wherein Zimadzhunt L 340 belongs to the 4th family (HSL family) and the 5th family (thermophilic enzyme family) more.Currently, studied both at home and abroad Mainly have about thermophilic esterase/fat enzyme source thermophilus strain: Fervidobacterium belongs to, and Thermotoga belongs to, Aureobasidium belongs to, and Pseudomonas belongs to, and Geobacillus belongs to, and Sulfolobus belongs to etc..Esterase is as a kind of efficient Biocatalyst, since thermophilic esterase has pyroreaction activity and good thermal stability, and to organic solvent, detergent With the stronger resistance of denaturant, keep its a very wide range of applied to food processing, medical industry, leather manufacture, animal feed, makeup The industries such as conduct industry and waste water control.

With the progress of science and technology, the scientists gradually isolated micro- life of hyperthermophilic from some special high-heat environments Object, also to make people further go deep into the understanding of Zimadzhunt L 340.However, most of Thermophilic Bacteria speeds of growth are slower, item is cultivated Part is stringent, therefore extremely difficult to obtain a large amount of Zimadzhunt L 340s by culture wild mushroom.However, with the hair of technique for gene engineering Exhibition, people start the target gene found in Thermophilic Bacteria to express in some common easy culture hosts, thus mild Under the conditions of obtain a large amount of Zimadzhunt L 340.Currently, having some thermophilic esterase genes realizes the successful expression in Escherichia coli.But In industrialized production and application, it is contemplated that the processing of enzyme preparation downstream separation and safety problem, the selection of heterogenous expression host is especially It is important.Pichia pastoris and bacillus megaterium are all food-grade expressive hosts, and can be with exocytosis recombinant protein.

It is still in the primary stage at present to the research of Zimadzhunt L 340, finds that novel Zimadzhunt L 340 is current from hyperthermophilic microorganism The main method of novel enzyme preparation.In addition, facing the extensive use of thermophilic esterase, finding novel thermophilic esterase and selecting suitably Expressive host is particularly important.

Summary of the invention

To solve the above-mentioned problems, present invention finds a kind of new thermophilic esters from Aquifex aeolicus Enzyme.The present invention demonstrates gene function by realizing the heterogenous expression of newfound esterase gene.

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

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

2) missing on the basis of amino acid sequence 1) limited through amino acid, substitution, insertion or mutation form and have Encode the amino acid sequence of active 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, is found in the hot spring of American National Yellowstone, with H₂/CO₂/O₂And other inorganic substances are Somatomedin, maximum growth temperature belong to several most one of the thermoduric bacterias being currently known at 95 DEG C.

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

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

2) amino acid sequence missing through amino acid, substitution, insertion or is mutated in the sequence basis 1) limited And there is the sequence for encoding active esterase.

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

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

4) missing on the basis of nucleotide sequence 3) limited through base, substitution, insertion or mutation form and have and compile The nucleotide sequence of the active esterase of code.

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

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

In one embodiment of the invention, the genetic engineering bacterium is Pichia yeast engineering, and first building recombination is finished Red Yeast expression carrier is that host expresses with Pichia pastoris；The restructured Pichia pastoris in expression carrier can be Building obtains 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 recombination of pPIC9K Female expression vector is what host constructed with 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 building recombination is big Enterobacteria expression vector is that host expresses with Escherichia coli；The expression of recombinant e. coli carrier can be Building obtains on the basis of pET28T, MBP3, pBR series, pUC series or pAT153 carrier etc..

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

In one embodiment of the invention, the nucleotides sequence of the colibacillus engineering expression is listed in SEQ ID It is slightly modified on the basis of NO:6, (eliminate the 678th 's to the 705th on SEQ ID NO:6 without containing histidine tag 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 building weight Group bacillus megaterium expression vector is that host expresses with bacillus megaterium；The recombination bacillus megaterium expression Carrier can be structure on the basis ofs pHIS1525, pHIS1522, pSTREP1525, pSTREPHIS1525, pSTOP 1622 etc. It builds.

In one embodiment of the invention, the genetic engineering bacterium is huge using pHIS1525 as vector construction recombination Bacillus expression vector is what host constructed with 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 third problem be to provide a kind of recombinant vector for expressing thermophilic esterase, the recombinant vector contains 1) or 2) there is amino acid sequence gene as shown in:

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

In one embodiment of the invention, the recombinant vector is in pPIC9, pPIC3K, pPIC9K, PAO815 Or building obtains on the basis of pPICZ α etc..

In one embodiment of the invention, the recombinant vector is in pET 28T, MBP3, pBR series, pUC system Building obtains on the basis of column or pAT153 carrier etc..

In one embodiment of the invention, the recombinant vector be with pHIS1525, pHIS1522, Building obtains on the basis of pSTREP1525, pSTREPHIS1525 or pSTOP 1622 etc..

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

In one embodiment of the invention, the method is that the sequence of SEQ ID NO.6 is connected to pPIC9K to obtain To recombinant expression carrier pPIC9K-Aaeo2, then by recombinant expression carrier electrotransformation into Pichia pastoris GS115, screening is obtained Positive transformant is recombinant yeast pichia pastoris engineering bacteria；Obtained positive restructuring Pichia pastoris GS115/pPIC9K-Aaeo2 is existed 30 DEG C, under the conditions of 200rpm culture to OD₆₀₀=2.0-6.0, then thallus is transferred in fresh culture by centrifugation, 28 DEG C, Final concentration of 1.0% methanol, inducing expression 30-120h are cultivated and are added under the conditions of 200rpm.

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

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

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

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

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 glycerolipid, 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 esterases, and recombinate and express the thermophilic esterase.The present invention obtains 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 influence recombination enzyme stability, metal ion is to recombination enzyme stability shadow It rings, surfactant influences recombination enzyme stability, inhibitor influences recombination enzyme stability.The result shows that recombinase Aaeo2 Most suitable substrate is p-NPC₄, optimum temperature and pH are 90 DEG C and 8.0, and have good thermal stability, to metal ion There is certain tolerance with organic solvent etc..

Detailed description of the invention

Fig. 1 is the chadogram structure figures 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 figure of the embodiment of the present invention 2；

Fig. 3 is the thermophilic ester 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 structure diagram；

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

Fig. 5 is the pPIC9K-Aaeo2 recombinase SDS-PAGE electrophoresis result figure 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 purifying recombinase albumin A aeo2；

Fig. 6 is the A.aeolicus recombination thermophilic esterase Aaeo2 substrate specificity analysis of purifying；

Fig. 7 is A.aeolicus recombination thermophilic esterase Aaeo2 optimum temperature (Fig. 7 A) and temperature stability of purifying (Fig. 7 B) analysis；

Fig. 8 is the Tm value analysis of the A.aeolicus recombination thermophilic esterase Aaeo2 of purifying；

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

Specific embodiment

Term as used in the present invention generally has those of ordinary skill in the art usual unless otherwise specified The meaning of understanding.

Below with reference to specific preparation embodiment and Application Example, and this hair is described in further detail referring to data It is bright.It should be understood that these embodiments are of the invention solely for the purpose of illustration, rather than limit the scope of the invention in any way.

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

Embodiment 1:A.aeolicus recombinates acquisition and the gene sequencing of thermophilic esterase gene

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 hypothesis protein sequences of gene group selection of A.aeolicus bacterial strain, in SWISS-PROT data Blast analysis is carried out to it in library, obtain Aaeo1 sequence and derives from a kind of methyl of Nitrosomonas europaea Ester esterase (Reference sequence:Q82SL8.2) has 27% similitude.Aaeo2 sequence 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, carry out sequence alignment point with two kinds of thermophilic esterases of A.aeolicus Analysis, esterase/lipase known to 5 kinds be respectively the source Sulfolobus solfataricus esterase (GenBank NO.: CCQ48704), the lipolytic enzyme (GenBank NO.:AAC67392) in the source S.acidocaldarius, The carboxylesterase (GenBank NO.:WP_010879825) in the source Archaeoglobusfulgidus, The lipase (GenBank NO.:EF138832) in the source F.changbaicum and the source Thermotoga maritima lipase(GenBank NO.:AAD36421).As a result, it has been found that two kinds of hypothesis albumen of A.aeolicus are protected with typical esterase Defending zone domain and key enzyme activity site, in addition to this, there are also the presence in other same region.The key enzyme activity site of Aaeo1 It is conservative region for Ser62, Asp163, His190, sequence Gly60-Gly64.The key enzyme activity site of Aaeo2 be Ser82, Asp185, His212, sequence Gly80-Gly84 are conservative region.

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

Wherein, the amino acid sequence of Aaeo1 is as shown in SEQ ID NO:1, and nucleotide sequence is as shown in SEQ ID NO:3.

The amino acid sequence of Aaeo2 is as shown in SEQ ID NO:2, and nucleotide sequence is as shown in SEQ ID NO:4.

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

A kind of thermophilic esterase Gene A aeo1 length from A.aeolicus is 621bp, encodes 207 amino acid, point Son amount size is 23.4kDa, and the nucleic acid sequence and protein sequence of the albumen are shown in SEQ ID NO:3 and SEQ ID NO:1.It is another Thermophilic esterase Gene A aeo2 length is 678bp, encodes 226 amino acid, molecular size range 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 N-terminal of albumin A aeo2 without signal peptide there are a possibility that (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/) carries out homologous modeling, followed by Pymol Software analyzes (see Fig. 3) two kinds of homologous modeling structures of thermophilic esterase of A.aeolicus.Enzymatic activity position as seen from Figure 3 Point (red-label), negative oxygen ion hole (blue markings) and cap structure (white marking).The template of Aaeo2 albumen homology modeling The wheat bran esterase of rumen bacteria Butyrivibrio Proteoclasticus is derived from for 2wtmA, 2wtmA.Aaeo2's Sequence homology similitude between model (Fig. 3) and template is 25%, catalytic triads Ser82, Asp185, His212, structure At two the residues Leu19 and His83 in negative oxygen ion hole.

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

One, the building of carrier for expression of eukaryon

Two kinds of thermophilic esterase genes of A.aeolicus are according to Pichia pastoris codon preference, in the C-terminal of DNA encoding frame 6xHis label is introduced, and introduces AvrII and NotI restriction enzyme site respectively in 5 ' and 3 ' two sides, is 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 figure (AvrII and NotI), M is 15000bp Marker.

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

1) screening of electrotransformation pichia methanolica and positive transformant

According to pichia yeast expression system operation manual, correct recombinant plasmid pPIC9K-Aaeo2 will be sequenced in restricted After enzyme cutting SacI linearisation, Electroporation Pichia pastoris GS115 is coated on screening target gene integration on MD plate (containing G418) Transformant onto recipient bacterium chromosome.Picking transformant extracts total DNA, using it as template, using 5 ' AOX and 3 ' AOX as primer PCR identification is carried out, is compared with empty carrier transformed bacteria, verifying purpose gene has been integrated into Yeast genome.

2) inducing expression of the recombination pPIC9K-Aaeo2 carrier in Pichia pastoris

Recombinant yeast pichia pastoris in 25mL BMGY culture medium (250mL triangular flask) 30 DEG C culture to OD be 2.0~6.0, from The heart collects thallus, is added to after 100mL BMMY culture medium in 28 DEG C, continues to cultivate under the conditions of 200rpm, every 12-24h is added 1.0% (v/v) methanol, timing sampling measure cell density and protein concentration, while carrying out the SDS-PAGE and enzyme activity of culture solution Measurement.Protein concentration uses Bradford method.

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

Protein purification refers to GE Healthcare guide, and SDS-PAGE analysis is according to " Molecular Cloning:A Laboratory guide " (the Three editions), the gel resolving gel concentration used is 12%, and concentration gum concentration is 5%, and applied sample amount is 5-25 μ L.Protein is with examining horse This brilliant blue R-250 dyeing.In Fig. 5 as can be seen that the single purifying protein Aaeo2 that land2 band is shown, molecular weight are 25kDa or so.

4) thermophilic esterase Enzyme activity assay

Thermophilic esterase enzyme activity determination method referring to Wang Lele paper (gene cloning of rhizopus chinensis lipase, expression, purifying and Zymologic property research, Southern Yangtze University, 2008): use p-NPC4 as substrate, pH8.0Tris-HCl buffer, in 70 DEG C of conditions Lower measurement enzymatic activity.The enzyme amount that enzyme activity is defined as generating 1 μm of ol p-nitrophenol under certain reaction condition per minute is an enzyme International unit living.

Enzyme assay is shown with esterase active.

4 active site rite-directed mutagenesis of embodiment further identifies two kinds of hypothesis thermophilic esterases of A.aeolicus

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

The PCR primer (sequence is as shown in SEQ ID NO:7~SEQ ID NO: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 measured, discovery enzymatic activity disappears, and illustrates these three positions Point also further demonstrates the two and assumes that albumen is esterase to the importance of recombinase.

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

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

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

Act on the activity determination method of glyceride type substrate referring to jade Song at paper (stearothermophilus enzyme molecular clone and Zymologic property research, Jilin University, 2008).Select triacetic acid glycerolipid, tributyrin, tricaprylin, three certain herbaceous plants with big flowers acid 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, measure esterase activity under the conditions of 70 DEG C, determine recombinase to triglycerin esters be substrate when most Suitable substrate.

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

Embodiment 6: A.aeolicus thermophilic esterase optimum temperature and the temperature stability analysis of purifying

Thermophilic esterase enzyme activity determination method referring to Wang Lele paper (gene cloning of rhizopus chinensis lipase, expression, purifying and Zymologic property research, Southern Yangtze University, 2008)

Optimum temperature and temperature stability analysis have been carried out to the recombination thermophilic esterase that purifying obtains.As shown in fig. 7, The result shows that with p-NPC₄When for substrate, the optimum temperature of recombinase Aaeo2 is 90 DEG C, is still retained in 70 and 80 DEG C of placement 2h 65% residual enzymic activities.In 90 DEG C of half-life period 2h.

Experimental result sufficiently shows that recombinating thermophilic esterase Aaeo1 and Aaeo2 all has the high temperature being satisfied in industrial production Condition.

Embodiment 7: the T of the A.aeolicus thermophilic esterase of purifying_mValue measurement

Using the method for circular dichroism spectra, the albumen thermal stability of measurement purifying recombinase, i.e. T_mIt is worth (melting temperature), dissolution The corresponding temperature of the maximum 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 is aobvious Show that curvilinear motion is most fast at 88 DEG C, gradually tends to balance in the temperature lower curve higher than 90 DEG C.Therefore T_mValue is 88 DEG C, this Sufficiently further demonstrate the thermal stability experiment of recombinase Aaeo2.

Embodiment 8: the most suitable action pH of the A.aeolicus thermophilic esterase of purifying and pH stability analysis

The recombination thermophilic esterase Aaeo2 obtained to purifying has carried out most suitable action pH and pH stability analysis such as Fig. 9 A institute Show, in pH8.0, opposite enzyme activity highest, therefore optimal reaction pH value is 8.0.The pH stability of recombinase Aaeo2 such as Fig. 9 B institute Show, measure stability of recombinase under the conditions of pH7.0-9.0 respectively, with the increase of standing time, opposite enzyme activity sharply under Drop, half-life period are followed successively by 54min, 50min and 46min.

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

Thermophilic esterase enzyme activity determination method referring to Wang Lele paper (gene cloning of rhizopus chinensis lipase, expression, purifying and Zymologic property research, Southern Yangtze University, 2008).

As can be seen from Table 1, its enzymatic activity can be inhibited for recombinase Aaeo2, EDTA, illustrate its for metal ion according to Rely property enzyme.For Aaeo2, low concentration Fe³⁺、Co²⁺、Mn²⁺Enzymatic activity, low concentration Ca can be improved²⁺Enzymatic activity is influenced less, And high concentration Ca²⁺There is slight inhibiting effect to recombinase.

Analysis of each metal ion species of table 1 to the A.aeolicus recombination thermophilic esterase stability of purifying

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

Analysis of the various organic solvents of table 2 to the A.aeolicus recombination thermophilic esterase stability of 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 inhibiting effect to recombinase.The organic solvent pair of low concentration Enzymatic activity influences little.

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

As can be seen from Table 3, surfactant has certain inhibiting effect to recombinase Aaeo2, with surfactant The increase of concentration, inhibiting effect enhancing, but low concentration influence enzymatic activity smaller.

Analysis of the various surfactants of table 3 to the A.aeolicus recombination thermophilic esterase stability of purifying

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

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

Analysis of the various inhibitor of table 4 to the A.aeolicus recombination thermophilic esterase stability of purifying

The building of embodiment 13:A.aeolicus thermophilic esterase coli expression carrier, recombinant expression and its albumen table It reaches

One, the building of coli expression carrier

1) design of primers: from the gene design primer after optimization, C-terminal 6xHis label (sequence such as SEQ ID is not included Shown in NO:19~SEQ ID NO:22)

g1:5’-GCTCATATGATGCTGAAGAACCCTTTGT-3’

g2:5’-GCTGGATCCTTAATACCCCTTTAAAATAGCTTT-3’

g3:5’-GCCCATATGATGTACTTCTACGATATTAACAA-3’

g4:5’-GGCGGATCCTTACAACAATTCCTCAATATATTTTTG-3’

2) PCR reacts, and using carrier pPIC9K-Aaeo1 as template, degenerate temperature is 50 DEG C by 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 initial denaturation；Denaturation, 98 DEG C, 30s, annealing, 50 DEG C (or 55 DEG C), 1min, renaturation, 72 DEG C, 45s, second step to the 4th step carries out 30 circulations；72℃,10min；10 DEG C of heat preservations.Electrophoresis detection pcr amplification product, is obtained The DNA fragmentation that size is 650 and 700bp or so is obtained, the gel purification kit for utilizing omega company to provide purifies target DNA Segment.

3) PCR product of NdeI and BamHI double digestion Aaeo1 and Aaeo2 and plasmid MBP3,37 DEG C of digestion 4h.It utilizes The gel plastic recovery kit recycling target DNA fragment and carrier that omega company provides

4) it connects

Take 1 μ l of T4DNA ligase, 1 μ l of 10x T4DNAligase Buffer, target gene+carrier (n₁:n₂=1: 3) 8 μ l is uniformly mixed, in 16 DEG C of connection 16h.

5) it converts

It takes 10 μ l connection products to be added in competent cell BL21 and Origami 2, is placed in 30min on ice, 42 DEG C of heat shocks It is immediately placed at after 90s on ice, after placing 2min, the not antibiotic LB culture medium of 1ml is added, 37 DEG C of shaking table culture 1h will turn The bacterium solution of change is applied on LB (Amp) plate, 37 DEG C of overnight incubations.Multiple bacterium colonies are picked them separately on plate is transferred to test tube, 37 DEG C culture 8h.

6) plasmid is extracted

Plasmid is extracted with the plasmid extraction kit of omega company, send to biotech firm and carries out sequencing identification.

Two, table of the recombinant expression carrier MBP3-Aaeo1 and MBP3-Aaeo2 in e. coli bl21 and Origami 2 It reaches

1) inducing expression of the recombinant vector in two kinds of hosts

Picking positive restructuring bacterial strain transformant is inoculated in 5mL LB liquid medium on LB plate (containing Amp) (containing Amp) In, it is incubated overnight 8h in 37 DEG C, 200rpm, obtains seed liquor.

Seed liquor is inoculated in 50-100mL LB culture medium (250mL triangular flask) with 1% inoculum concentration, 37 DEG C, 200rpm Culture to OD600 be 0.6 or so when, the IPTG of final concentration of 0.2mM is added, while being cooled to 17 DEG C of cultures, carries out recombination egg White inducing expression, culture 12h or so.

2) collection and purifying of recombinase

Thalline were collected by centrifugation, and thallus is resuspended using BufferA, carries out ultrasonication.The broken thallus of low-temperature centrifugation is molten Liquid collects supernatant and carries out protein purification.Purification process refers to GE Healthcare guide.First time protein purification obtains MBP-Aaeo1 and MBP-Aaeo2 fusion protein removes imidazoles by ultrafiltration, and appropriate purified TEV enzyme is added and carries out protease It cuts, 16 DEG C of digestion 16h, carries out second of protein purification, collection penetrates peak, the recombinant protein A aeo1 and Aaeo2 as purified. SDS-PAGE carries out electrophoretic analysis.

Respectively to host's extracellular component, periplasmic space component, cytoplasm fraction, whole-cell component, cell precipitation component into Row electrophoretic analysis, it is seen that recombinant protein is primarily present in cytoplasm fraction.Pichia pastoris protein expression quantity is 0.32mg/mL, E. coli protein expression quantity is 1.2mg/mL.Compared with Pichia pastoris, expression quantity 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 16h is placed, measures the enzymatic activity before and after the digestion of TEV enzyme, as the result is shown Origami2/MBP3-Aaeo2 recombinant bacterial strain table respectively The recombinase reached, specific enzyme activity power increases after digestion.

The building of two kinds of thermophilic esterase bacillus megaterium expression vectors of embodiment 14:A.aeolicus, recombinant expression and Its protein expression

One, the building of bacillus megaterium expression vector

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

G5:5 '-TAGAGATCTATATGCTGAAGAACCCTTTGTT-3’

G6:5 '-TCAGGATCCTTAGTGATGATGATGGTGGT-3’

G7:5 '-TAGAGATCTATATGTACTTCTACGACATCAACAA-3’

G8:5 '-TGCGGATCCTTAGTGGTGATGATGGTGGTG-3’

2) according to routine PCR reaction, g5 and g6 using carrier pPIC9K-Aaeo1 as template, degenerate temperature is 51 DEG C；G7 and For g8 using carrier pPIC9K-Aaeo2 as template, degenerate temperature is 57 DEG C.Electrophoresis detection pcr amplification product, acquisition size are 650 Hes The DNA fragmentation of 700bp or so purifies target DNA fragment using the gel purification kit that omega company provides.

3) PCR product of BglII and BamHI double digestion Aaeo1 and Aaeo2 and plasmid pHIS1525,37 DEG C of digestion 4h.Benefit The gel plastic recovery kit recycling target DNA fragment and carrier provided with omega company.

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

Two, 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 into bacillus megaterium YYBm1 protoplasm somatocyte using protoplast transformation, it is former The preparation of raw plastid and protoplast transformation method refer to " Bacillus megaterium Protein Production System " handbook.Screening obtains positive transformant on the LB plate containing tetracyclin resistance, extracts the identification of plasmid double digestion.

2) induction table of the recombinant vector pHIS1525-Aaeo1 and pHIS1525-Aaeo2 in bacillus megaterium YYBm1 It reaches

It is inoculated in 5mL LB liquid medium (containing Tet) from picking positive transformant in 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 liquid medium of 200mL (containing Tet), 37 DEG C, 220rpm shaken cultivation, recombinant bacterium culture grow to OD600 between 0.3-0.4, and 0.5% (w/v) (D)-xylose conduct is added Inducer still keeps 37 DEG C, 220rpm shake culture, and every 30-60min takes a sample, measures OD600nm and protein concentration, 6h terminates to cultivate after induction.

3) purifying of recombinase and SDS-PAGE analysis

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

4) enzyme assay

Enzyme assay is shown with esterase active.

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

Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with this skill The people of art can do various change and modification, therefore protection model of the invention without departing from the spirit and scope of the present invention Enclosing subject to the definition of the claims.

<110>Southern Yangtze University

<120>a kind of thermophilic esterase and its functional verification from Aquifex aeolicus bacterial strain

<160> 22

<170> PatentIn version 3.3

<210> 1

<211> 207

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

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

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

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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

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

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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

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

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

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

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

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

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

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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

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

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

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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

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

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

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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

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

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

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

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Leu Leu

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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

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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

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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

1. a kind of genetic engineering bacterium or transgenic cell line for expressing thermophilic esterase, which is characterized in that the genetic engineering bacterium 1) or 2) or transgenic cell line express amino acid sequence gene as shown in:

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

2) amino acid sequence is that missing, substitution, insertion or mutation in the sequence basis 1) limited through amino acid form and have There is the sequence for encoding active esterase.

2. genetic engineering bacterium according to claim 1, which is characterized in that the genetic engineering bacterium is with Pichia pastoris, greatly Enterobacteria or bacillus megaterium are what host constructed.

3. genetic engineering bacterium according to claim 2, which is characterized 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 express thermophilic esterase recombinant vector, which is characterized in that the recombinant vector contain amino acid sequence for example 1) or 2) gene shown in:

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

5. recombinant vector according to claim 4, which is characterized in that the recombinant vector be in pPIC9, pPIC3K, PPIC9K, PAO815, pPICZ α, pET 28T, MBP3, pBR series, pUC series, pAT153 carrier, pHIS1525, Building obtains on the basis of pHIS1522, pSTREP1525, pSTREPHIS1525 or pSTOP 1622.

6. a kind of method for producing thermophilic esterase, which is characterized in that the method is thermophilic using expression as claimed in claim 4 The recombinant vector of esterase, the genetic engineering bacterium of expression thermophilic esterase described in claim 1 or expression described in claim 1 The transgenic cell line of thermophilic esterase.

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

8. application according to claim 7, which is characterized in that the application is to be applied to food processing, biocatalysis, system Standby drug, leather manufacture, animal feed, cosmetics or field of sewage treatment.

9. a kind of method of hydrolysis p-nitrophenyl phenols or glycerolipid substrate, which is characterized in that the method is exploitation right Benefit require 4 described in expression thermophilic esterase recombinant vector, it is described in claim 1 expression thermophilic esterase genetic engineering bacterium or 1) or 2) transgenic cell line express amino acid sequence gene as shown in of person's expression thermophilic esterase described in claim 1, It is hydrolyzed by catalyst of gene expression product；Wherein gene:

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

10. according to the method described in claim 9, it is characterized 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 glycerolipid, tributyrin, Tricaprylin, three certain herbaceous plants with big flowers acid glycerides, three sour nutmeg glyceride, tripalmitin or olive oil.