CN103695361B - A kind of genetic engineering bacterium and construction process thereof producing proline aminopeptidase - Google Patents
A kind of genetic engineering bacterium and construction process thereof producing proline aminopeptidase Download PDFInfo
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Abstract
The invention discloses a kind of genetic engineering bacterium and the construction process thereof of producing proline aminopeptidase, does is belonging to technical field of bioengineering, to derive from aspergillus oryzae JN-412(CGMCC? No.8474) encoding mature proline aminopeptidase channel genes E.coli? the genetic engineering bacterium that BL21 (DE3) obtains.Said gene engineering bacteria energy high expression proline aminopeptidase, the enzyme in fermented liquid supernatant is lived as 25.87U/mL, lives as 40.87U/mg than enzyme, for the characteristic, structure etc. of later stage research proline aminopeptidase provide desirable proteins.
Description
Technical field
The present invention relates to a kind of genetic engineering bacterium and the construction process thereof of producing proline aminopeptidase, belong to genetically engineered and enzyme engineering research field.
Background technology
Aspergillus oryzae (Aspergillusoryzae) is a kind of aerobic fungi, and taxonomy belongs to Deuteromycotina, Aspergillus, can secrete a large amount of enzymes, is a kind of very useful fermentation industry bacterium, is widely used in production leavened food.2005, the full-length genome of A.oryzaeRIB40 bacterial strain checked order (http://www.bio.nite.go.jp/dogan/project/view/AO).Researchist, according to complete genome sequence, infers 134 kinds of protease-like genes, wherein has 69 genes encoding exopeptidases.
It is that aminoterminal or carboxyl terminal can be divided into aminopeptidase and carboxypeptidase that exopeptidase shears peptide chain end according to it.Wherein, aminopeptidase (aminopeptidases, be called for short APs, EC3.4.11.) is a class from the enzyme of the N end order of polypeptide chain hydrolysis amino acid one by one.In the food industry, aminopeptidase usually and proteolytic enzyme compound use, is widely used in the aspects such as the production of seasonings and cheese, the debitterize of protein hydrolyte, protein depth hydrolysis and polypeptide preparation.Along with the development of foodstuffs industry, the application of aminopeptidase presents more and more wide prospect.
Proline aminopeptidase (prolylaminopeptidase in the present invention expressed by recombination bacillus coli, be called for short PAP, EC3.4.11.5) the nitrogen end proline residue of protolysate and polypeptide specifically, this characteristic makes proline aminopeptidase play an important role in the peptide and protein (as collagen protein and gelatin) of digestion proline rich.
Summary of the invention
Technical problem to be solved by this invention is to provide: a kind of genetic engineering bacterium of expressing proline aminopeptidase, is to derive from A.oryzaeJN-412(CGMCCNo.8474) correct coding proline aminopeptidase gene full length cDNA sequence import E.coliBL21 (DE3) obtain genetic engineering bacterium.Utilize the engineering bacterium fermentation preparation restructuring proline aminopeptidase built, and the basic zymologic property of research restructuring proline aminopeptidase.
Described A.oryzaeJN-412(CGMCCNo.8474), screen in Japanese Cheng Qu by this research department, on November 15th, 2013, be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is CGMCCNo.8474, preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica.
The full length cDNA sequence of described coding proline aminopeptidase gene is as shown in SEQIDNO.1.
Present invention also offers a kind of construction process of said gene engineering bacteria, concrete grammar is as follows:
1) from A.oryzaeJN-412(CGMCCNo.8474) extract high-quality total serum IgE, and use reverse transcription technology to utilize total serum IgE for templated synthesis double-strand cDNA;
2) according to the specific designs primer of pap gene, and utilize the method for pcr amplification to clone to obtain pap gene, pap gene and pMD19-T being spent the night in 16 DEG C is connected, connection product conversion E.coliDH5 α, obtain recombinant plasmid pMD19-pap, serve Hai Shenggong order-checking;
3) recombinant plasmid pMD19-pap and unloaded pET-28a (+) is carried out double digestion respectively, and glue reclaims digestion products.Glue is reclaimed product in 16 DEG C of connections of spending the night, Transformed E .coliBL21 (DE3).Select positive colony.
Concrete technical scheme is:
1) according to the specific designs primer of pap gene, with double-strand cDNA for template, clone's goal gene; PCR system is: 10 μMs of primer P1 and P2 2 μ L, template 2 μ L, PrimeSTARMaxDNA polysaccharase 25 μ L, distilled water polishing 50 μ L; PCR condition: 98 DEG C of denaturation 5min; 98 DEG C of sex change 10s, 59 DEG C of annealing 5s, 72 DEG C extend 90s, 38 circulations; 10min is extended after 72 DEG C; Goal gene PCR primer glue reclaims, and end adds " A ", and reaction system is: glue reclaims DNA fragmentation 25 μ L, 10 × PCRbuffer5 μ L, dNTPs4 μ L, and distilled water supplies 50 μ L; Reaction conditions is: 72 DEG C, 30min; The DNA fragmentation after " A " will be added mix with TVectorpMD19-T (Simple), in 16 DEG C of connections of spending the night, Transformed E .coliDH5 α, 20 transformants are chosen at the LB flat board containing kalamycin resistance, also just plasmid PCR and double digestion serve Hai Shenggong order-checking after verifying, recombinant plasmid and pMD19-pap to extract recombinant plasmid;
2) recombinant plasmid pMD19-pap and unloaded pET-28a (+) is carried out double digestion respectively, and digestion products is carried out glue recovery.Glue reclaims product in 16 DEG C of connections of spending the night, Transformed E .coliDH5 α, at the dull and stereotyped picking transformant of the LB containing kalamycin resistance, extracts recombinant plasmid and just checks order after performing PCR and double digestion checking, recombinant plasmid and pET-28a (+)-pap; By positive colony Transformed E .coliBL21 (DE3), do at the LB dull and stereotyped picking list bacterium colony containing kalamycin resistance and express checking.
The invention provides a kind of recombination bacillus coli energy high expression proline aminopeptidase producing proline aminopeptidase, enzyme in fermented liquid supernatant is lived as 25.87U/mL, live as 40.87U/mg than enzyme, for the characteristic, structure etc. of later stage research proline aminopeptidase provide desirable proteins.
Accompanying drawing explanation
Fig. 1 aspergillus oryzae proline aminopeptidase gene clone result (1: goal gene).
Fig. 2 recombinant plasmid pMD19-pap single endonuclease digestion and double digestion result (1:pMD19-pap, 2:pMD19,3: goal gene).
Fig. 3 recombinant plasmid pET-28a (+)-pap double digestion result (1:pET-28a (+), 2: goal gene).
Fig. 4 expression of recombinant e. coli SDS-PAGE result (M: low molecular weight protein (LMWP) standard, 1: supernatant after induction E.coliBL21 (DE3)/pET-28a (+) cytoclasis, 2: induction E.coliBL21 (DE3)/pET-28a (+) cytoclasis postprecipitation, supernatant after 3: non-induced E.coliBL21 (DE3)/pET-28a (+)-pap cytoclasis, 4: non-induced E.coliBL21 (DE3)/pET-28a (+)-pap cytoclasis postprecipitation, 5: supernatant after induction E.coliBL21 (DE3)/pET-28a (+)-pap cytoclasis, 6: induction E.coliBL21 (DE3)/pET-28a (+)-pap cytoclasis postprecipitation, 7: target protein).
Fig. 5 recombinates proline aminopeptidase optimal reaction pH
Fig. 6 recombinates proline aminopeptidase pH stability
Fig. 7 recombinates proline aminopeptidase optimal reactive temperature
Fig. 8 recombinates proline aminopeptidase thermostability
Embodiment:
Material and detection method
YPD substratum: 1% yeast extract paste, 2% Tryptones, 2% glucose, pH6.0.
LB substratum: 1% Tryptones, 0.5% yeast extract, 1% sodium-chlor, pH7.0.
This research department's preservation of E. coli DH5 α and E.coliBL21 (DE3), A.oryzaeJN-412(CGMCCNo.8474) screen from soy sauce koji.
RNAisoPlus and cDNASynthesisKit (M-MLVVersion) is purchased from precious biotechnology (Dalian) company limited.
PMD19T-simplevector, T4DNA ligase enzyme and 10 × T4 ligase enzyme Buffer are purchased from precious biotechnology (Dalian) company limited, and this research department of pET-28a (+) preserves.
Restriction enzyme Not Ι and BamH Ι and shared Buffer is purchased from precious biotechnology (Dalian) company limited.
Other raw materials and reagent are commercially available domestic or Import Analysis straight product.
Proline aminopeptidase activity determination method: with L-PROLINE-p-Nitroaniline for substrate, in the Tris-HCl damping fluid of 50mMpH7.5, add enzyme liquid and the substrate 2.5mM of dilution certain multiple, 50 DEG C of water-bath 10min, measure absorbancy under 405nm.Enzyme is lived and is defined: at 50 DEG C, and it is a Ge Meihuo unit (1U) that 1min decomposes L-PROLINE-p-Nitroaniline enzyme amount produced needed for 1 μM of p-Nitroaniline.
Embodiment 1: the clone of proline aminopeptidase gene and recombinant bacterium E.coliBL21 (DE3)/pET-28a (+)-pap
With A.oryzaeJN-412(CGMCCNo.8474) total serum IgE be template, according to the operational condition synthetic double chain cDNA that Reverse Transcription box provides.
With the double-strand cDNA of above-mentioned synthesis for template, the method of PCR clone is used to obtain proline aminopeptidase gene pap, and be connected with carrier pMD19T-simple and obtain carrier pMD19-pap, cloning vector is transformed into competent escherichia coli cell E.coliDH5 α, select positive colony, sequence verification, its base sequence: as shown in SEQIDNO.1.
Carry out double digestion and glue recovery with BamH Ι and Not Ι to recombinant plasmid pMD19-pap and empty plasmid pET-28a (+), glue reclaims product in 16 DEG C of connections of spending the night.Transformation of E. coli competent cell E.coliDH5 α, picking positive colony, upgrading grain, with BamH Ι and the checking of Not Ι double digestion, serves Hai Shenggong order-checking.By recombinant plasmid pET-28a (+)-pap Transformed E .coliBL21 (DE3) built, select positive colony, upgrading grain, successfully constructs with BamH Ι and Not Ι double digestion checking E.coliBL21 (DE3)/pET-28a (+)-pap recombinant bacterium.
Embodiment 2: the expression checking of recombinant bacterium
Picking list bacterium colony in 5mLLB substratum (kantlex 50 μ g/mL), 37 DEG C, 200rpm incubated overnight, then by 1% inoculum size to be forwarded in 50mLLB substratum (kantlex 50 μ g/mL) 37 DEG C, 200rpm is cultured to OD
600value, between 0.8 ~ 1.0, adds IPTG to final concentration 0.5mM, 25 DEG C, 200rpm cultivates after 6h, and 10, the centrifugal 3min of 000rpm, abandons supernatant, the Tris-HCl damping fluid Eddy diffusion of cell precipitation 40mL50mMpH7.5, ultrasonication, collected by centrifugation supernatant, carries out enzyme and lives and SDS-PAGE checking.The enzyme recording supernatant is lived as 25.87U/mL, lives as 40.87U/mg than enzyme.Control group with BL21 (DE3) bacterial strain containing empty carrier pET-28a (+) and do not add IPTG induction containing pET-28a (+)-pap recon BL21 (DE3) bacterial strain, all the other conditions are the same, and SDS-PAGE the results are shown in Figure 4.
Embodiment 3: the zymologic property research of restructuring proline aminopeptidase
The pure enzyme liquid of the purified acquisition of the crude enzyme liquid in embodiment 2, this enzyme liquid is used for zymologic property research, found that:
1) optimal reaction pH and pH stability
At four kinds of buffer solution systems (100mM citrate buffer solution pH3-6; 100mM phosphate buffered saline buffer pH6-8; 100mMTris-HCl pH of buffer 7-9; 100mM Glycine-NaOH pH of buffer 9-11) under detect enzyme live.The results are shown in Figure 6, upper as can be seen from figure, restructuring proline aminopeptidase shows the highest enzyme and lives when 100mMTris-HClpH7.5.
(100mM citrate buffer solution pH3-6 under three kinds of buffer solution systems; 100mM phosphate buffered saline buffer pH6-8; 100mM Glycine-NaOH pH of buffer 9-11), by pure enzyme liquid 30 DEG C of insulation 1h in buffer system, the enzyme work that standard method records is 100%.The results are shown in Figure 7, as can be seen from the figure, this enzyme has good stability between pH5-11, and relative enzyme is lived all more than 80%.
2) optimal reactive temperature and thermostability
Under 30 DEG C, 40 DEG C, 50 DEG C, 60 DEG C and 70 DEG C of conditions, detect enzyme live, the enzyme soprano that lives is 100%, the results are shown in Figure 8, upper as can be seen from figure, and 60 DEG C is the optimal reactive temperature of this enzyme.
Under 30 DEG C, 40 DEG C, 50 DEG C and 60 DEG C of conditions, pure enzyme liquid insulation 1h, standard method detects enzyme and lives, with the pure enzyme liquid of 4 DEG C of insulations for contrast.This enzyme is at 50 DEG C of insulation 1h, and residual enzyme work is lower than 20%, and at 60 DEG C of insulation 1h, this enzyme completely loses catalytic activity.And having good thermostability 30 DEG C and 40 DEG C, residual enzyme work is more than 80%.
Claims (2)
1. producing a genetic engineering bacterium for proline aminopeptidase, is be the genetic engineering bacterium that encoding mature proline aminopeptidase channel genes E.coliBL21 (DE3) of the aspergillus oryzae JN-412 of CGMCCNo.8474 obtains by deriving from deposit number;
The construction step of described engineering bacteria is as follows: be 1) extract total serum IgE the aspergillus oryzae JN-412 mycelium of CGMCCNo.8474 from deposit number, and recommends condition synthetic double chain cDNA according to Reverse Transcription box;
2) according to the specificity of proline aminopeptidase gene, design primer clones proline aminopeptidase gene from double-strand cDNA, by described proline aminopeptidase gene transformation E.coliDH5 α, obtains recombinant plasmid pMD19-pap;
3) by step 2) the proline aminopeptidase gene clone that obtains to carrier pET-28a (+), Transformed E .coliBL21 (DE3), obtain genetic engineering bacterium;
The construction process concrete steps of described engineering bacteria are as follows: 1) according to the specificity of proline aminopeptidase gene, design upstream primer P1:5'CGGGATCCATGGCTGCCAAACTAGTAGAC3'; Downstream primer P2:5'ATAAGAATGCGGCCGCCTAATCAATAGAGTCG3', with double-strand cDNA for template, clone's goal gene; PCR system is: 10 μMs of each 2 μ L of primer P1 and P2, template 2 μ L, PrimeSTARMaxDNA polysaccharase 25 μ L, distilled water polishing 50 μ L; PCR condition: 98 DEG C of denaturation 5min; 98 DEG C of sex change 10s, 59 DEG C of annealing 5s, 72 DEG C extend 90s, 38 circulations; 10min is extended after 72 DEG C; Goal gene PCR primer glue reclaims, and end adds " A ", and reaction system is: glue reclaims DNA fragmentation 25 μ L, 10 × PCRbuffer5 μ L, dNTPs4 μ L, and distilled water supplies 50 μ L; Reaction conditions is: 72 DEG C, 30min; The DNA fragmentation that will add after " A " mixes with TVectorpMD19-T, in 16 DEG C of connections of spending the night, Transformed E .coliDH5 α, at the dull and stereotyped picking of the LB containing kalamycin resistance 20 transformants, extraction recombinant plasmid also serves Hai Shenggong order-checking, recombinant plasmid and pMD19-pap after carrying out plasmid PCR and double digestion checking;
2) recombinant plasmid pMD19-pap and unloaded pET-28a (+) carried out double digestion respectively and cut glue recovery, glue reclaims product in 16 DEG C of connections of spending the night, Transformed E .coliDH5 α, at the dull and stereotyped picking transformant of LB containing kalamycin resistance, extract recombinant plasmid go forward side by side performing PCR and double digestion checking after order-checking, recombinant plasmid and pET-28a (+)-pap; By positive colony Transformed E .coliBL21 (DE3), do at the LB dull and stereotyped picking list bacterium colony containing kalamycin resistance and express checking.
2. the method for a fermentative production proline aminopeptidase, it is characterized in that with genetic engineering bacterium described in claim 1 for producing bacterial strain, picking list bacterium colony is in 5mLLB substratum, wherein containing kantlex 50 μ g/mL, 37 DEG C, 200rpm incubated overnight, 50mLLB substratum is forwarded to again by the inoculum size of 1%, wherein containing in kantlex 50 μ g/mL 37 DEG C, 200rpm is cultured to OD600 value between 0.8 ~ 1.0, add IPTG to final concentration 0.5mM, 25 DEG C, after 200rpm cultivates 6h, 10, the centrifugal 3min of 000rpm, abandon supernatant, the Tris-HCl damping fluid Eddy diffusion of cell precipitation 40mL50mMpH7.5, ultrasonication, collected by centrifugation supernatant, obtain proline aminopeptidase.
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