CN103695361B - A kind of genetic engineering bacterium and construction process thereof producing proline aminopeptidase - Google Patents

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

A kind of genetically engineered bacteria producing proline aminopeptidase and its construction method

technical field

The invention relates to a genetically engineered bacterium for producing proline aminopeptidase and a construction method thereof, belonging to the research fields of genetic engineering and enzyme engineering.

Background technique

Aspergillus oryzae (Aspergillus oryzae) is an aerobic fungus, which belongs to the subphylum Deuteromycetes and the genus Aspergillus in taxonomy. It can secrete a large amount of enzymes. It is a very useful fermentation industrial bacterium and is widely used in the production of fermented food. In 2005, the whole genome of A.oryzaeRIB40 strain was sequenced (http://www.bio.nite.go.jp/dogan/project/view/AO). Based on the whole gene sequence, the researchers deduced 134 protease-like genes, of which 69 genes encode exopeptidases.

Exopeptidases can be divided into aminopeptidases and carboxypeptidases according to whether the end of the peptide chain they cut is amino-terminal or carboxyl-terminal. Among them, aminopeptidases (APs for short, EC3.4.11.) are a class of enzymes that hydrolyze amino acids one by one from the N-terminal sequence of the polypeptide chain. In the food industry, aminopeptidase is usually used in combination with protease, and is widely used in the production of condiments and cheese, debittering of protein hydrolyzate, deep hydrolysis of protein and preparation of peptides. With the development of food industry, the application of aminopeptidase presents more and more broad prospects.

The prolylaminopeptidase (PAP for short, EC3.4.11.5) expressed by recombinant Escherichia coli in the present invention specifically hydrolyzes the nitrogen-terminal proline residues of proteins and polypeptides, which makes proline Aminopeptidase plays an important role in the digestion of proline-rich peptides and proteins such as collagen and gelatin.

Contents of the invention

The technical problem to be solved by the present invention is to provide: a genetically engineered bacterium expressing proline aminopeptidase, which is derived from the correct coding proline aminopeptidase gene derived from A.oryzaeJN-412 (CGMCCNo.8474). The full-length cDNA sequence was introduced into E.coliBL21(DE3) to obtain genetically engineered bacteria. The recombinant proline aminopeptidase was prepared by fermentation of the constructed engineering bacteria, and the basic enzymatic properties of the recombinant proline aminopeptidase were studied.

The said A.oryzaeJN-412 (CGMCCNo.8474) was screened by the Japanese-style koji by our laboratory, and was deposited in the General Microorganism Center of China Committee for the Collection of Microorganisms on November 15, 2013. The preservation number is CGMCCNo. 8474, and the preservation address is No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

The full-length cDNA sequence of the gene encoding proline aminopeptidase is shown in SEQ ID NO.1.

The present invention also provides a method for constructing the above-mentioned genetically engineered bacteria, the specific method is as follows:

1) Extract high-quality total RNA from A.oryzaeJN-412 (CGMCCNo.8474), and use reverse transcription technology to synthesize double-stranded cDNA using total RNA as a template;

2) Design primers according to the specificity of the pap gene, clone the pap gene by PCR amplification, connect the pap gene and pMD19-T overnight at 16°C, transform the ligation product into E.coliDH5α, and obtain the recombinant plasmid pMD19-pap, Sent to Shanghai Sangong for sequencing;

3) The recombinant plasmid pMD19-pap and the empty pET-28a(+) were subjected to double enzyme digestion, and the digested products were recovered by gel. The gel-recovered product was ligated overnight at 16°C to transform E.coliBL21(DE3). Pick positive clones.

The specific technical solutions are:

1) Design primers according to the specificity of the pap gene, and use double-stranded cDNA as a template to clone the target gene; the PCR system is: 2 μL of 10 μM primers P1 and P2, 2 μL of template, 25 μL of PrimeSTARMax DNA polymerase, and 50 μL of double-distilled water; PCR Conditions: pre-denaturation at 98°C for 5 minutes; denaturation at 98°C for 10 s, annealing at 59°C for 5 s, extension at 72°C for 90 s, 38 cycles; extension at 72°C for 10 min; gel recovery of the PCR product of the target gene, adding "A" at the end, the reaction system is: Gel recovery DNA fragment 25 μL, 10×PCR buffer 5 μL, dNTPs 4 μL, double distilled water to make up 50 μL; reaction conditions: 72°C, 30min; DNA fragment after adding “A” was mixed with TVectorpMD19-T (Simple), ligated overnight at 16°C , transform E.coliDH5α, pick out 20 transformants on the LB plate containing kanamycin resistance, extract the recombinant plasmid and perform plasmid PCR and double enzyme digestion verification, and send it to Shanghai Sangong for sequencing. The recombinant plasmid is pMD19-pap;

2) The recombinant plasmid pMD19-pap and the empty pET-28a(+) were subjected to double enzyme digestion, and the digested products were recovered by gel. The products recovered from the gel were ligated overnight at 16°C, transformed into E.coliDH5α, and the transformants were picked on the LB plate containing kanamycin resistance, and the recombinant plasmid was extracted and sequenced after verification by PCR and double enzyme digestion. The recombinant plasmid was pET-28a (+)-pap; transform the positive clone into E.coliBL21(DE3), and pick a single colony on the LB plate containing kanamycin resistance for expression verification.

The invention provides a proline aminopeptidase-producing recombinant Escherichia coli capable of efficiently expressing proline aminopeptidase, the enzyme activity in the supernatant of the fermentation broth is 25.87U/mL, and the specific enzyme activity is 40.87U/mg. Provide the required protein for later research on the characteristics and structure of proline aminopeptidase.

Description of drawings

Fig. 1 Cloning result of Aspergillus oryzae proline aminopeptidase gene (1: target gene).

Figure 2 Results of single and double digestion of recombinant plasmid pMD19-pap (1: pMD19-pap, 2: pMD19, 3: target gene).

Fig. 3 Results of double digestion of recombinant plasmid pET-28a(+)-pap (1: pET-28a(+), 2: target gene).

Figure 4 SDS-PAGE results of recombinant E. coli expression (M: low molecular weight protein standard, 1: supernatant after inducing E.coliBL21(DE3)/pET-28a(+) cell disruption, 2: inducing E.coliBL21(DE3)/ pET-28a(+) cell pellet after disruption, 3: non-induced E.coliBL21(DE3)/pET-28a(+)-pap cell supernatant after disruption, 4: non-induced E.coliBL21(DE3)/pET-28a (+)-pap cell crushed pellet, 5: induced E.coliBL21(DE3)/pET-28a(+)-pap cell supernatant after crushed, 6: induced E.coliBL21(DE3)/pET-28a(+) -pap cells are broken and precipitated, 7: target protein).

Figure 5 Optimum reaction pH of recombinant proline aminopeptidase

Fig. 6 pH stability of recombinant proline aminopeptidase

Fig. 7 optimal reaction temperature of recombinant proline aminopeptidase

Fig. 8 thermal stability of recombinant proline aminopeptidase

detailed description:

Materials and Testing Methods

YPD medium: 1% yeast extract, 2% tryptone, 2% glucose, pH 6.0.

LB medium: 1% tryptone, 0.5% yeast extract, 1% sodium chloride, pH7.0.

Escherichia coli E.coliDH5α and E.coliBL21(DE3) were preserved in our laboratory, and A.oryzaeJN-412 (CGMCCNo.8474) was screened from soy sauce koji.

RNAisoPlus and cDNASynthesisKit (M-MLVVersion) were purchased from Bao Biological Engineering (Dalian) Co., Ltd.

pMD19T-simplevector, T4 DNA ligase and 10×T4 ligase Buffer were purchased from Treasure Bioengineering (Dalian) Co., Ltd., and pET-28a(+) was preserved in our laboratory.

Restriction enzymes NotΙ and BamHI and shared Buffer were purchased from Bao Bioengineering (Dalian) Co., Ltd.

Other raw materials and reagents are commercially available domestic or imported analytically pure products.

Proline aminopeptidase activity assay method: with L-proline-p-nitroaniline as the substrate, add enzyme solution and substrate 2.5mM diluted by a certain number of times in 50mM pH7.5 Tris-HCl buffer solution, 50 ℃ water bath for 10 min, and measure the absorbance at 405 nm. Enzyme activity definition: at 50°C, the amount of enzyme needed to decompose L-proline-p-nitroaniline for 1 minute to produce 1 μM p-nitroaniline is one enzyme activity unit (1U).

Example 1: Cloning of proline aminopeptidase gene and recombinant bacteria E.coliBL21(DE3)/pET-28a(+)-pap

Using the total RNA of A. oryzaeJN-412 (CGMCCNo.8474) as a template, double-stranded cDNA was synthesized according to the operating conditions provided by the reverse transcription kit.

Using the double-stranded cDNA synthesized above as a template, the proline aminopeptidase gene pap was cloned by PCR method, and connected with the vector pMD19T-simple to obtain the vector pMD19-pap, and the cloning vector was transformed into Escherichia coli competent cells E. coliDH5α, positive clones were selected and verified by sequencing, the base sequence of which is shown in SEQ ID NO.1.

Recombinant plasmid pMD19-pap and empty plasmid pET-28a(+) were double digested with BamHI and NotI and recovered by gel, and the recovered product was ligated overnight at 16°C. Transform Escherichia coli competent cells E.coliDH5α, pick positive clones, extract plasmids, double digest with BamHI and NotI for verification, and send to Shanghai Sangon for sequencing. Transform the constructed recombinant plasmid pET-28a(+)-pap into E.coliBL21(DE3), select positive clones, extract the plasmid, and double digest with BamHI and NotI to verify E.coliBL21(DE3)/pET-28a(+) -pap recombinant bacteria were successfully constructed.

Embodiment 2: Expression verification of recombinant bacteria

Pick a single colony in 5mL LB medium (kanamycin 50μg/mL), culture overnight at 37°C, 200rpm, and then transfer to 50mLLB medium (kanamycin 50μg/mL) at 1% inoculum 37 Cultivate at 200 rpm until the _OD600 value is between 0.8 and 1.0, add IPTG to a final concentration of 0.5 mM, culture at 25 °C and 200 rpm for 6 hours, centrifuge at 10,000 rpm for 3 minutes, discard the supernatant, and use 40 mL of 50 mM Tris-HCl pH 7.5 for cell sedimentation The buffer was resuspended, sonicated, and the supernatant collected by centrifugation for enzyme activity and SDS-PAGE verification. The measured enzyme activity of the supernatant was 25.87U/mL, and the specific enzyme activity was 40.87U/mg. The control group consisted of the BL21(DE3) strain containing the empty vector pET-28a(+) and the BL21(DE3) strain containing the pET-28a(+)-pap recombinant without IPTG induction, and the rest of the conditions were the same as above, and the SDS-PAGE results were shown in Figure 4.

Embodiment 3: Research on the Enzymatic Properties of Recombinant Proline Aminopeptidase

The pure enzyme liquid that the crude enzyme liquid in embodiment 2 obtains through purification, this enzyme liquid is used for enzymatic property research, it is found that:

1) Optimum reaction pH and pH stability

Enzyme activity was detected in four buffer systems (100mM citric acid buffer pH3-6; 100mM phosphate buffer pH6-8; 100mM Tris-HCl buffer pH7-9; 100mM glycine-sodium hydroxide buffer pH9-11) . The results are shown in Figure 6. It can be seen from the figure that the recombinant proline aminopeptidase exhibits the highest enzyme activity at 100mM Tris-HCl pH7.5.

Under three buffer systems (100mM citric acid buffer pH3-6; 100mM phosphate buffer pH6-8; 100mM glycine-sodium hydroxide buffer pH9-11), incubate the pure enzyme solution in the buffer system at 30°C 1h, the enzyme activity measured by the standard method is 100%. The results are shown in Figure 7. It can be seen from the figure that the enzyme has good stability between pH5-11, and the relative enzyme activity is above 80%.

2) Optimum reaction temperature and thermal stability

The enzyme activity was detected under the conditions of 30°C, 40°C, 50°C, 60°C and 70°C, and the highest enzyme activity was 100%. The results are shown in Figure 8. It can be seen from the figure that 60°C is the optimum reaction for the enzyme temperature.

Under the conditions of 30°C, 40°C, 50°C and 60°C, the pure enzyme solution was incubated for 1 hour, and the enzyme activity was detected by the standard method, and the pure enzyme solution incubated at 4°C was used as a control. When the enzyme is incubated at 50°C for 1 hour, the residual enzyme activity is less than 20%, and at 60°C for 1 hour, the enzyme completely loses its catalytic activity. However, it has good thermal stability at 30°C and 40°C, and the residual enzyme activity is above 80%.

Claims (2)

1. A genetically engineered bacterium producing proline aminopeptidase, which is obtained by introducing the mature proline aminopeptidase gene encoded by Aspergillus oryzae JN-412 with a preservation number of CGMCCNo.8474 into E.coliBL21 (DE3) Genetically engineered bacteria; The construction steps of the engineering bacteria are as follows: 1) Extract total RNA from the mycelium of Aspergillus oryzae JN-412 with the preservation number CGMCCNo.8474, and synthesize double-stranded cDNA according to the conditions recommended by the reverse transcription kit; 2) According to the specificity of the proline aminopeptidase gene, design primers to clone the proline aminopeptidase gene from the double-stranded cDNA, transform the proline aminopeptidase gene into E.coliDH5α, and obtain the recombinant plasmid pMD19 -pap; 3) Cloning the proline aminopeptidase gene obtained in step 2) into the vector pET-28a(+), transforming E.coliBL21(DE3), and obtaining genetically engineered bacteria; The specific steps of the construction method of the engineering bacteria are as follows: 1) According to the specificity of the proline aminopeptidase gene, design an upstream primer P1: 5'CGGGATCCATGGCTGCCAAACTAGTAGAC3'; a downstream primer P2: 5'ATAAGAATGCGGCCGCCTAATCAATAGAGTCG3', using double-stranded cDNA as a template , clone the target gene; PCR system: 10 μM primers P1 and P2 2 μL each, template 2 μL, PrimeSTARMax DNA polymerase 25 μL, double distilled water to make up 50 μL; PCR conditions: 98°C pre-denaturation for 5 minutes; 98°C denaturation for 10 seconds, 59°C annealing for 5 seconds , extended at 72°C for 90s, 38 cycles; extended at 72°C for 10min; the PCR product of the target gene was recovered by gel, and "A" was added to the end. The reaction system was: 25 μL of DNA fragment recovered by gel, 5 μL of 10×PCR buffer, 4 μL of dNTPs, and 50 μL of double distilled water ;Reaction conditions: 72°C, 30min; Mix the DNA fragment after adding "A" with TVectorpMD19-T, ligate overnight at 16°C, transform E.coliDH5α, pick 20 on the LB plate containing kanamycin resistance Transformants, extract the recombinant plasmid, carry out plasmid PCR and double enzyme digestion verification, and send it to Shanghai Sangong for sequencing. The recombinant plasmid is pMD19-pap; 2) The recombinant plasmid pMD19-pap and the empty pET-28a(+) were subjected to double enzyme digestion and gel recovery, and the gel recovery products were ligated overnight at 16°C, transformed into E.coliDH5α, and kanamycin-resistant Pick the transformant from the LB plate, extract the recombinant plasmid and carry out PCR and double-enzyme digestion verification and then sequence, the recombinant plasmid is pET-28a(+)-pap; transform the positive clone into E. Pick a single colony from the resistant LB plate for expression verification. 2. A method for fermentative production of proline aminopeptidase, characterized in that taking the genetically engineered bacterium as claimed in claim 1 as the production strain, picking a single bacterium colony in 5mLLB medium, wherein containing kanamycin 50 μg/mL , cultivated overnight at 37°C and 200rpm, and then transferred to 50mL LB medium at 1% inoculum size, which contained 50μg/mL of kanamycin. The final concentration is 0.5mM, cultured at 25°C and 200rpm for 6h, centrifuged at 10,000rpm for 3min, discarding the supernatant, resuspending the cell pellet with 40mL of 50mM Tris-HCl buffer at pH 7.5, ultrasonically crushing, and centrifuging to collect the supernatant to obtain proline ammonia peptidase.