CN103275882B - Gene engineering bacteria highly expressing transglutaminase, and applications thereof - Google Patents

Abstract

The present invention discloses gene engineering bacteria highly expressing transglutaminase, and applications thereof, wherein a cleavage site Lys-Arg of yeast endogenous Kex2 protease is introduced between a proenzyme region gene and a maturase region gene of the expressed streptomyces hygroscopicus CCTCC M203062 transglutaminase proenzyme, and the proenzyme is expressed in Yarrowia lipolytica by using the expression vector. According to the present invention, the gene engineering bacteria is adopted to carry out fermentation to produce transglutaminase so as to achieve independent activation of the transglutaminase without an exogenous protease effect, effectively reduce production cost, and simplify production steps.

Description

A kind of genetic engineering bacterium of high expression Transglutaminase EC2.3.2.13 and application thereof

Technical field

The present invention relates to a kind of genetic engineering bacterium and application thereof of high expression Transglutaminase EC2.3.2.13, belong to technical field of enzyme engineering.

Technical background

Microorganism paddy ammonia phthalein amine transaminase (TransglutaminaseEC2.3.2.13 full name R-glutaminyl-peptide:amine-γ-glutayle-transferase is called for short TGase); can the γ-carboxamide groups of catalytic proteins peptide chain GLN residue and various acyl acceptor generation amide group shift reaction; form ε-(γ-glutamyl) Methionin covalent linkage, to cause in protein molecule, to be intermolecularly cross-linked, the hydrolysis of paddy ammonia phthalein amido in connection between protein and amino acid and protein molecule.At present, streptomycete TGase is widely used in foodstuffs industry, and appropriate interpolation TGase significantly can improve functional property and the nutritive value of varieties of food items protein, and range of application comprises meat product, fishery products, milk-product, plant protein preparation etc.In addition, TGase also has larger application prospect, as organizational project, textile industry and biological study etc. in some non-food industries fields.But, rely on the raising of the traditional zymotic Technique on T Gase output of dominant bacteria seed selection and process optimization very limited, far can not meet the need of market.

Streptomycete TGase is a kind of extracellular enzyme, usually with the secretion of the proenzyme of non-activity (pro-TGase) form, after the cut N-of pro-TGase holds proenzyme district, changes into active TGase.In secretion process, metalloprotease (TAMEP) and serine protease take part in the activation of TGase.In the TGase maturing enzyme process of research expression activity, run into all difficulties.And different from the expression of TGase maturing enzyme, efficient soluble-expression in the pro-TGase yeast of each Streptomyces and streptomycete, bar bacterium, intestinal bacteria.Pro-TGase needs after expressing just can change into active Tgase through proteolytic cleavage except behind proenzyme district, if directly can effectively reduce production cost by expression-secretion paddy ammonia phthalein amine transaminase, simplifies production stage.

Summary of the invention

The technical problem to be solved in the present invention be to provide a kind of can the genetic engineering bacterium of high expression Transglutaminase EC2.3.2.13, its Transglutaminase EC2.3.2.13 can autonomous active, does not need the effect of exogenous protease.

In early-stage Study, this research department is used for the Transglutaminase EC2.3.2.13 proenzyme (proTG) of Absorb Water streptomycete (Streptomyces hygroscopicus) CCTCCM203062, build recombinant expression vector pINA1297-proTG, and transformed Yarrowia lipolytica Yarrowia lipolytica WSH-Z06, obtain genetic engineering bacterium pINA1297-proTG/Y.lipolyticaWSH-Z06, this bacterial strain can express prochymosin gene.On this basis molecular modification is carried out to it, between proenzyme district gene and maturing enzyme district gene, add the cleavage site Lys-Arg of yeast entogenous Kex2 proteolytic enzyme, obtain the mutant strain that can produce Transglutaminase EC2.3.2.13 TGase maturing enzyme.

Described Transglutaminase EC2.3.2.13 proenzyme synthesizes according to Genebank:EU477523.

The preferred pINA1297 of described expression vector.

The present invention also provides a kind of and applies the method that said gene engineering bacterium fermentation produces Transglutaminase EC2.3.2.13, between streptomyces hygroscopicus CCTCC M203062 Transglutaminase EC2.3.2.13 proenzyme proenzyme district's gene and maturing enzyme district gene, introduced the cleavage site Lys-Arg of yeast entogenous Kex2 proteolytic enzyme by PCR method, improved proenzyme uses expression vector to express in Yarrowia lipolytica.

Concrete steps are as follows:

1) by chemical complete synthesis acquisition Transglutaminase EC2.3.2.13 prochymosin gene fragment;

2) the Transglutaminase EC2.3.2.13 prochymosin gene fragment of recovery and plasmid pINA1297 are carried out SfiI single endonuclease digestion respectively, then add KpnI enzyme to cut, endonuclease bamhi carries out post recovery, digested plasmid pINA1297 carries out glue recovery, the two connection is spent the night, transformation of E. coli competent cell, uses kalamycin resistance plate screening positive transformant, obtains plasmid pINA1297-proTG;

3) introduce cleavage site Lys-Arg according to the design of plasmid pINA1297-proTG gene order, corresponding nucleotides sequence is classified as PCR mutant primer Kex1 and Kex2 of AAGCGA; The Transglutaminase EC2.3.2.13 prochymosin gene fragment of transformation is cloned into again pINA1297 and obtains pINA1297-proKexTG;

4) recombinant plasmid pINA1297-proKexTG NotI linearizing empirical tests successfully constructed, glue recovery fragment is converted into Yarrowia lipolytica and obtains recombinant bacterium;

5) recombinant bacterium fermentative production Transglutaminase EC2.3.2.13 is utilized.

Described mutant primer Kex1 and Kex2 is:

Kex1:5 '- cGAgACGCTGCCGACGAGAGGG-3 ', dashed part is for introducing base sequence;

Kex2:5 '- cTTgGGGGCCCGGAAGAGCG-3 ', dashed part is for introducing base sequence.

Substratum used by the present invention:

LB substratum: Tryptones 10g/L, yeast powder 5g/L, NaCl10g/L, pH7.0;

YPD substratum: glucose 2.0g, peptone 2.0g, yeast powder 1.0g, be dissolved in 100.0ml distilled water, solid medium separately adds agar 2.0g.

YNB substratum YNB-N ₅₀₀₀(w/v): 0.17% yeast extract (not containing amino acid and vitriol), glucose 1.0%, ammonium sulfate 0.5%.The wherein independent sterilizing of glucose, then fully mixes.Solid medium needs the agar powder adding 2.0%.

PPB substratum (w/v): glucose 2.0%, yeast powder 0.132%, NH ₄cl0.132%, KH ₂pO ₄0.032%, MgSO ₄7H ₂o0.024%, VitB1 0.33mg/ml, being dissolved in final concentration is in the citrate-phosphate disodium hydrogen damping fluid (pH6.0) of 0.02M.

The activation of pro-TGase:

Get 100 μ L to mix with Proteinase K (20mg/mL) solution of 0.6 μ L containing the restructuring fermentation supernatant of pro-TGase, 37 ° of C constant temperature 15min.

The mensuration of GLN transaminase vigor of the present invention:

Colorimetric method for determining enzyme is lived: with N-α-CBZ-GLN-GLY for substrate specificity, and the single Hydroxylamine HCL of Pidolidone-γ does typical curve.1 unit Transglutaminase EC2.3.2.13 enzyme is lived and is defined as: during 37 ° of C, per minute catalysis forms the enzyme amount (U/mL) of the single Hydroxylamine HCL of 1 μm of ol Pidolidone-γ.

N α-the CBZ-GLN-GLY of reagent A: 100mg is dissolved in the NaOH solution of 2mL0.2moL/L, adds the Tris-HC damping fluid 4mL of 0.2mol/L pH6.0, the reduced glutathion 2mL of 0.1mol/L azanol 2mL, 0.01mol/L, and regulates pH to 6.0.

The HCL of reagent B:3mol/L, 12%TCA, 5%FeCL ₃mix by 1:1:1.

Pidolidone-γ-mono-hydroxamic acid standardized solution of preparation 0-4 μm of ol/mL.Get 1mL reagent A to mix with Pidolidone-γ-mono-hydroxamic acid standardized solution of 0.4mL different concns, 37 ° of C water-baths 10 minutes.Add 0.4mL reagent B termination reaction, in 525nm colorimetric, draw out typical curve.Replace standardized solution with 0.4mL through the enzyme liquid of suitably dilution, insulation and colorimetric under the same conditions, obtain enzyme from typical curve and live.The supernatant liquor after 10 minutes centrifugal is heated for blank with 100 ° of C.Enzyme activity (u/mL)=(6.8548 × OD ₅₂₅-0.0164) × extension rate

Provided by the invention can the genetic engineering bacterium of efficient high expression Transglutaminase EC2.3.2.13, its Transglutaminase EC2.3.2.13 can autonomous active, does not need the effect of exogenous protease, can effectively reduce production cost, simplify production stage.

Accompanying drawing explanation

The SDS-PAGE electrophoretic analysis of the fermented supernatant fluid of Fig. 1 recombinant bacterium Y.l-pINA1297-proKexTG

Swimming lane 1:Maker

Swimming lane 2: recombinant bacterium Y.l-pINA1297-proTG fermented supernatant fluid, in contrast

Swimming lane 3: recombinant bacterium Y.l-pINA1297-proTG fermented supernatant fluid activates 15min through Proteinase K, in contrast

Swimming lane 4: recombinant bacterium Y.l-pINA1297-proKexTG fermented supernatant fluid

Swimming lane 5: recombinant bacterium Y.l-pINA1297-proKexTG fermented supernatant fluid activates 15min through Proteinase K

Embodiment

The acquisition of embodiment 1proTG proenzyme

ProTG can increase and to publish an article from this laboratory carrier construction pET22b in early stage (+)/proTG() or according to Genebank:EU477523 relevant information synthetic.

Specifically, recombinant bacterial strain construction process is as follows:

(1) proTG design of primers

According to PCR primer P1 and the P2 of pET22b (+)/proTG gene order design proTG gene.

P1：5’-TTG GGCCGTTCTGGCCGCCAGCGGCGGCGACG-3’(SfiI)

P2:5’-CGC GGATCCTTACGACCAGCCCTGCTTCACCTC-3’(BamHI)

(2) structure of recombinant expression vector pINA1297-proTG

Utilize primer P1,2, with pET22b (+)/proTG plasmid for template, amplification obtains the PCR primer of proTG, reclaim test kit specification sheets according to the glue of Fermentas and reclaim PCR primer, the fragment be recovered to and plasmid pINA1297 are carried out SfiI single endonuclease digestion respectively, 50 DEG C, 1h, then add KpnI enzyme to cut, 37 DEG C, after 45min, endonuclease bamhi carries out post recovery, digested plasmid pINA1297 carries out glue recovery, the two is connected by a certain percentage and spends the night, Transformed E .coli JM109 competent cell, use kalamycin resistance plate screening positive transformant.Extract transformant plasmid, recombinant plasmid is through SfiI single endonuclease digestion, and 50 DEG C, 1h, then cut through KpnI enzyme, 37 DEG C, 45min, discharges the gene fragment that size is 5.2kb and 1.1kb, proves that construction of recombinant plasmid successfully, recombinant plasmid called after p INA1297-proTG.

Embodiment 2: the design of primers introducing the cleavage site Lys-Arg of Kex2 proteolytic enzyme

Introduce the corresponding nucleotides sequence of cleavage site Lys-Arg(according to the design of plasmid pINA1297-proTG gene order and be classified as AAGCGA) PCR mutant primer Kex1 and Kex2.

Kex1:5 '- cGAgACGCTGCCGACGAGAGGG-3 ' (dashed part is for introducing base sequence)

Kex2:5 '- cTTgGGGGCCCGGAAGAGCG-3 ' (dashed part is for introducing base sequence)

Embodiment 3: the amplification of mutator gene

Utilize primer Kex1 and Kex2, with plasmid pINA1297-proTG for template, amplification condition is: 95 DEG C of denaturations, 5min, a circulation; 95 DEG C of sex change, 30s, 65 DEG C of annealing, 30s, 72 DEG C of extensions, 6min35s, 34 circulations; 72 DEG C, 10min, a circulation; 12 DEG C, 10min, a circulation.PCR amplification system: 5 × PCR buffer(Mg ²⁺plus) 10 μ L, dNTP Mix4 μ L, template 1 μ L, each 0.1 μ L of upstream and downstream primer, the distilled water 35 μ L of sterilizing, Prime Star archaeal dna polymerase 0.5 μ L.Adopt gel to reclaim test kit and carry out purifying and recovery to PCR primer, the concentration of product is reclaimed in electrophoresis inspection.Reclaiming product leaves in the centrifuge tube of 1.5mL, and-20 DEG C of Refrigerator stores are for subsequent use.

Embodiment 3: the structure of recombinant expression vector pINA1297-proKexTG

The operation such as the fragment be recovered to Takara mutagenesis kit is carried out phosphorylation, connect, concrete steps are as follows: the fragment that 4.25ul reclaims mixed with 0.5ul10 × Blunting Kination Buffer, 0.25ul Blunting Kination Enzyme Mix, 37 DEG C of reaction 1h, 70 DEG C, 10min, then in mixed solution, add 5ul Ligation Solution I, 16 DEG C of connections are spent the night, Transformed E .coli JM109 competent cell, uses kalamycin resistance plate screening positive transformant.Extract transformant plasmid, serve the order-checking of Hai Shenggong order-checking portion, correct through sequence alignment.The success of proof construction of recombinant plasmid, recombinant plasmid called after pINA1297-proKexTG.

Embodiment 4: recombinant plasmid pINA1297-proKexTG transforms bacterial strain Y.lipolytica WSH-Z06

Recombinant plasmid pINA1297-proKexTG NotI linearizing empirical tests successfully constructed, glue reclaims fragment and is converted in bacterial strain Y.lipolytica WSH-Z06.Method for transformation is Li-acetate method.Step of converting is as follows:

(1). bacterial strain Y.lipolytica WSH-Z06 rules on YPD solid medium, cultivates 2d for 28 DEG C;

(2). choose a ring list bacterium colony and be dissolved in 1mlTE, 10000rpm, 1min, remove supernatant;

(3). add 600 μ l0.1mol/L pH6.0 Lithium Acetate LiAc, 28 DEG C of water-bath 1h, note not rocking sample; After water-bath terminates, under 3000rpm, centrifugal 2min, abandons supernatant;

(4). gently by thalline Eddy diffusion in the Lithium Acetate (pH6.0) of 80.0 μ l0.1mol/l;

So far, prepared by yeast competent cell.

(5). get the above-mentioned yeast competence of 40.0 μ l, add 2.0 μ l strand milt DNA and 3.0 μ l through the linearizing DNA fragmentation of NotI; 28 DEG C of water-bath 15min, note not rocking sample in water-bath process;

(6). add 350.0 μ l40%(w/v in above-mentioned sample) PEG4000(be dissolved in 0.1mol/l pH6.0 lithium acetate) with the DDT of 16.0 μ l1mol/l; 28 DEG C of water-bath 1h, note not rocking sample;

(7). dropwise add 40.0 μ l DMSO, mix gently, be then placed in 39 DEG C of water-bath heat-shocked 10min;

(8). in above-mentioned sample, add the Lithium Acetate (pH6.0) of 600.0 μ l0.1mol/l, rock mixing gently;

(9). yeast suspension is diluted to different concns coating YNB dull and stereotyped, cultivates 5d, obtain single bacterium colony for 28 DEG C.

Embodiment 5: transformant genomic insert detects

On picking YNB flat board, single colony inoculation is in 25mL YPD liquid nutrient medium, 28 DEG C, 200rpm, and cultivate 24h, get 500 μ l bacterium liquid in being equipped with in the sterilized glycerine pipe of 500 μ l, the final concentration of glycerine is (v/v) 18% ,-80 DEG C of Storage in refrigerator are for subsequent use.Get bacterium liquid a little, for extracting the genomic dna of bacterial strain, concrete operation step is carried Yeast genome test kit specification sheets according to sky root and is carried out simultaneously.Redesign checking primer P1:5 '-TTG GGCCGTTCTGGCCGCCAGCGGCGGCGACG-3 ', P2:5 '-CGC GGATCCTTACGACCAGCCCTGCTTCACCTC-3 ', genomic dna is template, carries out PCR checking.Amplification program: 95 DEG C of denaturations, 5min, a circulation; 95 DEG C of sex change, 30s, 58 DEG C of annealing, 30s, 72 DEG C of extensions, 80s, 29 circulations; 72 DEG C, 10min, a circulation; 12 DEG C, 10min, a circulation.By the positive transformant called after Y.l-pINA1297-proKexTG obtained.

Embodiment 6: recombinant bacterium Y.l-pINA1297-proKexTG fermentation culture, enzyme activity determination.

The positive bacteria Y.l-pINA1297-proKexTG100 μ l be stored in glycerine pipe that goes bail for is connected in 25mL YPD liquid nutrient medium, 28 DEG C, 200rpm, cultivates 18h, is then connected in 25mL PPB liquid nutrient medium by the switching amount of 10%, 28 DEG C, 200rpm, cultivates 4,5,6 days, gets fermentation supernatant, direct survey enzyme is lived, and separately gets a part of enzyme of surveying again after protease activated and lives.Record result as table 1.

Embodiment 7:SDS-PAGE detects albumen

Adopt green skies SDS-PAGE gel electrophoresis test kit to prepare, specific experiment operation steps is shown in product description.According to concentrated glue 5%, the concentration glue of separation gel 12%.Get the sample that processed and albumen Marker and boil loading after 10min, carry out electrophoresis, by coomassie brilliant blue staining 1h after terminating, then decolour.SDS-PAGE electrophoresis result as shown in Figure 1.

The enzyme work that table 1 recombinant bacterium Y.l-pINA1297-proKexTG fermentation supernatant is activated and not activated

Claims (3)

1. the genetic engineering bacterium of a high expression Transglutaminase EC2.3.2.13, it is characterized in that, the encoding sequence of the cleavage site Lys-Arg of yeast entogenous Kex2 proteolytic enzyme is introduced between the proenzyme district of its streptomyces hygroscopicus CCTCC M203062 Transglutaminase EC2.3.2.13 prochymosin gene of expressing and maturing enzyme district, described prochymosin gene uses expression vector to express in Yarrowia lipolytica, described expression vector is pINA1297, and described Transglutaminase EC2.3.2.13 prochymosin gene synthesizes according to Genbank:EU477523.

2. application rights requires that described in 1, engineering bacteria fermentation produces the method for Transglutaminase EC2.3.2.13, it is characterized in that, between the proenzyme district and maturing enzyme district of streptomyces hygroscopicus CCTCC M203062 Transglutaminase EC2.3.2.13 prochymosin gene, introduced the encoding sequence of the cleavage site Lys-Arg of yeast entogenous Kex2 proteolytic enzyme by PCR method, improved prochymosin gene uses expression vector to express in Yarrowia lipolytica.

3. method according to claim 2, it is characterized in that, step is as follows:

1) by chemical complete synthesis acquisition Transglutaminase EC2.3.2.13 prochymosin gene fragment;

2) the Transglutaminase EC2.3.2.13 prochymosin gene fragment of recovery and plasmid pINA1297 are carried out SfiI single endonuclease digestion respectively, then add KpnI enzyme to cut, endonuclease bamhi carries out post recovery, digested plasmid pINA1297 carries out glue recovery, the two connection is spent the night, transformation of E. coli competent cell, uses kalamycin resistance plate screening positive transformant, obtains plasmid pINA1297-proTG;

3) introduce the encoding sequence of cleavage site Lys-Arg according to the design of plasmid pINA1297-proTG gene order, corresponding nucleotides sequence is classified as PCR mutant primer Kex1 and Kex2 of AAGCGA; The Transglutaminase EC2.3.2.13 prochymosin gene fragment of transformation is cloned into again pINA1297 and obtains pINA1297-proKexTG;

4) recombinant plasmid pINA1297-proKexTG NotI linearizing empirical tests successfully constructed, glue recovery fragment is converted into Yarrowia lipolytica and obtains recombinant bacterium;

5) recombinant bacterium fermentative production Transglutaminase EC2.3.2.13 is utilized;

Described mutant primer Kex1 and Kex2 is:

Kex1:5 '- cGAgACGCTGCCGACGAGAGGG-3 ', dashed part is for introducing base sequence;

Kex2:5 '- cTTgGGGGCCCGGAAGAGCG-3 ', dashed part is for introducing base sequence.