CN101633897B - Recombinant bacterium for expressing glutamine transaminage and application thereof - Google Patents
Recombinant bacterium for expressing glutamine transaminage and application thereof Download PDFInfo
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Abstract
The invention discloses a recombinant bacterium which is a recombinant methanol nutritional type microzyme containing a glutamine transaminage prosoma gene and a subtilisin type hydrolase gene SAM-P45. The invention also protects the recombinant bacterium as Pichia pastoris GSMTGB, the recombinant bacterium is preserved in CGMCC (address: Datun Road, Chaoyang district, Beijing city, China) on August 25th, 2009, the preserving number is CGMCC No.3249, and the enzyme activity of the glutamine transaminage reaches 0.43u/ml when the recombinant bacterium is induced and fermented for 72 hours.
Description
Technical field
The present invention relates to biological technical field, particularly express the reorganization bacterium and the application thereof of Transglutaminase EC2.3.2.13.
Background technology
Transglutaminase EC2.3.2.13 (Transglutaminase; Claim trans-glutaminases again; EC 2.3.2.13 is called for short TG) extensively be present in human body, animal, plant and the mikrobe, can the catalytic proteins molecule between or within the acyl group shift reaction; Through these reactions, can between the range protein molecule or within cause polymerization and crosslinked.Wherein microbe-derived Transglutaminase EC2.3.2.13 because of its to Ca2+ no dependence, substrate scope and relative low production cost widely, the focus that becomes Transglutaminase EC2.3.2.13 research and use.
Transglutaminase EC2.3.2.13 is widely used in food-processing: 1. crosslinked through what take place between the catalytic proteins molecule, improve key propertys such as proteinic solvability, whipability and emulsifying property; 2. some essential amino acid (like Methionin) is covalently bonded on the protein, prevents that Maillard reaction to amino acid whose destruction, improving proteinic nutritive value; 3. heat-resisting, water-proof film can be formed after the casein dehydration with the crosslinked mistake of this enzyme, packaging material for food can be used as; 4. be used for embedding lipid or lipid-soluble substance, the catalytic proteins molecule go amidation.Transglutaminase EC2.3.2.13 also has good application prospects at field of medicaments: Transglutaminase EC2.3.2.13 (being human body Hageman factor I) can the reinforced fibers structure in coagulation process; In epidermis keratinization process, form cutinized layer; Accelerating wound healing; Participate in the endocytosis of receptor-mediated protein and polypeptide hormone etc.These physiological functions explain that also Transglutaminase EC2.3.2.13 has become the focus that the medicine industry investigator is attracted attention.Also constantly there is new application to occur recently in fields such as makeup, textile industry and organizational projects.
The original bacterial strain of Transglutaminase EC2.3.2.13 mainly contains streptomyces mobaraensis, streptomyces hygroscopicus etc.In order to bring into play the mode trickle biology; Like intestinal bacteria easy cultivations such as (Escherichia coli), zymotechnique maturation and advantage with short production cycle; The report of continuous in recent years useful pattern host's heterogenous expression Transglutaminase EC2.3.2.13 is like intestinal bacteria, lactic acid chain lactococcus spp (Lactococcus lactis), corynebacterium glutamicum (Corynebacteriumglutamicum).But the Transglutaminase EC2.3.2.13 that intestinal bacteria produce does not have activity, still needs external folding renaturation.The glutamine transaminase output of lactic acid chain lactococcus spp and corynebacterium glutamicum is too low, can't compare with original streptomyces strain.
Methyl alcohol nutritional type yeast pichia spp is a kind of efficient heterologous protein expression system that recent two decades grows up, and it has following good characteristics: the posttranslational modification function that 1. has eukaryotic expression system and had is particularly suitable for the eucaryon proteic expression of originating; 2. sophisticated carrier and promoter systems can be selected corresponding promotor as required, and wherein the application of alcohol oxidase gene promoter is maximum, and its startup intensity is big and regulated and control by the methyl alcohol strictness; 3. sophisticated high-density culture technology, process is prone to amplify; 4. PE efficiently, bacterial strain self secretion extracellular protein amount is few, is easy to the separation and purification of foreign protein; 5) exogenous origin gene integrator is in yeast chromosomal, thereby gene genetic stability is high, need not to add resistance pressure.Based on above-mentioned advantage, existing at present nearly 600 kinds of albumen successful expression in this system, pichia spp has become one of first-selected system of PE expression.Hiroya Yurimoto etc. once reported the Transglutaminase EC2.3.2.13 with pichia yeast expression system secreting, expressing streptomyces mobaraensis source; No matter be directly to express ripe Transglutaminase EC2.3.2.13, Transglutaminase EC2.3.2.13 precursor or between pro sequence and ripe Transglutaminase EC2.3.2.13, add Methionin-l-arginine introns, it shakes bottle enzyme and lives all less than 0.025u/ml.
Summary of the invention
The object of the present invention is to provide a kind of reorganization bacterium.
Reorganization bacterium provided by the invention is the reorganization methyl alcohol nutritional type yeast that contains Transglutaminase EC2.3.2.13 precursor-gene and subtilisin class hydrolase gene SAM-P45.
The encoding sequence of above-mentioned Transglutaminase EC2.3.2.13 precursor-gene as be for GenBank number AF531437 from shown in the 671-1798 position of 5 ' end; The encoding sequence of said subtilisin class hydrolase gene SAM-P45 be GenBank number for D83672 from 5 ' end the 14-3322 position shown in.
Above-mentioned subtilisin class hydrolase gene SAM-P45 imports in the methyl alcohol nutritional type yeast through recombinant expression vector A, and methyl alcohol nutritional type yeast A obtains recombinating;
Above-mentioned recombinant expression vector A inserts the recombinant expression vector that the MCS of pPICZ α obtains with said subtilisin class hydrolase gene SAM-P45.
Above-mentioned Transglutaminase EC2.3.2.13 precursor-gene is to import among the reorganization methyl alcohol nutritional type yeast A through recombinant expression vector B, obtains the reorganization bacterium of the present invention's protection;
Above-mentioned recombinant expression vector B inserts the recombinant expression vector that the MCS of carrier pAO815alpha obtains with said Transglutaminase EC2.3.2.13 precursor-gene;
Said carrier pAO815alpha is connected the recombinant vectors that obtain with the small segment that the EcoRI double digestion obtains with the pAO815 that crosses through same double digestion with SacI with pPICZ α.
Arbitrary above-mentioned methyl alcohol nutritional type yeast is the pichia pastoris phaff bacterium.
Said pichia pastoris phaff bacterium is pichia pastoris phaff bacterium GS115.
Above-mentioned reorganization bacterium is pichia pastoris phaff (Pichia pastoris) GSMTGB; Be preserved in Chinese microorganism strain preservation board of trustee reason person on August 25th, 2009 and understand common micro-organisms center (abbreviation CGMCC; The address is: Da Tun road, Chaoyang District, BeiJing, China city), preserving number is CGMCC № .3249.
The application of above-mentioned reorganization bacterium in producing Transglutaminase EC2.3.2.13 also belongs within protection scope of the present invention.
Above-mentioned application is meant arbitrary described reorganization bacterium fermentation culture in the BMMY substratum; Said fermentation culture conditions is 28-30 ℃, and pH is 4-5, and rotating speed is 200r/min.
Arbitrary described reorganization bacterium also belongs within protection scope of the present invention in Application in Food Processing.
The present invention adopts pichia spp as host strain secreting, expressing Transglutaminase EC2.3.2.13.Ripe also generation is active necessary because the pro sequence is a Transglutaminase EC2.3.2.13; Therefore Transglutaminase EC2.3.2.13 precursor protein sequence nucleotide sequence is placed under the regulation and control of strong promoter AOX1; And be connected with the sequence of secreting signal peptide α-mating factor, make yeast cell secreting, expressing Transglutaminase EC2.3.2.13 precursor at first in a large number.Precursor protein also need be outside the yeast born of the same parents through under the lytic enzyme effect, self pro sequence is cut away, finally be converted into activated sophisticated enzyme.Though pichia spp self secretion lytic enzyme, the enzymic activity of specificity cutting pro sequence is very low.In order to improve the cutting efficiency of pichia spp external (in the fermented liquid) Transglutaminase EC2.3.2.13 precursor; The present invention efficiently enzyme subtilisin class hydrolase gene SAM-P45 that cuts the pro sequence is connected to secretion expression carrier; And carrier transformed pichia spp, make it to express simultaneously with the Transglutaminase EC2.3.2.13 precursor.Like this, the Transglutaminase EC2.3.2.13 precursor is cut by the SAM-P45 lytic enzyme of while secreting, expressing in fermented liquid, thereby can efficiently express sophisticated Transglutaminase EC2.3.2.13.The experiment proof: pichia pastoris phaff (Pichia pastoris) GSMTGB is when inducing fermentation 72h, and the enzyme work of Transglutaminase EC2.3.2.13 reaches 0.43u/ml.
Description of drawings
Fig. 1 is the structure synoptic diagram of secreted expression carrier pAO815alpha.
Fig. 2 is the structure synoptic diagram of recombinant expression vector pAO α-promtg.
Fig. 3 is the structure synoptic diagram of recombinant expression vector pPICZ-SAMP45.
Embodiment
Below in conjunction with specific embodiment the present invention is described further, but the present invention is not limited to following examples.
Among the following embodiment,, be ordinary method like no specified otherwise.
Preparation and the Function detection thereof of the yeast strain of embodiment 1, expression Transglutaminase EC2.3.2.13
One, expresses the preparation of the bacterial strain of Transglutaminase EC2.3.2.13
1, the structure that contains the recombinant expression vector of Transglutaminase EC2.3.2.13 precursor-gene
1) extracting of streptomyces mobaraensis (Streptomyces mobaraensis) chromosomal DNA
Streptomyces mobaraensis CPCC 200365 (available from the China medicinal microbial strains preservation administrative center) spore suspension or the inclined-plane mycelium of-70 ℃ of preservations are seeded to (20g/L Zulkovsky starch, 20g/L peptone, 2g/L yeast powder, 2g/L sal epsom, 2g/L potassium primary phosphate, 2g/L potassium hydrogenphosphate) in the 5mlTSB substratum, and 30 ℃ of concussions were cultivated 48 hours.Get the centrifugal collection mycelium of 3ml bacterium liquid, and with 500 μ lSTE damping fluids (10mM Tris-Cl, pH8.0,0.1mM NaCl, 1mM EDTA, pH8.0) washed twice.With 500 μ lSTE mycelium is fully suspended, adding N,O-Diacetylmuramidase, to make its final concentration be 2mg/ml, and this moment, thallus suspension liquid became translucent glue, added 250 μ l2%SDS, the concussion mixing.Add the neutral phenol/chloroform of 250 μ l (phenol: abundant mixing chloroform=25: 24) then.Centrifugal 5 minutes of 10000rpm is transferred to supernatant in the new Eppendorf pipe, and adding Rnase, to make its final concentration be 20-40 μ g/ml, 37 ℃ of insulations 30 minutes.Repeat with neutral phenol/chloroform extracting 3-4 time, no longer include metaprotein up to two-phase interface and exist, remove phenol twice with the chloroform extracting again.In the aqueous solution, add 1/10 volume 3mol/LNaAC and isopyknic Virahol, room temperature was placed 30 minutes behind the mixing.Centrifugal 5 minutes of 10000rpm, 70% washing with alcohol once, be dissolved in after vacuum is drained 100-200 μ lTE damping fluid (10mM Tris-Cl, pH8.0,0.1mM NaCl, 1mM EDTA, pH8.0) subsequent use.
2) pcr amplification of Transglutaminase EC2.3.2.13 precursor-gene
According to the Transglutaminase EC2.3.2.13 precursor-gene in streptomyces mobaraensis source among the GenBank (nucleotide sequence as be for GenBank number AF531437 from shown in the 671-1798 position of 5 ' end), design the PCR primer:
Primer 1:5 '
CTCGAGAAAAGAGACAATGGCGCGGGGGAAGA 3 '
Primer 2: 5 ' TCTA
GAATTCACGGCCAGCCCTGCTTTACC 3 '
In α-mating factor signal peptide-coding sequence downstream an XhoI restriction enzyme site (CTCGAG is arranged; Corresponding amino acid is Leu-Glu); Thereafter immediately following a Kex2 proteolytic enzyme recognition site (AAAAGA; Corresponding aminoacid sequence Lys-Arg), therefore, the design of primer 1 is before the Transglutaminase EC2.3.2.13 gene, to introduce XhoI site and Kex2 proteolytic enzyme recognition site sequence.Through this design; Can the Transglutaminase EC2.3.2.13 gene directly be connected after the signal peptide sequence; Like this behind the protein translation; Under the effect of pichia spp self Kex2 proteolytic enzyme, can cut and give expression to the constant target protein of amino acid (and can not introduce extra amino-acid residue) because of the reason of restriction enzyme site.Primer 2 comprises terminator codon TGA, and introduces an EcoRI restriction enzyme site.
The streptomyces mobaraensis chromosomal DNA that extracts with step 1) is a template, adds primer 1, primer 2, Taq archaeal dna polymerase and dNTP mixture, carries out touchdown PCR (touchdown PCR), and reaction conditions is following: 94 ℃ of 5min; 94 ℃ of 30s, 65-55 ℃ of 30s, 72 ℃ of 50s, 1 ℃ of each cycle down, totally 10 circulations; 94 ℃ of 30s, 60 ℃ of 30s, 72 ℃ of 50s, totally 25 circulations.
Pcr amplification obtains the fragment of 1.1kb; Called after promtg; Behind Omega purification kit purifying, connect into pMD18-T carrier (available from TaKaRa company), obtain recombinant vectors pMD18-promtg; Send Beijing AudioCodes to check order pMD18-promtg, sequencing result show fragment that pcr amplification obtains have GenBank number for AF531437 from the nucleotide sequence shown in the 671-1798 position of 5 ' end.
3) structure of intermediate carrier
PPICZ α (available from Invitrogen company) is used the SacI/EcoRI double digestion; Obtain small segment; Small segment is connected with the pAO815 carrier (available from Invitrogen company) that digested with SacI/EcoRI equally, promptly obtains excretion vector pAO815alpha (building process is as shown in Figure 1) based on pAO815.
4) contain the structure of the recombinant expression vector of Transglutaminase EC2.3.2.13 precursor-gene
With step 2) checking is after correct pMD18-promtg cuts with the XhoI/EcoRI enzyme, is connected with the excretion vector pAO815alpha that step 3) obtains, and can obtain containing the recombinant expression vector pAO α-promtg (Fig. 2) of Transglutaminase EC2.3.2.13 precursor-gene.With the recombinant expression vector pAO α that obtains-promtg transformed into escherichia coli DH5 α competent cell; LB screening of medium with containing 10 μ g/ml sulphuric acid kanamycins goes out required recon and carries out sequencing analysis; Identify through order-checking, prove that sequence and the structure of recombinant expression vector pAO α-promtg is correct.
2, the structure that contains the recombinant expression vector of subtilisin class hydrolase gene SAM-P45
1) extracting of white light gray streptomycete (Streptomyces albogriseolus) chromosomal DNA
Extraction is from white light gray streptomycete chromosome DNA, and process for extracting is with the extracting of streptomyces mobaraensis chromosomal DNA in the step 1.
2) pcr amplification of subtilisin class hydrolase gene SAM-P45
According to the subtilisin class hydrolase gene in white light gray streptomycete source among the GenBank (GenBank number be D83672 from shown in the 14-3322 position of 5 ' end), design the PCR primer:
Primer 1:5 '
CTCGAGAAAAGAAACGGGGAGAACAGCACG 3 '
Primer 2: 5 ' CTACTTGCCGTAGTAGGCGTTGTGG 3 '
The principle of design of primer 1 is with step 1; Through this design; Can the Transglutaminase EC2.3.2.13 gene directly be connected after the signal peptide sequence; Behind the protein translation, under the effect of pichia spp self Kex2 proteolytic enzyme, can cut and give expression to the constant target protein of amino acid (and can not introduce extra amino-acid residue) like this because of the reason of restriction enzyme site.Primer 2 comprises terminator codon TAG.
With white light gray streptomycete CGMCC 4.565 (available from China Committee for Culture Collection of Microorganisms common micro-organisms center) chromosomal DNA is template; Add primer 1, primer 2, Taq archaeal dna polymerase and dNTP mixture; Carry out touchdown PCR (touchdown PCR), reaction conditions is following: 94 ℃ of 5min; 94 ℃ of 30s, 65-55 ℃ of 30s, 72 ℃ of 50s, 1 ℃ of each cycle down, totally 10 circulations; 94 ℃ of 30s, 60 ℃ of 30s, 72 ℃ of 50s, totally 25 circulations.
Pcr amplification obtains the fragment of 3.2kb; Called after SAM-P45; Behind Omega purification kit purifying, connect into the pMD18-T carrier, obtain recombinant vectors pMD18-SAM-P45; Send Beijing AudioCodes to check order pMD18-SAM-P45, sequencing result show fragment that pcr amplification obtains have GenBank number for D83672 from the nucleotide sequence shown in the 14-3322 position of 5 ' end.
3) contain the structure of the recombinant expression vector of subtilisin class hydrolase gene SAM-P45
With step 2) in the correct pMD18-SAM-P45 of checking cut back (introducing the segmental XbaI of purpose site) with the XhoI/XbalI enzyme by the pMD18-T carrier, be connected with the pPICZ α that cut through enzyme equally, get final product recombinant expression vector pPICZ-SAMP45 (Fig. 3).With the recombinant expression vector pPICZ-SAMP45 transformed into escherichia coli DH5 α competent cell that obtains; LB screening of medium with containing 25 μ g/ml Zeocin goes out required recon and carries out sequencing analysis; Identify through order-checking, prove that the sequence of recombinant expression vector pPICZ-SAMP45 and structure are correct.
3, express the acquisition of the yeast strain of Transglutaminase EC2.3.2.13
1) electricity transforms the preparation of pichia pastoris phaff cell
(Invitrogen, Ca.18100) in 500ml YPD substratum (10 grams per liter yeast extract pastes, 20 grams per liter peptones, 20 grams per liter glucose, 15 grams per liter agaroses), 30 ℃, 200r/min is cultured to OD600=1.3-1.5 to inoculation Pichi strain GS115.4 ℃, 1, the centrifugal 5min of 500g collects thalline, respectively washes once with the aqua sterilisa of 500ml, 250ml precooling and the 1mol/L sorbyl alcohol of 20ml precooling respectively.After washing at every turn all at 4 ℃, 1, centrifugal 5min collects thalline under the 500g, the 1mol/L sorbyl alcohol with the 1ml precooling suspends at last, promptly obtains the competent cell that shocks by electricity.
2) recombinant expression plasmid pPICZ-SAMP45 electricity transformed yeast cell
Use the BstXI linearization for enzyme restriction with making up the correct about 10ug of recombinant expression plasmid pPICZ-SAMP45 in the step 2, ethanol sedimentation reclaims linear DNA and is dissolved in the 10ul sterilized water.Above-mentioned linearizing DNA is mixed with 80ul GS115 competent cell, change in the 0.2cm electricity revolving cup of precooling.Adopt U.S. BIO-RAD company electricity conversion instrument, the pichia spp parameter preset according to making electricity consumption conversion instrument shocks by electricity.After finishing, electric shock adds immediately in 1M sorbyl alcohol to the electric shock cup of 1ml precooling; To shock by electricity then the cup in solution all be transferred in the aseptic centrifuge tube; Hatch 1-2h for 37 ℃; Get the YPD flat board that the 500ul coating is added with 100 μ g/ml microbiotic Zeocin, be inverted cultivation in 30 ℃ and occurred until bacterium colony in 2-4 days.So far, obtain containing the recombinant yeast pichia pastoris of pPICZ-SAMP45.
3) the electric transformed yeast cell of recombinant expression vector pAO α-promtg obtains single copy bacterial strain
Use the StuI linearization for enzyme restriction with 10ug approximately with making up correct recombinant expression vector pAO α-promtg in the step 1, ethanol sedimentation reclaims linear DNA and is dissolved in the 10ul sterilized water.With step 2) recombinant yeast pichia pastoris that contains pPICZ-SAMP45 that obtains is that starting strain prepares competence.Above-mentioned linearizing DNA is mixed with the recombinant yeast pichia pastoris competent cell that contains pPICZ-SAMP45, and it is the same that electricity changes operation.Add immediately after the electric shock in 1M sorbyl alcohol to the electric shock cup of 1ml precooling, the solution in the cup that will shock by electricity then all is transferred in the aseptic centrifuge tube, directly gets dull and stereotyped (the 13.4 grams per liter YNB, 4 * 10 of 200-300ul bacterium liquid coating MD
-4Grams per liter vitamin H, 10 grams per liter glucose, 15 grams per liter agaroses).Be inverted to cultivate for 30 ℃ and occurred until bacterium colony in 2-4 days.So far, obtain the bacterial strain of single copy promtg of expression Transglutaminase EC2.3.2.13.
4) structure of multi-copy strains
The acquisition of the bacterial strain of A, 2 copy promtg
The recombinant expression vector pAO α-promtg that obtains in the step 1 is used the BamHI/BglII double digestion; Reclaim the BamHI-promtg-BglII fragment; Connect into the BamHI site of expression vector pAO α-promtg, get final product 2 the copy promtg expression vector pAO α-promtg2; The method electricity step of converting 2 that pAO α-promtg2 is provided according to the step 3) in the step 3) recombinant yeast pichia pastoris that contains pPICZ-SAMP45 that obtains so far, obtains to express the bacterial strain of 2 copy promtg of Transglutaminase EC2.3.2.13.
The acquisition of the bacterial strain of B, 3 copy promtg
Recombinant expression vector pAO α-promtg2 that steps A is obtained uses the BamHI/BglII double digestion; Reclaim the BamHI-promtg-promtg-BglII fragment; Connect into the BamHI site of expression vector pAO α-promtg, get final product 3 the copy promtg expression vector pAO α-promtg3; The method electricity step of converting 2 that pAO α-promtg3 is provided according to the step 3) in the step 3) recombinant yeast pichia pastoris that contains pPICZ-SAMP45 that obtains so far, obtains to express the bacterial strain of 3 copy promtg of Transglutaminase EC2.3.2.13.
Two, Function detection
1, the fermentation culture of reorganization bacterium
The single bacterial strain of promtg, the bacterial strain of 2 copy promtg and inoculation (10 grams per liter yeast extracts, 20 grams per liter peptones in 25ml BMGY liquid nutrient medium of 3 copy promtg of copying with the expression Transglutaminase EC2.3.2.13 that obtains in the step 1; Behind 121 ℃ of sterilization 20min, when reducing to room temperature, temperature adds 13.4 grams per liter YNB, 4 * 10
-4Grams per liter vitamin H, 10 grams per liter glycerine), in 30 ℃, the 200r/min shaking table is cultivated 48h, OD
600Reach 10-20, the centrifugal 5min of 3000r/min collects thalline, abandons supernatant, washs thalline 1-2 time with sterile purified water.(10 grams per liter yeast extracts, 20 grams per liter peptones behind 121 ℃ of sterilization 20min, add 13.4 grams per liter YNB, 4 * 10 when temperature is reduced to room temperature with the BMMY inducing culture with thalline
-4Grams per liter vitamin H, 0.5% methyl alcohol), be diluted to OD
600=10, replace cotton plug with 4 layers of gauze, in 30 ℃, the 200r/min shaking table is cultivated.Adding methyl alcohol to final concentration every day is 0.5% (V/V).
2, extracellular enzyme mensuration alive
After 3 days, draw fermented liquid 1mL 30 ℃ of cultivations in centrifuge tube,, draw supernatant in test tube with the centrifugal 10min of 10000r/min.(Tutofusin tris 12.110g, hydroxylamine chloride 3.475g, gsh 1.624g, CBZ-GLN-GLY (Nalpha-Carbobenzyloxy-Glutamine Glycine) 5.060g put into the 500ml beaker to add 0.1ml sample solution and 0.4mL reaction solution; Add 350ml water; The magnetic agitation mixing; With 1mol/L hydrochloric acid adjustment pH value to 6, constant volume is to 500ml then then), 37 ℃ of insulation 10min.Add 0.5mL terminator (3M HCL, 12%TCA (trichoroacetic acid(TCA)), 5%FeCl
36H
2O (being dissolved among the 0.1N HCl), three kinds of solution mix with 1: 1: 1 equal-volume), whirlpool mixing, the centrifugal 10min of 1500r/min.After adding stop buffer during 30min, get supernatant at 525nm place the mensuration light absorption value.The enzyme liquid of making a living to go out is done blank, and the treatment process of blank is the same with the processing of sample.Under 37 ℃ of conditions with pH=6.0, PM forms the required enzyme amount of 1 μ mol CBZ-Glu-Gly hydroxamic acid product and is defined as the enzyme unit that lives.
The result is as shown in table 1, induce fermentation 72h after, the 1-3 copy bacterial strain shake flask fermentation liquid enzyme best result Bie Keda 0.12,0.28 that lives, 0.43u/ml.With inducing the work of 72h enzyme to reach the 3 copy bacterial strain called after GSMTGB of 0.43u/ml.
The enzyme of the different copy of table 1. bacterial strain is lived
| Fermentation time (h) | Single copy bacterial strain enzyme (u/ml) alive | 2 copy bacterial strains (u/ml) | 3 copy bacterial strains (u/ml) |
| 72h | 0.12 | 0.28 | 0.43 |
Bacterial strain GSMTGB is preserved in Chinese microorganism strain preservation board of trustee reason person on August 25th, 2009 to be understood the common micro-organisms center (be called for short CGMCC, the address is: Da Tun road, Chaoyang District, BeiJing, China city), preserving number is CGMCC № .3249.For the bacterial strain GSMTGB of preservation, continue to cultivate, when 96h and 120h, taking a sample, it is alive to record enzyme; Experiment repetition 3 times; The result is as shown in table 2, serves as the fermentation culture that similarity condition is carried out in contrast with pichia pastoris phaff GS115 wherein, when 72h, 96h and 120h, does not all have enzyme to live.
The enzyme of the Transglutaminase EC2.3.2.13 of table 2. bacterial strain GSMTGB is lived
| Fermentation time (h) | Bacterial strain GSMTGB (u/ml) |
| 72 | 0.43 |
| 96 | 0.41 |
| 120 | 0.37 |
Claims (8)
1. a reorganization bacterium is the reorganization methyl alcohol nutritional type yeast that contains Transglutaminase EC2.3.2.13 precursor-gene and subtilisin class hydrolase gene SAM-P45;
Said methyl alcohol nutritional type yeast is the pichia pastoris phaff bacterium; Said pichia pastoris phaff bacterium is pichia pastoris phaff bacterium GS115;
The encoding sequence of said Transglutaminase EC2.3.2.13 precursor-gene as be for GenBank number AF531437 from shown in the 671-1798 position of 5 ' end.
2. reorganization bacterium as claimed in claim 1 is characterized in that: the encoding sequence of said subtilisin class hydrolase gene SAM-P45 be GenBank number for D83672 from 5 ' end the 14-3322 position shown in.
3. according to claim 1 or claim 2 reorganization bacterium, it is characterized in that: said subtilisin class hydrolase gene SAM-P45 imports in the methyl alcohol nutritional type yeast through recombinant expression vector A, and methyl alcohol nutritional type yeast A obtains recombinating;
Said recombinant expression vector A inserts the recombinant expression vector that the MCS of pPICZ α obtains with said subtilisin class hydrolase gene SAM-P45.
4. reorganization bacterium as claimed in claim 3 is characterized in that: said Transglutaminase EC2.3.2.13 precursor-gene is to import among the reorganization methyl alcohol nutritional type yeast A through recombinant expression vector B, obtains the described reorganization of claim 1 bacterium;
Said recombinant expression vector B inserts the recombinant expression vector that the MCS of carrier pAO815alpha obtains with said Transglutaminase EC2.3.2.13 precursor-gene;
Said carrier pAO815alpha is connected the recombinant vectors that obtain with the small segment that the EcoRI double digestion obtains with the pAO815 that crosses through same double digestion with SacI with pPICZ α.
5. reorganization bacterium as claimed in claim 1 is characterized in that: said reorganization bacterium is pichia pastoris phaff (Pichia pastoris) GSMTGB CGMCC № .3249.
6. the application of the arbitrary described reorganization bacterium of claim 1-5 in producing Transglutaminase EC2.3.2.13.
7. application as claimed in claim 6 is characterized in that: said application is meant the arbitrary described reorganization bacterium of claim 1-5 fermentation culture in the BMMY substratum; Said fermentation culture conditions is 28-30 ℃, and pH is 4-5, and rotating speed is 200r/min.
8. the arbitrary described reorganization bacterium of claim 1-5 is in Application in Food Processing.
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| CN102154140B (en) * | 2011-01-20 | 2012-11-07 | 江苏锐阳生物科技有限公司 | High-efficiency preparation method of beta-amylase |
| CN109749948A (en) * | 2019-03-05 | 2019-05-14 | 天津科技大学 | A kind of recombinant pichia yeast strain and construction method sticking the expression of corn glutamine transaminage using TEF1 promoter regulation |
| CN112980759B (en) * | 2021-03-08 | 2022-08-26 | 上海交通大学 | Method for improving TG enzyme fermentation level by enhancing transcription level of Subtilisin gene |
| CN113817619B (en) * | 2021-10-21 | 2023-03-21 | 河南省科学院生物研究所有限责任公司 | High-temperature-resistant transglutaminase yzk producing strain and application thereof |
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