CN104212776A - Method for producing recombinant alpha-cyclodextrin glucosyltransferase by using bacillus subtilis - Google Patents

Method for producing recombinant alpha-cyclodextrin glucosyltransferase by using bacillus subtilis Download PDF

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CN104212776A
CN104212776A CN201410440327.0A CN201410440327A CN104212776A CN 104212776 A CN104212776 A CN 104212776A CN 201410440327 A CN201410440327 A CN 201410440327A CN 104212776 A CN104212776 A CN 104212776A
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cgt
bacillus
enzyme
bacillus subtilis
plasmid
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张佳瑜
吴敬
吴丹
陈晟
陈坚
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Jiangnan University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
    • C12N9/1074—Cyclomaltodextrin glucanotransferase (2.4.1.19)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/01—Hexosyltransferases (2.4.1)
    • C12Y204/01019—Cyclomaltodextrin glucanotransferase (2.4.1.19)

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Abstract

The invention relates to a method for producing recombinant alpha-cyclodextrin glucosyltransferase (alpha-CGTase for short) by using bacillus subtilis, belonging to the technical fields of genetic engineering and fermentation engineering. The method specifically comprises the following steps: (1) constructing a recombinant bacillus subtilis genetically engineered bacterium of Bacillus macerans alpha-CGTase; and (2) optimizing fermentation conditions to obtain the optimal concentrations of key components of the medium, namely maltose, corn starch and yeast powder are respectively 15.5g/L, 13g/L and 20g/L, inoculating the recombinant bacillus subtilis in the medium, and carrying shaking culture for 36 hours, wherein the activity of the alpha-CGTase is 17.6U/ml. The method has positive meaning for early achievement of production of the CGTase by using the bacillus subtilis so as to satisfy application of the CGTase in foods.

Description

A kind of method utilizing Bacillus subtilus Restruction alpha-cyclodextrin glucosyl transferase
Technical field
Utilize a method for Bacillus subtilus Restruction alpha-cyclodextrin glucosyl transferase, belong to genetically engineered and field of fermentation engineering.
Background technology
Cyclomaltodextrin glucanotransferase (be called for short CGT enzyme, EC2.4.1.19) be a kind of can the glucose polymer such as catalytic starch, glycogen, Fructus Hordei Germinatus oligosaccharide and obtain the extracellular enzyme of cyclodextrin, can be divided into α-, β-and γ-three types.Cyclodextrin molecular has unique hydrophobic cavity structure, and energy inclusion hydrophobic guest molecules, therefore has a wide range of applications in fields such as food, medicine, agriculturals.The suitability for industrialized production of cyclodextrin all adopts enzymatic clarification, cyclodextrin research early stage, cause the productivity of cyclodextrin very low owing to not filtering out the bacterial strain of High-efficient Production CGT enzyme, the application of cyclodextrin is very limited.At present, more to the research of CGT enzyme abroad, and China is still in the starting stage to the research of this enzyme, the research of genetic engineering bacterium is few.
In order to overcome the low CGT enzyme throughput of Natural strains, utilizing DNA recombinant technology CGT enzyme encoding gene to be imported overexpression in Escherichia coli and Bacillus subtilis and being considered to one of most effective way.Although also lag far behind intestinal bacteria about the research Bacillus subtilus of exogenous protein expression, Bacillus subtilus belongs to the safe Host Strains of foodstuffs industry, by albumen direct secretion to outside born of the same parents, can have great importance in the engineered research of food.Along with engineered develop rapidly, believe that Bacillus subtilus expression system will be more perfect, one of best host becoming prokaryotic protein expression.
Summary of the invention
The object of this invention is to provide a kind of method utilizing Bacillus subtilus Restruction alpha-cyclodextrin glucosyl transferase.
Technical scheme of the present invention:
Utilize a method for Bacillus subtilus Restruction α-CGT enzyme, it is characterized in that comprising the steps:
(1) bacillus macerans α-CGT enzyme recombination bacillus subtilis genetic engineering bacterium is built;
(2) optimization of fermentation conditions obtains substratum key ingredient maltose, and W-Gum and yeast powder optimum concn are respectively: 15.5g/L, 13g/L and 20g/L, recombination bacillus subtilis are inoculated in this substratum, collected by centrifugation supernatant liquor after shake-flask culture 36h.
Wherein the method for step (1) is:
I. the fragment of cgt gene and native signal peptide thereof is cloned:
Being template with P.macerans JFB05-01 (culture presevation at national Type Culture Collection, preserving number: CCTCC M203062) genomic dna, is the fragment that primer amplification obtains cgt gene and native signal peptide thereof with P1, P2;
Forward primer P1:5 '-ACGAGGAATTCATGAAATCGCGGTAC-3 ' (containing EcoR I restriction enzyme site)
Reverse primer P2:5 '-CGCGGATCCTTAATTTTGCCAGTCCAC-3 ' (containing BamH I restriction enzyme site)
PCR reaction system: dNTPs 5 μ L, 10 × Ex Taq buffer (Mg2+Plus) 5 μ L, forward primer P1 (10 μMs) 1 μ L, reverse primer P2 (10 μMs) 1 μ L, template 1 μ L, Ex Taq HS (5U/ μ L) 0.25 μ L, adds ddH2O and supplies 50 μ L.
PCR reaction conditions: 94 DEG C of denaturation 4min, 94 DEG C of sex change 45s, 60 DEG C of annealing 45s, 72 DEG C extend 2.2min, after 30 circulations, extend 10min in 72 DEG C again, amplification obtains goal gene PCR fragment, and rubber tapping is reclaimed, reclaim fragment to be connected with pMD18-T simple carrier, connect product conversion e. coli jm109, converted product coating is dull and stereotyped containing the LB of 100mg/L penbritin, through 37 DEG C of overnight incubation, choosing colony, access LB liquid nutrient medium, extracts plasmid after 8-10h, this plasmid is carried out sequencing.
II. construction recombination plasmid pGJ-cgt:
Plasmid for construction of expression vector is pGJ103, containing maltose inducible promoter, the goal gene of this plasmid and amplification is carried out EcoR I and BamH I double digestion, digestion products rubber tapping to connect in 16 DEG C with T4 ligase enzyme after reclaiming again spends the night, connect product conversion E.coli competent cell, through 37 DEG C of overnight incubation, select transformant and carry out liquid culture in 100mg/L penbritin LB, then extracting plasmid, obtains the recombinant plasmid pGJ-cgt of enrichment.
III. the bacillus subtilis gene engineering bacteria containing recombinant plasmid pGJ-cgt is built:
Recombinant plasmid pGJ-cgt transforming B bacillus WB600 is obtained the recombination bacillus subtilis genetic engineering bacterium containing bacillus macerans α-CGT enzyme gene.
In step (2), the optimum concn of inductor maltose is 15.5g/L.
In step (2), carbon source is W-Gum, and optimum concn is 13g/L.
In step (2), nitrogenous source is yeast powder, and optimum concn is 20g/L.
The present invention is starting point from the safe Host Strains of foodstuffs industry, construct bacillus macerans α-CGT enzyme recombination bacillus subtilis genetic engineering bacterium, adopt experiment of single factor determination remarkable affecting genes, and adopt response surface center form the interaction of laboratory method research remarkable affecting genes and determine the best of breed of each factor, search out the top condition that engineering bacteria fermentation produces enzyme, in such optimised conditions, recombinase active is 17.6U/mL, has positive effect to realizing Bacillus subtilus production CGT enzyme early to meet its application in food.
Embodiment
Embodiment 1 construction recombination plasmid pGJ-cgt
With bacillus macerans (Peanibacillus macerans) JFB05-01 for template, be the fragment that primer amplification obtains cgt gene and native signal peptide thereof with P1, P2;
Forward primer P1:5 '-ACGAGGAATTCATGAAATCGCGGTAC-3 ' (containing EcoR I restriction enzyme site)
Reverse primer P2:5 '-CGCGGATCCTTAATTTTGCCAGTCCAC-3 ' (containing BamH I restriction enzyme site)
PCR reaction system: dNTPs 5 μ L, 10 × Ex Taq buffer (Mg2+Plus) 5 μ L, forward primer P1 (10 μMs) 1 μ L, reverse primer P2 (10 μMs) 1 μ L, template 1 μ L, Ex Taq HS (5U/ μ L) 0.25 μ L, adds ddH2O and supplies 50 μ L.
PCR reaction conditions: 94 DEG C of denaturation 4min, 94 DEG C of sex change 45s, 60 DEG C of annealing 45s, 72 DEG C extend 2.2min, after 30 circulations, then extend 10min in 72 DEG C.Amplification obtains goal gene PCR fragment, rubber tapping is reclaimed, reclaim fragment to be connected with pMD18-T simple carrier, connect product conversion e. coli jm109, converted product coating is dull and stereotyped containing the LB of 100mg/L penbritin, through 37 DEG C of overnight incubation, choosing colony, access LB liquid nutrient medium, extracts plasmid after 8-10h, this plasmid is carried out sequencing.
EcoR I and BamH I double digestion is carried out by containing the plasmid pGJ103 of maltose inducible promoter and the goal gene of amplification, digestion products rubber tapping to connect in 16 DEG C with T4 ligase enzyme after reclaiming again spends the night, connect product conversion E.coli competent cell, through 37 DEG C of overnight incubation, select transformant and carry out liquid culture in 100mg/L penbritin LB, then extracting plasmid, obtains the recombinant plasmid pGJ-cgt of enrichment.
Embodiment 2 builds recombination bacillus subtilis genetic engineering bacterium and secreting, expressing α-CGT enzyme
By recombinant plasmid pGJ-cgt by chemical transformation transforming B bacillus WB600, containing on paraxin (10 μ g/mL) LB flat board through 37 DEG C of overnight incubation, select transformant 37 DEG C of liquid culture in LB substratum to spend the night, then 4% inoculum size access TB fermention medium, after 1.5% maltose induction 48h, produce enzyme reach 6.1U/mL, fermented supernatant fluid is identified through SDS-PAGE, at about 72kDa place appearance protein band.
Embodiment 3 experiment of single factor optimizes CGT enzyme fermentation condition
Generally, the possible factor affecting recombinant bacterium enzymatic production is a lot, comprises carbon source, nitrogenous source, inorganic salt, substratum initial ph value, leavening temperature, kind age, inoculum size, liquid amount etc.By carrying out on the basis of initial optimization in previous work to recombination bacillus subtilis enzyme ferment condition, the principal element can determining to affect in this research this recombinant bacterium enzymatic production is inductor, Carbon and nitrogen sources, thus inducer concentrations (5 has been investigated by experiment of single factor, 10, 15, 20, 25g/L), carbon source (glucose, sucrose, Zulkovsky starch, dextrin and W-Gum, concentration is by containing carbon mass fraction equal principle), nitrogenous source (peptone, yeast powder, extractum carnis, corn steep liquor, concentration is by containing nitrogen content equal principle) impact on α-CGT enzyme fermentative production.
Result shows, along with the increase of maltose concentration, dry cell weight increases gradually, and when maltose concentration is 15g/L, enzyme is lived and reached maximum value 6.1U/mL, then increases maltose concentration, and enzyme is lived not to be increased.Very big-difference is there is in different carbon source to product enzyme, glucose is to thalli growth and to produce enzyme all unfavorable, and W-Gum is conducive to thalli growth and produces enzyme, and produces enzyme peak period and be advanced to 36h, when interpolation concentration is 10g/L, dry cell weight and α-CGT enzyme enzyme are lived and are respectively 10g/L and 8.3U/mL.Nitrogenous source yeast powder can obtain higher enzyme and lives and be conducive to thalli growth, although peptone can not promote to produce enzyme, but it is suitable with yeast powder to live than enzyme, on the basis of adding yeast powder, thus adds 4g/L peptone make restructuring B.subtilis product enzyme be increased to 12.5U/mL.
The composition experiment of embodiment 4 response surface center optimizes CGT enzyme fermentation condition further
According to above result of trophic factor being carried out to experiment of single factor, select inductor-maltose, carbon source-W-Gum, nitrogenous source-yeast powder responsively face design factor, each selecting factors three concentration levels, table 1 is the horizontal calendar of response surface empirical factor.Table 2 lists the α-experimental value of CGT enzyme activity and the predictor of model under Box-Behnken experimental design.
Carry out equation model to experimental data and carry out variance analysis, result is comparatively remarkable, obtains second order regression equation Y=16.26667+3.2375 × X 1+ 1.53375 × X 2-0.25375 × X 3-3.7 × X 1 2-2.75 × X 2 2-2.04 × X 3 2-0.0825 × X 1x 2-0.1275 × X 1x 3-0.425 × X 2x 3, wherein Y is that α-CGT enzyme enzyme is lived.
Table 1 response surface center combination design level of factor table
Table 2 response surface experimental design and result
Carry out variance analysis to above-mentioned regression model, result shows, model coefficient of determination R 2=0.99, show that model can explain the change of the α-CGT enzyme output of 99%, regression fit degree is fine.Usually carry out the degree of confidence of judgment models by CV value, this experiment show that CV value is 2.5, and p value is less than 0.001 simultaneously, shows that the data that experimental model draws are genuine and believable.
In order to determine the optimum concn of three further, adopt SAS software to carry out Ridge analysis to equation, obtain substratum key ingredient maltose, W-Gum and yeast powder three optimum concn are respectively 15.5g/L, 13g/L and 20g/L.With this understanding, the enzyme of α-CGT enzyme predictor alive is 17.3U/mL.In order to whether verification model prediction is accurate, carry out three fermentations with optimal medium, its result mean value is 17.6U/mL.Illustrate that this model equation and practical situation matching are better thus, can be used for prediction recombinant bacterium enzymatic production, after optimization enzyme live improve 1.5 times before optimizing.

Claims (5)

1. utilize the method for Bacillus subtilus Restruction alpha-cyclodextrin glucosyl transferase (being called for short α-CGT enzyme), it is characterized in that comprising the steps:
(1) bacillus macerans α-CGT enzyme recombination bacillus subtilis genetic engineering bacterium is built;
(2) optimization of fermentation conditions obtains substratum key ingredient maltose, and W-Gum and yeast powder optimum concn are respectively: 15.5g/L, 13g/L and 20g/L, recombination bacillus subtilis are inoculated in this substratum, collected by centrifugation supernatant liquor after shake-flask culture 36h.
2. the method utilizing Bacillus subtilus Restruction α-CGT enzyme according to claim 1, is characterized in that the method for the recombination bacillus subtilis genetic engineering bacterium that step (1) builds containing bacillus macerans α-CGT enzyme gene cgt is:
I. the fragment of cgt gene and native signal peptide thereof is cloned:
With bacillus macerans (Peanibacillus macerans) JFB05-01 for template, be the fragment that primer amplification obtains cgt gene and native signal peptide thereof with P1, P2;
Forward primer P1:5 '-ACGAGGAATTCATGAAATCGCGGTAC-3 ' (containing EcoR I restriction enzyme site)
Reverse primer P2:5 '-CGCGGATCCTTAATTTTGCCAGTCCAC-3 ' (containing BamH I restriction enzyme site)
PCR reaction system: dNTPs 5 μ L, 10 × Ex Taq buffer (Mg2+Plus) 5 μ L, forward primer P1 (10 μMs) 1 μ L, reverse primer P2 (10 μMs) 1 μ L, template 1 μ L, Ex Taq HS (5U/ μ L) 0.25 μ L, adds ddH2O and supplies 50 μ L.
PCR reaction conditions: 94 DEG C of denaturation 4min, 94 DEG C of sex change 45s, 60 DEG C of annealing 45s, 72 DEG C extend 2.2min, after 30 circulations, then extend 10min in 72 DEG C.Amplification obtains goal gene PCR fragment, rubber tapping is reclaimed, reclaim fragment to be connected with pMD18-T simple carrier, connect product conversion e. coli jm109, converted product coating is dull and stereotyped containing the LB of 100mg/L penbritin, through 37 DEG C of overnight incubation, choosing colony, access LB liquid nutrient medium, extracts plasmid after 8-10h, this plasmid is carried out sequencing.
II. construction recombination plasmid pGJ-cgt:
Plasmid for construction of expression vector is pGJ103, containing maltose inducible promoter, the goal gene of this plasmid and amplification is carried out EcoR I and BamH I double digestion, digestion products rubber tapping to connect in 16 DEG C with T4 ligase enzyme after reclaiming again spends the night, connect product conversion E.coli competent cell, through 37 DEG C of overnight incubation, select transformant and carry out liquid culture in 100mg/L penbritin LB, then extracting plasmid, obtains the recombinant plasmid pGJ-cgt of enrichment;
III. the bacillus subtilis gene engineering bacteria containing recombinant plasmid pGJ-cgt is built:
Recombinant plasmid pGJ-cgt transforming B bacillus WB600 is obtained the recombination bacillus subtilis genetic engineering bacterium containing bacillus macerans α-CGT enzyme gene.
3. the method utilizing Bacillus subtilus Restruction α-CGT enzyme according to claim 1, is characterized in that the optimum concn of inductor maltose in step (2) is 15.5g/L.
4. the method utilizing Bacillus subtilus Restruction α-CGT enzyme according to claim 1, it is characterized in that in step (2), carbon source is W-Gum, optimum concn is 13g/L.
5. the method utilizing Bacillus subtilus Restruction α-CGT enzyme according to claim 1, it is characterized in that in step (2), nitrogenous source is yeast powder, optimum concn is 20g/L.
CN201410440327.0A 2014-09-01 2014-09-01 Method for producing recombinant alpha-cyclodextrin glucosyltransferase by using bacillus subtilis Pending CN104212776A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104611284A (en) * 2015-02-03 2015-05-13 江南大学 Strain for production of cyclodextrin glucosyltransferase and application of strain
CN108384741A (en) * 2018-02-12 2018-08-10 江南大学 A kind of genetic engineering bacterium of high yield cyclodextrin glycosyltransferase

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101831414A (en) * 2010-05-25 2010-09-15 江南大学 Process for extracellularly producing recombinant alpha-cyclodextrin glucosyltransferase
CN102796712A (en) * 2012-09-11 2012-11-28 云南师范大学 Method for producing extracellular production and recombination beta-cyclodextrin transferase

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101831414A (en) * 2010-05-25 2010-09-15 江南大学 Process for extracellularly producing recombinant alpha-cyclodextrin glucosyltransferase
CN102796712A (en) * 2012-09-11 2012-11-28 云南师范大学 Method for producing extracellular production and recombination beta-cyclodextrin transferase

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
张佳瑜: "软化芽孢杆菌α-环糊精葡萄糖基转移酶在毕赤酵母和枯草杆菌中的表达", 《中国优秀硕士学位论文全文数据库 基础科学辑》, 15 February 2012 (2012-02-15) *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104611284A (en) * 2015-02-03 2015-05-13 江南大学 Strain for production of cyclodextrin glucosyltransferase and application of strain
CN108384741A (en) * 2018-02-12 2018-08-10 江南大学 A kind of genetic engineering bacterium of high yield cyclodextrin glycosyltransferase
CN108384741B (en) * 2018-02-12 2020-10-09 江南大学 Genetically engineered bacterium for high-yield cyclodextrin glucosyltransferase

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Application publication date: 20141217