CN114958701A - High-yield gamma-cyclodextrin glucosyltransferase gene engineering bacteria and construction method thereof - Google Patents
High-yield gamma-cyclodextrin glucosyltransferase gene engineering bacteria and construction method thereof Download PDFInfo
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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)
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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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
-
- 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)
Abstract
The invention discloses a high-yield gamma-cyclodextrin glucosyltransferase gene engineering bacterium and a construction method thereof, belonging to the technical field of microbial engineering. The invention provides gamma-cyclodextrin glucosyltransferase containing a sequence of SEQ ID NO.1, and successfully adopts a food-safe strain bacillus subtilis to carry out heterologous expression. Moreover, by adopting the method, after the bacillus subtilis expression system is optimized, the extracellular expression level of the enzyme is obviously improved, the extracellular enzyme activity can reach 30.18U/mL, and is improved by 7.4 times compared with the original expression system; the gamma-cyclodextrin glucosyltransferase can directly act on a starch substrate, the conversion rate can reach about 34 percent under the condition of not adding a complexing agent, the gamma-cyclodextrin proportion can reach 56 percent at most, and the gamma-cyclodextrin glucosyltransferase has industrial production potential.
Description
Technical Field
The invention relates to a high-yield gamma-cyclodextrin glucosyltransferase gene engineering bacterium and a construction method thereof, belonging to the technical field of genetic engineering and enzyme engineering.
Background
Cyclodextrins are cyclic oligomers formed by linking D-glucopyranose units via α -1, 4-glycosidic bonds, and the spatial structure thereof is a hollow cylindrical structure, thus having both a hydrophilic surface and a hydrophobic cavity structure, and being an encapsulating material with a wide application prospect, which has been used in the fields of medicine, food, cosmetics, materials, and the like. Cyclodextrins can be classified according to the number of glucose units constituting the cyclodextrin, and the common cyclodextrins composed of 6, 7, 8 glucose units are named α -cyclodextrin, β -cyclodextrin and γ -cyclodextrin, respectively.
Compared with alpha-cyclodextrin and beta-cyclodextrin, gamma-cyclodextrin has the best solubility and the largest cavity, can be basically and completely decomposed by alpha-amylase in vivo, can be quickly degraded and absorbed in gastrointestinal tracts, and is metabolized and discharged, so that the gamma-cyclodextrin has the lowest toxicity, does not have the limitation of the lowest daily intake at present, has wide application prospect in the fields of food, medicine and the like, and mainly shows that: 1) because of the unique cavity structure and excellent solubility of gamma-cyclodextrin, the gamma-cyclodextrin inclusion carrier is an ideal inclusion carrier, and a large cavity structure of the gamma-cyclodextrin inclusion carrier can include macromolecular substances which cannot be included by alpha-cyclodextrin and beta-cyclodextrin, so that the gamma-cyclodextrin inclusion carrier is widely applied to inclusion of medicines and food additives with poor solubility. 2) Because the gamma-cyclodextrin can be rapidly metabolized and discharged in vivo, the gamma-cyclodextrin serving as an inclusion carrier can obviously improve the bioavailability and stability of a guest molecule to achieve the purpose of slow release, and is commonly used as a stabilizer of medicines, spices, sweeteners or pigments and the like to reduce the irritation of the gamma-cyclodextrin or mask the bitter taste. 3) The unique spatial structure of the gamma-cyclodextrin ensures that the gamma-cyclodextrin is widely researched as an organic metal framework material, the organic metal framework material taking the gamma-cyclodextrin as a framework can be used for gas adsorption, separation and purification of substances, capture of high-activity intermediates and the like, and has wide application prospect as a novel material. 4) The structure of the gamma-cyclodextrin is modified and reformed, so that the gamma-cyclodextrin is widely applied to synthetic materials, a series of derivative materials with high added values can be prepared, and the gamma-cyclodextrin can be used in high and new technical fields such as liquid crystal, electronics and the like.
At present, the market scale of the global cyclodextrin industry reaches 13 billion yuan, mainly beta-cyclodextrin is taken as the main material, the yield of gamma-cyclodextrin only accounts for about 5 percent of the global cyclodextrin yield, and the price of the gamma-cyclodextrin is high and is about 80 times of that of the beta-cyclodextrin. The cyclodextrin industry in China starts late, the industrial development of beta-cyclodextrin production is focused, and a mature gamma-cyclodextrin industrial production system does not exist at present. There are two major problems faced in the production of gamma-cyclodextrin: firstly, the gamma-cyclodextrin glucosyltransferase with strong product specificity, high stability and good catalytic efficiency is lacked; secondly, mature and efficient gamma-cyclodextrin separation and purification technology is lacked.
Researchers at home and abroad try to find gamma-cyclodextrin glucosyltransferase with good product specificity and high catalytic efficiency and perform heterologous expression so as to greatly improve the yield of the gamma-cyclodextrin glucosyltransferase and break through the secretion limitation of natural enzyme, but the currently reported gamma-cyclodextrin glucosyltransferase has very limited sources and generally has the problems of low enzyme activity and poor stability, and most of the existing researches adopt escherichia coli for cloning expression and are not beneficial to the application of the enzyme in the fields of food, medicine and the like. Even if the gamma-cyclodextrin glucosyltransferase is secreted and expressed in the bacillus subtilis, the secretion amount is relatively low, so that a food-grade bacillus subtilis secretion expression system which is safe, efficient and high in yield of the gamma-cyclodextrin glucosyltransferase is required to be established.
Disclosure of Invention
In order to solve the problems of low expression level, poor stability and the like of the conventional gamma-cyclodextrin glucosyltransferase, the invention provides a bacillus subtilis system construction method of the gamma-cyclodextrin glucosyltransferase, and the expression level of the gamma-cyclodextrin glucosyltransferase in bacillus subtilis is improved.
The invention provides gamma-cyclodextrin glucosyltransferase, wherein a nucleotide sequence for coding the gamma-cyclodextrin glucosyltransferase is shown as SEQ ID NO. 3.
In one embodiment of the invention, the gamma-cyclodextrin glucosyltransferase is the signal peptide sequence of the parent enzyme of the gamma-cyclodextrin glucosyltransferase which is shown in SEQ ID No.1 after removal of the nucleotide sequence of the signal peptide shown in SEQ ID No. 2.
The invention also provides a recombinant vector carrying the gene of the gamma-cyclodextrin glucosyltransferase and a recombinant cell containing the recombinant vector.
The invention also provides a recombinant Bacillus subtilis which expresses the gamma-cyclodextrin glucosyltransferase derived from Bacillus sp.G-825-6, wherein a nucleotide sequence for coding the gamma-cyclodextrin glucosyltransferase is shown as SEQ ID NO. 3.
In one embodiment of the invention, the recombinant Bacillus subtilis takes Bacillus subtilis WB600, Bacillus subtilis WB800 or Bacillus subtilis 168 as an expression host.
In one embodiment of the present invention, the recombinant bacillus subtilis uses pST plasmid, pHT43 plasmid or pP43NMK plasmid as expression vector.
In one embodiment of the invention, the recombinant Bacillus subtilis takes Bacillus subtilis WB600 as an expression host and pP43NMK plasmid as an expression vector.
The invention also provides a method for improving the expression quantity of the gamma-cyclodextrin glucosyltransferase in the recombinant bacillus subtilis, which is to remove a signal peptide sequence of a gamma-cyclodextrin glucosyltransferase parent enzyme with a coding nucleotide sequence shown as SEQ ID NO.1, wherein the nucleotide sequence of the signal peptide is shown as SEQ ID NO. 2.
In one embodiment of the invention, the recombinant Bacillus subtilis takes Bacillus subtilis WB600 as an expression host and pP43NMK plasmid as an expression vector.
The invention also provides a method for preparing gamma-cyclodextrin by using an enzyme method, which comprises the steps of fermenting the recombinant bacillus subtilis to prepare gamma-cyclodextrin glucosyltransferase, and adding the gamma-cyclodextrin glucosyltransferase into a reaction system containing soluble starch for reaction to prepare the gamma-cyclodextrin.
In one embodiment of the invention, the gamma-cyclodextrin glucosyltransferase is added in an amount of at least: 2U/g (based on the dry mass of the starch).
In one embodiment of the present invention, the reaction conditions in the reaction system are: the concentration of the substrate is 5-10% (w/w), the pH is 8.0-9.0, the reaction temperature is 45-55 ℃, the stirring speed is 200-300 rpm, and the reaction time is 10-12 h.
In one embodiment of the present invention, the reaction substrate may be: corn starch, soluble potato starch, maltodextrin, and the like.
In one embodiment of the invention, gamma-cyclodextrin is prepared by allowing a gamma-cyclodextrin glucosyltransferase obtained by fermentation or a purified enzyme obtained by purification to act on 5% (w/w) of soluble starch at 55 ℃.
The invention also provides a method for preparing gamma-cyclodextrin by using whole cells, which comprises the step of adding the recombinant bacillus subtilis into a reaction system containing soluble starch for reaction to prepare the gamma-cyclodextrin.
In one embodiment of the present invention, the recombinant bacillus subtilis is added in an amount of at least: the final concentration of the wet thallus is 20 g/L.
In one embodiment of the present invention, the reaction conditions in the reaction system are: the concentration of the substrate is 5-10% (w/w), the pH is 8.0-9.0, the reaction temperature is 30-40 ℃, the speed of a water bath shaker is 200-300 rpm, and the reaction time is 10-12 h.
In one embodiment of the invention, the reaction substrate may be: corn starch, soluble potato starch, maltodextrin, and the like.
The invention also provides the recombinant bacillus subtilis or the application of the method in preparing products containing gamma-cyclodextrin.
The invention also provides a method for preparing gamma-cyclodextrin glucosyltransferase, which comprises the following steps: inoculating the recombinant cells or recombinant bacillus subtilis into a seed culture medium to prepare a seed solution, inoculating the seed solution into a fermentation culture medium according to the proportion of 2-4% (v/v), culturing at 30 ℃ for 60-72 h, centrifuging the fermentation liquor, and taking the supernatant to obtain the crude enzyme solution of the gamma-cyclodextrin glucosyltransferase.
Advantageous effects
(1) The invention provides a Bacillus subtilis genetic engineering bacterium for high yield of gamma-cyclodextrin glucosyltransferase, which improves the expression quantity of the gamma-cyclodextrin glucosyltransferase from Bacillus sp.G-825-6 by 7.43 times compared with an original expression system, adopts the Bacillus subtilis expression system to be safe and efficient, can be applied to the production of food and medicines, ensures that the catalytic activity of the expressed gamma-cyclodextrin glucosyltransferase crude enzyme liquid can reach 30.18U/mL, is obviously higher than the heterologous expression level of other reported gamma-cyclodextrin glucosyltransferases at present, and provides a reliable enzyme source for the industrial production of gamma-cyclodextrin.
(2) The gamma-cyclodextrin glucosyltransferase provided by the invention can act on various starches and maltodextrins as substrates, has higher conversion rate when acting on the starches, directly acts on the starches, not only removes the inhibition effect of micromolecule sugars on the generation of the gamma-cyclodextrin, but also reduces the production cost of the cyclodextrin, has the conversion rate of about 34% under the condition of not adding organic solvents as complexing agents, and has the highest proportion of the gamma-cyclodextrin of 56%, thereby providing a production method of the gamma-cyclodextrin with high conversion rate and low cost, and providing guidance for the industrial production of the gamma-cyclodextrin.
(3) Compared with the Bacillus subtilis before optimization, the transformation rate and the product proportion of the genetically engineered Bacillus subtilis strain prepared by the invention are both obviously improved, the total transformation rate is improved by 10.32 percent, and the highest proportion of the gamma-cyclodextrin can reach 61 percent.
Drawings
FIG. 1: the construction process of recombinant plasmid carrying fine gene sequence containing gamma-cyclodextrin glucosyltransferase.
FIG. 2: SDS-PAGE analysis of crude bacillus subtilis enzyme solutions carrying different vectors containing gamma-cyclodextrin glucosyltransferase gene sequences, wherein M: marker; lane 1: pST-cgt; lane 2: pHT 43-cgt; lane 3: pP43 NMK-cgt; lane 4: Δ SP-cgt.
FIG. 3: SDS-PAGE analysis of pure enzyme of γ -cyclodextrin glucosyltransferase, M: marker; lane 1: crude enzyme solution; lane 2: the pure enzyme was purified in two steps.
FIG. 4 is a schematic view of: the recombinant gamma-cyclodextrin glucosyltransferase acts on 5% (w/w) soluble starch to obtain a product HPLC (high performance liquid chromatography) map at different times; wherein, (A)2 h; (B)4 h; (C)8 h; (D) and (4) 12 h.
Detailed Description
The vector pST referred to in the following examples is disclosed in "secretion expression of linear maltooligosaccharide-producing enzyme in Bacillus subtilis, and its enzymatic properties and product synthesis studies, university of Jiangnan, Pan.
The media involved in the following examples are as follows:
LB liquid medium: 1% (w/v) tryptone, 0.5% (w/v) yeast extract, 1% (w/v) sodium chloride, pH 7.0.
LB solid medium: 1.5(w/v) agar was added to the LB liquid medium.
TB liquid medium: 24g/L yeast powder, 12g/L tryptone and KH 2 PO 4 2.32g/L，K 2 HPO 4 ·3H 2 O16.43 g/L, glycerol 5g/L, pH 7.0.
The detection methods referred to in the following examples are as follows:
the method for measuring the enzyme activity of the recombinant gamma-cyclodextrin glucosyltransferase comprises the following steps:
(1) method for measuring gamma-cyclization activity
The gamma-cyclization activity was determined using bromocresol green (BCG) method. 0.1mL of an appropriately diluted enzyme solution was taken, added to a centrifuge tube containing 0.9mL of 1% maltodextrin (DE ═ 4) prepared in advance with 20mM Tris-HCl buffer at pH 9.0, preheated for 5min, reacted at 55 ℃ for 10min, quenched with 100. mu.L of 1.0M hydrochloric acid solution, followed by addition of 2mL of 0.2M, pH 4.2.2 citric acid buffer and 100. mu.L of 5mM bromocresol green solution (BCG), developed at room temperature for 20min, and absorbance was measured at a wavelength of 630 nm. The blank was the inactivated enzyme solution.
One unit of enzyme activity is defined as the amount of enzyme required to produce 1. mu. mol of gamma-cyclodextrin per minute under the above conditions.
(2) Method for measuring hydrolytic activity
The hydrolytic activity was measured by iodometry. 0.1mL of appropriately diluted enzyme solution was added to a centrifuge tube containing 0.5mL of 1.0% soluble starch prepared in advance with 20mM Tris-HCl buffer solution at pH 9.0, and after reaction at 55 ℃ for 10min, enzyme was inactivated in a boiling water bath for 30 min. 0.1mL of the enzyme-inactivated reaction solution was added with 5mL of iodine solution (0.005% I) 2 + 0.05% KI) was developed in the dark for 15min and the absorbance was measured at 660 nm. The inactivated enzyme solution served as a blank.
One unit of enzyme activity is defined as a 10% reduction per minute in absorbance compared to control under the above conditions.
(3) Method for analyzing reaction product
The detection method of cyclodextrin adopts high performance liquid chromatography: inactivating enzyme in boiling water bath for 30min, centrifuging (10,000 × g, 10min), collecting supernatant, filtering with 0.22 μm water system filter membrane, and analyzing by High Performance Liquid Chromatography (HPLC). The HPLC conditions were as follows: waters 600HPLC System (with differential refractometer), column Hypersil GOLD TM Amino HPLC (4.6 mm. times.250 mm), column temperature 30 ℃ and mobile phase 70% (v/v) acetonitrile in water at a flow rate of 1 mL/min.
Example 1: construction of recombinant Bacillus subtilis secretion expression system
The method comprises the following specific steps:
(1) chemically synthesizing the nucleotide sequence of the parent enzyme of the gamma-cyclodextrin glucosyltransferase as shown in SEQ ID NO. 1.
(2) And (2) carrying out PCR amplification by taking the sequence of SEQ ID NO.1 as a template, wherein target gene amplification primers used for pST vector plasmids are P1/P2 in table 1, plasmid primers used for pHT43 vector are P3/P4 in table 1, and plasmid used for pP43NMK vector is P5/P6 in table 1.
TABLE 1 primers required for the construction of the Bacillus subtilis expression System
The PCR system is as follows: taq Buffer (Mg) 2+ Plus) 10. mu.L, dNTP mix (2.5 mM each) 4. mu.L, forward primer (10. mu.M) 1. mu.L, reverse primer (10. mu.M) 1. mu.L, template DNA 1. mu.L, Taq DNA Polymerase (1.25U/. mu.L) 1. mu.L, and double distilled water was added to 50. mu.L. The PCR amplification conditions were: pre-denaturation at 98 ℃ for 3 min; then 30 cycles (98 ℃ for 10s, 60 ℃ for 15s, 68 ℃ for 2min) are carried out; finally, the temperature is kept for 10min at 68 ℃.
(3) And (3) respectively connecting the genes of the gamma-cyclodextrin glucosyltransferase parent enzymes obtained in the step (2) with corresponding plasmid vectors (pST vector, pHT43 vector and pP43NMK vector) by using a homologous recombination method. The connecting system is as follows: 1 μ L of purified gamma-cyclodextrin glucosyltransferase PCR fragment (50 ng/. mu.L), 2 μ L of purified plasmid vector PCR fragment (50 ng/. mu.L), 4 μ L of 5 × CE II Buffer, 2 μ L of Exnase II, ddH 2 O11. mu.L. The homologous recombination conditions are as follows: incubating at 37 ℃ for 30 min; coli JM109 was then transformed, LB solid medium with 10. mu.g/mL ampicillin was applied, and transformants were picked for sequencing verification to obtain a recombinant vector containing the gamma-cyclodextrin glucosyltransferase parental enzyme gene: pST-cgt, pHT43-cgt, pP43 NMK-cgt;
and transforming the recombinant vector into B.subtilis WB600 to obtain the genetic engineering bacteria B.subtilis WB600/pST-cgt, B.subtilis WB600/pHT43-cgt and B.subtilis WB600/pP43NMK-cgt respectively.
Example 2: screening of recombinant Bacillus subtilis secretion expression system
The method comprises the following specific steps:
(1) inoculating single colonies of the genetically engineered bacteria B.subtilis WB600/pST-cgt, B.subtilis WB600/pHT43-cgt and B.subtilis WB600/pP43NMK-cgt prepared in example 1 into 50mL of LB liquid culture medium containing 5. mu.g/mL of kanamycin or 10. mu.g/mL of chloramphenicol, and culturing at 37 ℃ and 200rpm for 8-12 h to respectively prepare seed solutions;
respectively transferring the seed liquid obtained by the preparation to 50mL of TB liquid culture medium containing 5 mu g/mL kanamycin or 10 mu g/mL chloramphenicol according to the inoculation amount of 4% (v/v), and performing shake-flask culture at 30 ℃ and 200rpm for 60-72 h to prepare fermentation liquid; the obtained fermentation liquid is centrifuged at 10000rpm for 10min at 4 deg.C, and the supernatant is the crude extracellular enzyme solution (as shown in FIG. 2).
(2) The enzyme activity of the crude enzyme solution after the fermentation of the strain is respectively detected by measuring the activity of the gamma-cyclodextrin glucosyltransferase, and the results are shown in table 2:
table 2: comparison of enzyme activities of different Bacillus subtilis expression systems
As can be seen from table 2, strains carrying different vectors can successfully express the gamma-cyclodextrin glucosyltransferase with small difference in expression level, and the recombinant gamma-cyclodextrin glucosyltransferase prepared by fermentation has the highest activity by using pP43NMK as an expression vector and b.subtilis WB600 as an expression host, so that the subsequent optimization is continued by using the expression system.
Example 3: optimized modification of recombinant bacillus subtilis secretion expression system
The method comprises the following specific steps:
(1) synthesizing gamma-cyclodextrin glucosyltransferase delta SP-cgt.
The gamma-cyclodextrin glucosyltransferase delta SP-cgt with the nucleotide sequence shown as SEQ ID NO.3 is obtained by removing the signal peptide segment SEQ ID NO.2 by taking the gamma-cyclodextrin glucosyltransferase parent enzyme sequence with the nucleotide sequence shown as SEQ ID NO.1 as a template.
(2) And (2) performing PCR amplification by using the gamma-cyclodextrin glucosyltransferase delta SP-cgt sequence synthesized in the step (1) as a template, wherein the primer is P7/P8 in the table 3. The PCR conditions were the same as in example 1.
Table 3: primer required for optimizing bacillus subtilis expression system
(3) Connecting the PCR fragment obtained in the step (2) with a vector pP43NMK by using a homologous recombination method, and obtaining a recombinant vector containing a gamma-cyclodextrin glucosyltransferase delta SP-cgt gene by the following specific steps in the same manner as in example 1: pP43 NMK-. DELTA.SP-cgt (construction method shown in FIG. 1).
And transforming the recombinant vector into B.subtilis WB600 to finally obtain the genetic engineering bacterium B.subtilis WB600/pP43 NMK-delta SP-cgt.
Example 4: determination of expression level of recombinant gamma-cyclodextrin glucosyltransferase Δ SP-cgt
The method comprises the following specific steps:
(1) inoculating the B.subtilis WB600/pP43 NMK-delta SP-cgt single colony prepared in example 3 into 50mL LB liquid culture medium containing 5 mu g/mL kanamycin, and performing shake culture at 37 ℃ and 200rpm for 8-12 h to prepare a seed solution;
transferring the seed solution obtained by the preparation into 50mL of TB liquid culture medium containing 5 mu g/mL kanamycin according to the inoculation amount of 4% (v/v), and performing shake-flask culture at 30 ℃ and 200rpm for 60-72 hours to prepare a fermentation solution; the obtained fermentation liquid was centrifuged at 10000rpm at 4 deg.C for 10min, and the supernatant was obtained as crude extracellular enzyme (as shown in FIG. 2).
(2) The activity of the gamma-cyclodextrin glucosyltransferase is measured, the enzyme activity of crude enzyme solution after B.subtilis WB600/pP43 NMK-delta SP-cgt strain fermentation is detected, and the result shows that: the cyclization activity of the gamma-cyclodextrin glucosyltransferase delta SP-cgt is 30.18 +/-0.20U/mL, and the hydrolysis activity is 522.83 +/-1.06U/mL.
It can be seen that the expression level of the optimized B.subtilis WB 600/delta SP-cgt strain is remarkably increased, the crude enzyme activity gamma-cyclization activity is 7.4 times that of the control group (B.subtilis WB600/pP43NMK-cgt) before optimization, and the crude enzyme activity hydrolysis activity is 3.95 times that of the control group (B.subtilis WB600/pP43NMK-cgt) before optimization.
(3) Purifying the gamma-cyclodextrin glucosyltransferase delta SP-cgt extracellular crude enzyme solution prepared in the step (1) by adopting a two-step purification method:
the first step is as follows: Q-HP column anion exchange chromatography
The equilibration solution (solution A, pH 7.0) was 20mM Tris-HCl buffer, and the eluent (solution B, pH 7.0) was 1M NaCl +20mM Tris-HCl buffer. And (3) adjusting the pH of the crude enzyme solution to 7.0, performing gradient elution at the flow rate of 1mL/min, and collecting 40-70% of B solution elution solution for next hydrophobic column purification.
The second step is that: phenyl HP column hydrophobic chromatography
The equilibrium solution (solution A, pH 7.0) contained 30% (w/v) NH 4 (SO 4 ) 2 And (3) diluting the eluent collected in the first step to a proper concentration at a flow rate of 1mL/min by using 20mM Tris-HCl buffer solution and the eluent (solution B, pH 7.0) of 20mM Tris-HCl buffer solution, adopting gradient elution, collecting 70% B eluent, and concentrating to obtain the pure enzyme.
(4) The protein was identified by SDS-PAGE protein electrophoresis (results are shown in FIG. 3) and enzyme activity assay, and the protein concentration was measured to calculate the purification efficiency, the results are shown in Table 4.
Table 4: purification and identification of gamma-cyclodextrin glucosyltransferase
The molecular weight of the recombinant gamma-cyclodextrin glucosyltransferase was reported to be 78kDa, consistent with the molecular weight estimated by SDS-PAGE.
And detecting the enzyme activity of the pure enzyme solution, wherein the result shows that the enzyme activity of the cyclization reaction of the purified gamma-cyclodextrin glucosyltransferase is as follows: 67.92U/mg, the enzyme activity of the hydrolysis reaction is: 1176.37U/mg.
Example 5: preparation of gamma-cyclodextrin
Production of gamma-cyclodextrin was carried out using soluble potato starch as substrate and pure enzyme (added in the amount of cyclization enzyme activity) obtained in example 4, as follows:
(1) 100g of a 5% (w/w) soluble starch solution was prepared in 20mM Tris-HCl buffer, pH 9.0.
(2) Pouring the soluble starch solution obtained in the step (1) into a four-neck flask, placing the flask in a 55 ℃ water bath kettle, and preheating for 15min, wherein the rotating speed of a stirring head is 200-300 rpm, so as to obtain a reaction system;
adding the gamma-cyclodextrin glucosyltransferase pure enzyme solution prepared in example 4 into a reaction system; adding 5U/g (dry starch), reacting in a water bath kettle at 55 deg.C for 2h, 4h, 8h, and 12h, respectively, sampling, reacting for 12h, transferring to boiling water, inactivating enzyme for 20min to terminate the reaction;
in this case, the enzyme solution was replaced with an equal amount of Tris-HCl buffer as a blank.
(3) After enzyme deactivation, the obtained samples are respectively centrifuged for 10min under the condition of 10000rpm, and supernate is respectively taken and filtered through a water system film with the thickness of 0.22 mu m to remove impurities, and then the category and the concentration of cyclodextrin products in reaction liquid can be analyzed by high performance liquid chromatography. The results are shown in Table 5, and the results of the chromatographic analysis are shown in FIG. 4.
Table 5: production system result of gamma-cyclodextrin
nd: not detected, no result detected.
The result shows that the main reaction products are beta-cyclodextrin and gamma-cyclodextrin, the proportion of the beta-cyclodextrin and the gamma-cyclodextrin is almost maintained at 1:1 in the early stage of the reaction, the proportion of the gamma-cyclodextrin is highest when the gamma-cyclodextrin reacts for 2-4 hours, the highest proportion can reach 56.7%, a small amount of alpha-cyclodextrin is generated in the final stage of the reaction, the total conversion rate can reach about 34%, but because no organic solvent is added in the reaction process for complexing, the proportion of the gamma-cyclodextrin is reduced due to the inhibition effect of the product in the final stage of the reaction.
Example 6: preparation of gamma-cyclodextrin by whole cell transformation
The method comprises the following specific steps:
reaction system: 100g of soluble starch solution with the mass fraction of 5% (w/w) prepared by 20mM Tris-HCl buffer solution with the pH value of 9.0 is used as a substrate, wet bacteria (the final concentration of the wet bacteria is 20g/L) of the recombinant bacillus subtilis WB600/pP43NMK-cgt and the recombinant bacillus subtilis WB600/pP43 NMK-delta SP-cgt prepared in the embodiment 1-3 are respectively added, the reaction temperature is controlled to be 30 ℃, the reaction time is 12 hours, and after the reaction is finished, supernatant is obtained by centrifugation, namely the reaction product containing the gamma-cyclodextrin.
The result shows that compared with the B.subtilis WB600/pP43NMK-cgt before optimization, the conversion rate and the product ratio of the optimized thallus whole cell B.subtilis WB600/pP43 NMK-delta SP-cgt are both obviously improved.
Bacillus subtilis WB600/pP43 NMK-delta SP-cgt whole cell transformation is adopted to prepare the gamma-cyclodextrin, the total transformation rate is improved by 10.32%, and the highest proportion of the gamma-cyclodextrin can reach 61%.
However, the whole cell transformation needs to consider the cell state, so the reaction temperature is not the optimal reaction temperature of the gamma-cyclodextrin glucosyltransferase, so the transformation rate is integrally lower than that of the direct enzyme liquid reaction, but the result shows that the optimized B.subtilis WB 600/delta SP-cgt strain has better gamma-cyclodextrin production potential obviously.
Comparative example 1:
the specific implementation manner is the same as that of examples 1-2, except that host bacteria are adjusted to bacillus subtilis WB800 and BS168, namely: the method adopts pP43NMK as an expression vector, adopts bacillus subtilis WB800 and BS168 to express the recombinant gamma-cyclodextrin glucosyltransferase (the nucleotide sequence is shown as SEQ ID NO. 1), and detects the enzyme activity of the cyclization reaction of the recombinant gamma-cyclodextrin glucosyltransferase obtained by different hosts.
The result shows that the enzyme activity of the crude enzyme solution of the recombinant gamma-cyclodextrin glucosyltransferase prepared by taking the bacillus subtilis WB800 as a host can reach 3.99U/mL, and the highest enzyme activity of the crude enzyme solution can reach 3.86U/mL by taking the bacillus subtilis BS168 as the host.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
SEQUENCE LISTING
<110> university of south of the Yangtze river
<120> high-yield gamma-cyclodextrin glucosyltransferase gene engineering bacteria and construction method thereof
<130> BAA220562A
<160> 3
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ggagtcccaa cgatttatta tggtacggaa atttatatga caggtgatgg ggacccagat 1080
aatcggaaaa tgatgaatac atttgatcaa tcgacagttg cctatcaaat cattcaacgc 1140
ctttcatcac tgcggcaaga aaatagggcg attgcttatg gggatacgac ggaacgatgg 1200
ataaatgaag atgtattcat ttatgaacgt tcatttaatg gagaatatgc acttattgct 1260
gtgaaccgaa acttaaaccg ctcttatcag attagtagtt tggtaacgga tatgccttct 1320
caattatatg aagatgagct gtcaggtctt ttagacgggc aatcgataac cgtcgcacaa 1380
gatgggtctg ttcagccctt tttgttagcc ccaggtgaag taagtgtttg gcaatactca 1440
aatggtcaga atgtagcacc ggaaattggt caaattggtc ctcctattgg gaaaccagga 1500
gatgaagtga ggatcgatgg ttcaggtttt ggaaatagta tggggaatgt ttcttttgcg 1560
ggttcaacta tgaatgtatt gtcttggaat gacgagacaa ttatagccga actacctgtg 1620
cataatggtg gaaaaaatag tataactgta acgactaact caggtgaaag cagtaatggt 1680
tatccgtttg aattattaac tggttcacaa acatctgtaa gatttgtcgt gaatcaagcc 1740
gaaacgtctg ttggtgaaaa tctgtactta gttggtaatg tacctgaatt agggagctgg 1800
gatcctgata aagcaattgg tcctatgttt aatcaagttt tatactcata tcccacttgg 1860
tattatgatg tgagtgtacc tgctaatcaa gatatagagt acaaatatat tatgaaagat 1920
caaaatggaa atgtaagctg ggaaagtgga ggcaaccata tctatagaac accagaaaat 1980
tctactggaa tcgtagaagt gaattacaat caataa 2016
Claims (10)
1. A recombinant Bacillus subtilis expressing a gamma-cyclodextrin glucosyltransferase from Bacillus sp.G-825-6, wherein the nucleotide sequence encoding the gamma-cyclodextrin glucosyltransferase is shown in SEQ ID No. 3.
2. The recombinant Bacillus subtilis of claim 1 wherein the recombinant Bacillus subtilis is a Bacillus subtilis WB600, Bacillus subtilis WB800 or Bacillus subtilis 168 as an expression host.
3. The recombinant Bacillus subtilis of claim 1 or claim 2 wherein the recombinant Bacillus subtilis is an expression vector selected from the group consisting of the pST plasmid, the pHT43 plasmid and the pP43NMK plasmid.
4. A method for improving the expression quantity of gamma-cyclodextrin glucosyltransferase in recombinant bacillus subtilis is characterized in that a signal peptide sequence of a gamma-cyclodextrin glucosyltransferase parent enzyme with a coding nucleotide sequence shown as SEQ ID No.1 is removed, and the nucleotide sequence of the signal peptide is shown as SEQ ID No. 2.
5. The method of claim 4, wherein the recombinant Bacillus subtilis is a Bacillus subtilis WB600 expression host and a pP43NMK plasmid is an expression vector.
6. A method for preparing gamma-cyclodextrin by an enzymatic method, which is characterized in that the method comprises the steps of fermenting the recombinant Bacillus subtilis of any one of claims 1-3 to prepare gamma-cyclodextrin glucosyltransferase, adding the gamma-cyclodextrin glucosyltransferase into a reaction system containing starch or maltodextrin, and reacting to prepare the gamma-cyclodextrin.
7. The method of claim 6, wherein the γ -cyclodextrin glucosyltransferase is added in an amount of at least: 2U/g (based on the dry mass).
8. The method according to claim 6 or 7, wherein the reaction conditions in the reaction system are: the concentration of the substrate is 5-10% (w/w), the pH value is 8.0-9.0, the reaction temperature is 45-55 ℃, the stirring speed is 200-300 rpm, and the reaction time is 10-12 h.
9. A method for preparing gamma-cyclodextrin through whole cells, which is characterized in that the recombinant Bacillus subtilis of any one of claims 1 to 3 is added into a reaction system containing soluble starch or maltodextrin for reaction to prepare the gamma-cyclodextrin.
10. Use of a recombinant bacillus subtilis according to any one of claims 1 to 3 or a method according to any one of claims 6 to 9 for the preparation of a product containing γ -cyclodextrin.
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