CN112980754B - Method for preparing inositol by catalyzing starch with bacillus subtilis whole cells - Google Patents
Method for preparing inositol by catalyzing starch with bacillus subtilis whole cells Download PDFInfo
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- CN112980754B CN112980754B CN201911282987.XA CN201911282987A CN112980754B CN 112980754 B CN112980754 B CN 112980754B CN 201911282987 A CN201911282987 A CN 201911282987A CN 112980754 B CN112980754 B CN 112980754B
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- inositol
- thermostable
- heat
- expressing
- resistant
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- 229960000367 inositol Drugs 0.000 title claims abstract description 95
- CDAISMWEOUEBRE-GPIVLXJGSA-N inositol Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O CDAISMWEOUEBRE-GPIVLXJGSA-N 0.000 title claims abstract description 95
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Classifications
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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)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
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- 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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- 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.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
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Abstract
The invention discloses a method for preparing high-concentration inositol by using bacillus subtilis whole cell catalytic high-concentration starch, which comprises the steps of constructing engineering bacteria and/or engineering bacteria mixtures which co-express or independently express heat-resistant alpha-glucan phosphorylase, heat-resistant glucose phosphomutase, heat-resistant inositol-3-phosphate synthase and heat-resistant inositol monophosphorylase, carrying out cell membrane permeability treatment on the engineering bacteria and/or engineering bacteria mixtures, and then converting starch into inositol by using the permeable engineering bacteria and/or the permeable engineering bacteria mixtures. Compared with the existing method for producing inositol, the method provided by the invention has the advantages of full cell recycling, high safety performance, high yield, simple production process, low cost, easiness in large-scale preparation and the like.
Description
Technical Field
The invention relates to the technical field of bioengineering, in particular to a method for preparing high-concentration inositol by catalyzing high-concentration starch with bacillus subtilis whole cells, belonging to the field of inositol preparation.
Background
Inositol, also known as cyclohexanoyl alcohol, is one of the water-soluble vitamin B groups and is widely used in the industries of feed, food, medicine and the like. The inositol with the concentration of 0.2-0.5% is added into the feed, so that the livestock growth can be effectively promoted and the death can be prevented. The inositol is added in the goldfish cultivation process to obviously improve the swimming ability of goldfish, and the inositol with a certain amount is added in the feed for fish and shrimp to promote the growth of fish and shrimp, so that the growth speed of fish and shrimp is improved by more than 10%. Inositol is an essential substance for human, animal and microorganism growth, so that it is often used as a nutrition enhancer in health products, beverages (red ox functional beverage) and milk products, and it was recommended in 1987 that certain inositol be added to infant foods. Inositol-containing food and nutritional health product for reducing weight, reducing blood lipid and building body are popular in Europe and America. Inositol has good medicinal value, can promote metabolism of liver and fat, treat various vitamin deficiency diseases, is widely used for preparing compound vitamin preparation raw materials, can treat various diseases such as liver cirrhosis, fatty liver, vascular sclerosis, hypercholesteremia, obesity, diabetes, carbon tetrachloride poisoning and the like, and has the medical effects of promoting cell growth and preventing aging. Inositol is used as pharmaceutical intermediate for synthesizing inositol nicotinate, maitong, inositol selenate, etc. for treating hypercholesterolemia, arteriosclerosis, diabetes, cancer, etc. Wherein, inositol selenate is called as anticancer king in microelements, and can be used for preparing selenium-enriched anticancer drugs, foods and beverages. The new product of the fluoroinositol developed in recent years has the functions of resisting and treating cancers and improving immunity. Inositol and its derivatives can also treat depression and obsessive compulsive disorder by a complex amine reuptake inhibitor other than blood.
Currently, chemical production of inositol is mainly performed by conventional high-temperature pressurized hydrolysis of phytic acid (inositol hexaphosphate). The process equipment has strict material requirements, large one-time investment and operation pressure which can only be controlled within a certain range, the improvement of the utilization rate of raw materials is limited, the refining process of the crude product is complex, the loss is more, the production cost is higher, and the process can generate a large amount of phosphoric acid pollutants and has serious pollution to water sources and environment.
There are documents and patents reporting that inositol is produced by using glucose as a substrate by a microbial fermentation method. Introducing an inositol-1-phosphate synthase derived from Saccharomyces cerevisiae into Escherichia coli, and overexpressing an inositol monophosphorylase of Escherichia coli itself, thereby converting glucose to produce inositol. However, glucose in microbial cells acts as a substrate and is involved in multiple metabolic reactions for maintaining cellular metabolism; in addition to the low conversion rate of glucose to inositol, which is obtained by phosphorylating glucose, glucose-6-phosphate requires energy (ATP or phosphoenolpyruvate), E.coli contains endotoxin, and the use of E.coli as a host for the production of products in the food preparation industry is limited.
Zhang Yiheng and natans invents an enzymatic conversion method of inositol (CN 106148425A, preparation method of inositol), which provides an enzymatic conversion method of inositol, and a method for producing inositol by catalyzing starch or cellulose and derivatives thereof and glucose with multiple enzymes in vitro, which has the advantages of high yield and conversion rate of inositol, etc. However, in the method, the fermentation production host of the key enzyme is escherichia coli BL21 (DE 3), and escherichia coli contains endotoxin which is not suitable for industrial production as a production strain of the related enzyme for producing the food preparation. In addition, the concentration of the substrate starch is 10g/L, the substrate concentration is very low, the yield of the product inositol is lower, and the industrialized mass production application of the high-concentration product can not be realized.
Therefore, development of a new method for recycling whole cells, high safety, high yield, simple production process, low cost, suitability for high substrate concentration delivery and high product inositol output, and easy large-scale preparation of inositol is needed.
Disclosure of Invention
Aiming at the problems of the existing method for preparing inositol by multienzyme catalysis, such as that the endotoxin contained in escherichia coli is unfavorable for the industrial production of food preparations, the purification steps are complicated, the recycling rate of enzyme is low, the recycling is difficult, and the feeding of low-concentration substrate starch is difficult, the main purpose of the invention is to provide a method for preparing high-concentration inositol by using bacillus subtilis whole-cell catalytic high-concentration starch.
In order to solve the technical problems, the invention adopts the following technical scheme:
according to one aspect, the invention relates to a method for producing inositol by using bacillus subtilis whole cells to catalyze high-concentration starch, which is characterized by comprising the following steps:
(1) Constructing bacillus subtilis host bacteria for knocking out alpha-amylase;
(2) Constructing engineering bacteria which co-express heat-resistant alpha-glucan phosphorylase, heat-resistant glucose phosphomutase, heat-resistant inositol-3-phosphate synthase and heat-resistant inositol monophosphate enzyme and/or bacillus subtilis engineering bacteria which respectively express heat-resistant alpha-glucan phosphorylase, heat-resistant glucose phosphomutase, heat-resistant inositol-3-phosphate synthase and heat-resistant inositol monophosphate enzyme on the basis of the host bacteria constructed in the step (1);
(3) Fermenting the bacillus subtilis engineering bacteria constructed in the step (2) to obtain whole cells;
(4) Performing cell membrane permeability treatment on the bacillus subtilis whole cells obtained in the step (3) to obtain permeable whole cells;
(5) Preparing inositol by catalyzing starch with a mixture of the co-expressed thermostable α -glucan phosphorylase, thermostable glucose phosphomutase, thermostable inositol-3-phosphate synthase, and thermostable inositol monophosphorylase permeable whole cells obtained in step (4), the thermostable α -glucan phosphorylase-expressing permeable whole cells, the thermostable glucose phosphomutase-expressing permeable whole cells, the thermostable inositol-3-phosphate synthase-expressing permeable whole cells, and the thermostable inositol monophosphorylase-expressing permeable whole cells.
According to the invention, the amyE gene in the bacillus subtilis encodes alpha-amylase and can be secreted to extracellular degradation substrate starch in the fermentation process, and in order to avoid degrading the substrate starch in amylase produced by fermenting the genetic engineering bacteria so as to influence the application of substrate conversion to produce inositol, the invention knocks out the amyE gene of the alpha-amylase protein encoding gene in a bacillus subtilis host, and constructs bacillus subtilis host bacteria knocked out the alpha-amylase.
It will be appreciated by those skilled in the art that various strains of Bacillus subtilis known in the art can be used as starting strains in the present invention. Preferably, the bacillus subtilis starting strain is a protease knock-out bacillus subtilis strain, such as WB800, WB600, SCK6, 1a751 and the like. More preferably, the bacillus subtilis starting strain is SCK6.
According to the invention, the catalytic pathway comprises: converting the substrate starch into an intermediate glucose-1-phosphate (G1P) by a thermostable α -glucan phosphorylase in the presence of inorganic phosphorus; shifting an intermediate glucose-1-phosphate (G1P) to another intermediate glucose-6-phosphate (G6P) by a thermostable glucose phosphomutase; isomerising the intermediate glucose-6-phosphate (G6P) to another intermediate inositol-1-phosphate (I1P) by a thermostable inositol-3-phosphate synthase; the intermediate Inositol-1-phosphate (I1P) is dephosphorylated by thermostable Inositol monophosphorylase to produce Inositol (Inositol).
Preferably, according to the present invention, in step (2), the engineering bacterium comprises a vector co-expressing a thermostable α -glucan phosphorylase, a thermostable glucose phosphomutase, a thermostable inositol-3-phosphate synthase, and a thermostable inositol monophosphorylase, or comprises a vector expressing a thermostable α -glucan phosphorylase, or comprises a vector expressing a thermostable glucose phosphomutase, or comprises a vector expressing a thermostable inositol-3-phosphate synthase, or comprises a vector expressing a thermostable inositol monophosphorylase. It will be understood by those skilled in the art that the vector and engineering bacteria related to the present invention may be prepared by conventional methods known in the art, for example, by constructing by recombinant DNA technology, obtaining the coding gene αgp of α -glucan phosphorylase, the coding gene pgm of glucose phosphate mutase, the coding gene ips of inositol-3-phosphate synthase, the coding gene imp of inositol monophosphate phosphorylase, constructing recombinant expression vector, and transferring into host bacteria to obtain genetically engineered bacteria.
Preferably, according to the present invention, in step (2), "thermostable α -glucan phosphorylase" refers to an enzyme having a function of phosphorylating starch into glucose-1-phosphate (G1P) at 50 ℃ or higher, 55 ℃ or higher, 60 ℃ or higher, 65 ℃ or higher, 70 ℃ or higher, 75 ℃ or higher, or 80 ℃ or higher.
Further preferably, the thermostable α -glucan phosphorylase is derived from thermophilic microorganisms such as geobacillus stearothermophilus (Geobacillus kaustophilus), geobacillus stearothermophilus (Geobacillus stearothermophilus), thermomyces maritimus (Thermotoga maritima), thermococcus kodakarensis, etc.; or the amino acid sequence of the thermostable a-glucan phosphorylase has at least 70%, preferably at least 80%, more preferably at least 90%, most preferably at least 95% identity with the thermostable a-glucan phosphorylase derived from the thermophilic microorganism.
More preferably, the thermostable α -glucan phosphorylase is derived from Thermococcus kodakarensis; or the amino acid sequence of the thermostable a-glucan phosphorylase has at least 70%, preferably at least 80%, more preferably at least 90%, most preferably at least 95% identity with the thermostable a-glucan phosphorylase derived from Thermococcus kodakarensis.
Preferably, according to the present invention, in the step (2), the "thermostable glucose phosphomutase" refers to an enzyme having a function of shifting glucose-1-phosphate (G1P) to glucose-6-phosphate (G6P) at 50℃or higher, 55℃or higher, 60℃or higher, 65℃or higher, 70℃or higher, 75℃or higher, or 80℃or higher.
Further preferably, the thermostable glucose phosphomutase is derived from a thermophilic microorganism, such as Geobacillus stearothermophilus (Geobacillus kaustophilus), geobacillus stearothermophilus (Geobacillus stearothermophilus), thermotoga maritima (Thermotoga maritima), thermococcus kodakarensis, etc.; or the amino acid sequence of the thermostable glucose phosphomutase has at least 70%, preferably at least 80%, more preferably at least 90%, most preferably at least 95% identity with the thermostable glucose phosphomutase derived from the thermophilic microorganism.
More preferably, the thermostable glucose phosphomutase is derived from Thermococcus kodakarensis; or the amino acid sequence of the thermostable glucose phosphomutase has at least 70%, preferably at least 80%, more preferably at least 90%, most preferably at least 95% identity to the thermostable glucose phosphomutase derived from Thermococcus kodakarensis.
Preferably, according to the present invention, in the step (2), the "thermostable inositol-3-phosphate synthase" refers to an enzyme having a function of shifting glucose-6-phosphate (G1P) to inositol-1-phosphate (I1P) at 50℃or higher, 55℃or higher, 60℃or higher, 65℃or higher, 70℃or higher, 75℃or higher, or 80℃or higher.
Further preferably, the thermostable inositol-3-phosphate synthase is derived from thermophilic microorganisms such as Geobacillus stearothermophilus (Geobacillus kaustophilus), geobacillus stearothermophilus (Geobacillus stearothermophilus), thermotoga maritima (Thermotoga maritima), thermococcus kodakarensis, archaeoglobus fulgidus, etc.; or the amino acid sequence of said thermostable inositol-3-phosphate synthase has at least 70%, preferably at least 80%, more preferably at least 90%, most preferably at least 95% identity with the thermostable inositol-3-phosphate synthase derived from said thermophilic microorganism.
More preferably, the thermostable inositol-3-phosphate synthase is derived from Archaeoglobus fulgidus; or the amino acid sequence of the thermostable inositol-3-phosphate synthase has at least 70%, preferably at least 80%, more preferably at least 90%, most preferably at least 95% identity with the thermostable inositol-3-phosphate synthase derived from Archaeoglobus fulgidus.
Preferably, according to the present invention, in step (2), the "Inositol monophosphorylase" means an enzyme having a function of dephosphorylating Inositol-1-phosphate (I1P) to Inositol (inonitol) at 50 ℃ or higher, 55 ℃ or higher, 60 ℃ or higher, 65 ℃ or higher, 70 ℃ or higher, 75 ℃ or higher, or 80 ℃ or higher.
Further preferably, the inositol monophosphorylase is derived from a thermophilic microorganism such as geobacillus stearothermophilus (Geobacillus kaustophilus), geobacillus stearothermophilus (Geobacillus stearothermophilus), thermomyces maritimus (Thermotoga maritima), thermococcus kodakarensis, or the like; or the amino acid sequence of said inositol monophosphorylase has at least 70%, preferably at least 80%, more preferably at least 90%, most preferably at least 95% identity with the inositol monophosphorylase derived from said thermophilic microorganism.
More preferably, the inositol monophosphorylase is derived from Thermotoga maritima; or the amino acid sequence of the inositol monophosphorylase has at least 70%, preferably at least 80%, more preferably at least 90%, most preferably at least 95% identity with the inositol monophosphorylase derived from Thermotoga maritima.
Preferably, according to the present invention, the co-expressed vector includes a promoter, a thermostable α -glucan phosphorylase gene, a thermostable glucose phosphomutase gene, a thermostable inositol-3-phosphate synthase gene, a thermostable inositol monophosphorylase gene, and a terminator; the vector for expressing the heat-resistant alpha-glucan phosphorylase comprises a promoter, a heat-resistant alpha-glucan phosphorylase gene and a terminator; the vector for expressing the thermostable glucose phosphomutase comprises a promoter, a thermostable glucose phosphomutase gene and a terminator; the vector for expressing the heat-resistant inositol-3-phosphate synthase comprises a promoter, a heat-resistant inositol-3-phosphate synthase gene and a terminator; the vector for expressing the heat-resistant inositol monophosphate enzyme comprises a promoter, a heat-resistant inositol monophosphate enzyme gene and a terminator.
Those skilled in the art will appreciate that various promoters known in the art may be used as promoters in the present invention, including, but not limited to, the P43 promoter, the Pylb promoter, the PamyL promoter, the Plaps promoter, the PhpaII promoter, the PamyE promoter, the Pgrad promoter, and the like. Preferably, the promoter of the present invention is selected from the PhpaII promoter and the Pylb promoter in tandem.
According to the invention, in step (3), the preparation of the whole cells is carried out using methods known in the art. The fermentation may use any medium suitable for the expression of the foreign protein, including but not limited to LB medium, TB medium, and the like.
According to the present invention, in step (4), the cell membrane permeability treatment includes, but is not limited to, heat treatment, addition of an organic solvent, addition of a surfactant, and/or the like. Among them, the organic solvents include, but are not limited to, acetone, acetonitrile, etc. Surfactants include, but are not limited to, cetyltrimethylammonium bromide (CTAB), tween-80, and the like. Preferably, the cell membrane permeability treatment is a heat treatment. The purpose of the permeability treatment of the cell membrane is to allow extracellular starch to enter the cell through the cell membrane.
Preferably, the heat treatment temperature is 45-95 ℃; more preferably, the heat treatment temperature is 60-80 ℃.
Preferably, the heat treatment time is 1-60min; more preferably, the heat treatment time is 10 to 50 minutes.
Preferably, the cell concentration at the time of heat treatment is OD 600 =10-300; more preferably, the cell concentration is OD 600 ＝30-150。
According to the invention, the heat treatment can be carried out in a buffer-free system or in a buffer system; preferably, the heat treatment is performed in a buffer system, which may be HEPES buffer, phosphate buffer, tris buffer, acetate buffer, etc. Among them, phosphate buffers such as sodium phosphate buffer, potassium phosphate buffer, and the like.
Preferably, according to the present invention, in the reaction system for preparing inositol by catalyzing starch with permeable whole cells or a mixture of permeable whole cells in the step (5), the concentration of the substrate starch is 10-300g/L; more preferably, the concentration of substrate starch is 20-200g/L.
Preferably, according to the present invention, in step (4), the reaction conditions for preparing inositol using permeable whole cells or a mixture of permeable whole cells for catalyzing starch are: reacting for 0.5-96h at the pH of 6.0-8.0 and the temperature of 50-80 ℃; more preferably at pH 6.5-7.5, 55-75 ℃ for 12-60 hours; most preferably at pH 7.0, 60-65℃for 12-96h.
According to the invention, the catalytic reaction using the permeable whole cells or the mixture of permeable whole cells can be performed in a buffer-free system or a buffer system; preferably, the catalytic reaction using the permeable whole cells or the mixture of permeable whole cells is performed in a buffer system, and the buffer may be HEPES buffer, phosphate buffer, tris buffer, acetate buffer, etc. Among them, phosphate buffers such as sodium phosphate buffer, potassium phosphate buffer, and the like.
Preferably, according to the present invention, in step (5), the ratio of the permeant whole cells expressing thermostable α -glucan phosphorylase, the permeant whole cells expressing thermostable glucose phosphomutase, the permeant whole cells expressing thermostable inositol-3-phosphate synthase, the permeant whole cells expressing thermostable inositol monophosphorylase is 0.1:1:1:0.1 to 10:1:1:10 in the mixture of permeant whole cells; more preferably 0.5:1:1:0.5-5:1:1:5.
According to the invention, it is also possible to use a mixture of permeable whole cells to catalyze starch to prepare inositol, the reaction conditions being: reacting for 0.5-96h at the pH of 6.0-8.0 and the temperature of 50-80 ℃; more preferably at a pH of 6.5-7.5 at 55-75℃for 12-96h.
According to another aspect of the present invention, the present invention relates to the above-described vectors co-expressing thermostable α -glucan phosphorylase, thermostable glucose phosphomutase, thermostable inositol-3-phosphate synthase and thermostable inositol monophosphate phosphorylase, vectors expressing thermostable α -glucan phosphorylase, vectors expressing thermostable glucose phosphomutase, vectors expressing thermostable inositol-3-phosphate synthase and vectors expressing thermostable inositol monophosphate phosphorylase.
According to another aspect of the present invention, the present invention relates to the above-mentioned engineering bacterium co-expressing thermostable α -glucan phosphorylase, thermostable glucose phosphomutase, thermostable inositol-3-phosphate synthase and thermostable inositol monophosphorylase, engineering bacterium expressing thermostable α -glucan phosphorylase, engineering bacterium expressing thermostable glucose phosphomutase, engineering bacterium expressing thermostable inositol-3-phosphate synthase and engineering bacterium expressing thermostable inositol monophosphorylase. According to the present invention, the engineering bacteria co-expressing thermostable α -glucan phosphorylase, thermostable glucose phosphomutase, thermostable inositol-3-phosphate synthase and thermostable inositol monophosphorylase comprise vectors co-expressing thermostable α -glucan phosphorylase, thermostable glucose phosphomutase, thermostable inositol-3-phosphate synthase and thermostable inositol monophosphorylase, or comprise vectors expressing thermostable α -glucan phosphorylase, vectors expressing thermostable glucose phosphomutase, vectors expressing thermostable inositol-3-phosphate synthase and vectors expressing thermostable inositol monophosphorylase; the engineering bacteria expressing the thermostable alpha-glucan phosphorylase comprise a vector expressing the thermostable alpha-glucan phosphorylase; the engineering bacteria for expressing the thermostable glucose phosphomutase comprise a vector for expressing the thermostable glucose phosphomutase; the engineering bacteria expressing the thermostable inositol-3-phosphate synthase comprise a vector expressing the thermostable inositol-3-phosphate synthase; the engineering bacteria expressing the thermostable inositol monophosphate enzyme comprise a vector expressing the thermostable inositol monophosphate enzyme.
Compared with the prior art, the invention has the following beneficial effects:
(1) Bacillus subtilis is a food-grade microorganism generally considered to be safe (Generally Recognized As Safe, GRAS), does not produce endotoxin, knocks out an alpha-amylase coding gene, and is beneficial to the subsequent catalytic application of substrate starch;
(2) The invention firstly utilizes whole cell catalytic starch expressing heat-resistant alpha-glucan phosphorylase, heat-resistant glucose phosphomutase, heat-resistant inositol-3-phosphate synthase and heat-resistant inositol monophosphorylase to produce inositol, and develops a novel method which is simple and easy for preparing inositol in large scale.
(3) NAD is not required to be additionally added in the reaction system + Is beneficial to reducing the production cost and has important industrial application value.
(4) The preparation of inositol in the method can be carried out at a higher temperature, so that the solubility of substrate starch can be increased.
(5) The conversion reaction of inositol in the method can be carried out in a buffer-free system or a buffer system, and a culture medium containing a carbon source, a nitrogen source, inorganic salt and antibiotics is not needed, so that the production cost is reduced, and the separation and purification of the product inositol are facilitated.
Drawings
FIG. 1 is a schematic diagram of the permeable whole cell catalyzed starch production of inositol in accordance with the present invention.
FIGS. 2A-E are graphs of recombinant expression vectors pMA5-Pylb-aGP, pMA5-Pylb-PGM, pMA5-Pylb-IPS, pMA5-Pylb-IMP, pMA5-Pylb-aGP-PGM-IPS-IMP, respectively.
FIG. 3 is a graph showing the reaction time dependence of inositol production.
Detailed Description
The technical means adopted by the invention and the effects thereof are further described by the following specific embodiments. It should be understood that the embodiments described are exemplary only and should not be construed as limiting the scope of the invention in any way. It will be understood by those skilled in the art that various changes and substitutions can be made in the details and form of the technical solution of the present invention without departing from the spirit and scope of the invention, but these changes and substitutions fall within the scope of the present invention.
Example 1: construction of bacillus subtilis engineering bacteria for knocking out alpha-amylase
Competent preparation: the activated SCK6 strain was streaked onto LB solid plates containing 0.3. Mu.g/mL erythromycin and incubated overnight at 37 ℃. The following day the monoclonal was picked and inoculated in 5mL of LB liquid medium containing 0.3. Mu.g/mL erythromycin and incubated at 37℃for 8-12h at 200 rpm. Absorbance at 600nm was measured and then the culture was diluted to a600= -1.0 with fresh LB liquid medium containing 0.3 μg/mL erythromycin pre-warmed at 37 ℃. D-xylose was added at a final concentration of 1% (w/v), and the culture was continued at 37℃for 2 hours at 200rpm, thereby obtaining a product for transformation.
Conversion: 50ng PDG1730 plasmid was mixed with 200. Mu.L competent cells, incubated at 37℃for 1.5h at 200rpm, and then plated with LB solid plates containing 100. Mu.g/mL Qamycin, and incubated overnight at 37 ℃.
Colony PCR identification: a small amount of transformant cells were selected from the plate and diluted in 30. Mu.L of sterile water, boiled water for 5min, frozen at-20℃for 5min, then dissolved at room temperature, centrifuged at 12000rpm for 1min, 2. Mu.L of supernatant was taken as a template, and primer amyE-F was used: 5'-ACCTCTTTACTGCCGTTATTCG-3' and amyE-R:5'-TAGCACGTAATCAAAGCCAGG-3' PCR amplification was performed according to the following conditions: denaturation at 98℃for 2min was performed for 30 cycles as follows: denaturation at 98℃for 15s, annealing at 58℃for 15s, extension at 72℃for 30s, and finally extension at 72℃for 5min. And (3) according to the result of electrophoresis analysis, only amplifying a single band with the size of 2.6kb, further sequencing to verify that amyE is successfully knocked out, and marking the bacillus subtilis strain with the amyE gene successfully knocked out as SCK6/amyE.
Example 2: construction of recombinant vectors
(1) Construction of pMA5-Pylb-aGP
The thermostable α -glucan phosphorylase in this example is from Thermotoga maritima, KEGG accession No. TM1168; thermotoga maritima is purchased from China general microbiological culture collection center (CGMCC). The thermostable α -glucan phosphorylase encoding gene agp was obtained from genomic DNA by PCR using a pair of primers. Primer 299-F was used: 5'-AGAAACAACAAAGGGGGAGATTTGTatggtgaacgtttccaatgccgttg-3' and 300-R:5'-gcttgagctcgactctagaggatcctcagtcaagtcccttccacttgacca-3'; the pMA5-Pylb linear backbone was obtained by PCR using a pair of primers. Primer 301-F was used: 5'-tggtcaagtggaagggacttgactgaggatcctctagagtcgagctcaagc-3' and 302-R:5'-caacggcattggaaacgttcaccatACAAATCTCCCCCTTTGTTGTTTCT-3';
all primers were synthesized by su Jin Weizhi biotechnology limited. The PCR conditions of the gene were 94℃for 5min denaturation, and the cycle was 30 times according to the following parameters: denaturation at 94℃for 15s, annealing at 58℃for 15s, extension at 72℃for 1min, and extension at 72℃for 10min. The products obtained by the PCR reaction were analyzed by 0.8% agarose gel electrophoresis, respectively. After the correct size of the fragment is confirmed by imaging of a gel imaging system, a DNA purification recovery kit (Tiangen Biochemical technology Co., china) is adopted to recover the target fragment for constructing the recombinant expression vector.
The thermostable α -glucan phosphorylase gene fragment and the pMA5-Pylb vector backbone were then assembled using POE-PCR. POE-PCR system is as follows: 200ng of a purified pMA5-Pylb linear framework; 131ng of the purified heat-resistant alpha-glucan phosphorylase gene fragment; 2X PrimeSTAR MAX DNA Polymerase (Dalianbao organism, china), 25. Mu.L, and water in an amount of up to 50. Mu.L. POE-PCR conditions were 98℃for 2min denaturation, 30 cycles according to the following parameters: denaturation at 98℃for 15s, annealing at 58℃for 15s, extension at 72℃for 3.5min, and extension at 72℃for 5min. The connection product is transformed into competent E.coli Top10 by a calcium chloride method, colony PCR and double enzyme digestion identification are carried out on the selected transformants, 2-3 positive transformants are selected for further verification by sequencing, sequencing results show that the pMA5-Pylb-aGP recombinant co-expression vector is successfully obtained, and a plasmid map is shown in figure 2A.
(2) Construction of pMA5-Pylb-PGM
The thermostable glucose phosphomutase in this example is from Thermococcus kodakarensis, KEGG accession number TK1108; thermococcus kodakarensis is purchased from China general microbiological culture collection center (CGMCC). The thermostable glucose phosphomutase-encoding gene pgm is obtained from genomic DNA by PCR using a pair of primers. Primer 327-F was used: 5'-AGAAACAACAAAGGGGGAGATTTGTatgggcaaactgtttggtaccttcg-3' and 328-R:5'-agcttgagctcgactctagaggatccTTAacctttcagtgcttcttccagc-3'; the pMA5-Pylb linear backbone was obtained by PCR using a pair of primers. Primer 329-F was used: 5'-gctggaagaagcactgaaaggtTAAggatcctctagagtcgagctcaagct-3' and 330-R:5'-cgaaggtaccaaacagtttgcccatACAAATCTCCCCCTTTGTTGTTTCT-3';
all primers were synthesized by su Jin Weizhi biotechnology limited. The PCR conditions of the gene were 94℃for 5min denaturation, and the cycle was 30 times according to the following parameters: denaturation at 94℃for 15s, annealing at 58℃for 15s, extension at 72℃for 1min, and extension at 72℃for 10min. The products obtained by the PCR reaction were analyzed by 0.8% agarose gel electrophoresis, respectively. After the correct size of the fragment is confirmed by imaging of a gel imaging system, a DNA purification recovery kit (Tiangen Biochemical technology Co., china) is adopted to recover the target fragment for constructing the recombinant expression vector.
The thermostable glucose phosphomutase gene fragment and the pMA5-Pylb vector backbone were then assembled using POE-PCR. POE-PCR system is as follows: 200ng of a purified pMA5-Pylb linear framework; 131ng of the purified thermostable glucose phosphomutase gene fragment; 2X PrimeSTAR MAX DNA Polymerase (Dalianbao organism, china), 25. Mu.L, and water in an amount of up to 50. Mu.L. POE-PCR conditions were 98℃for 2min denaturation, 30 cycles according to the following parameters: denaturation at 98℃for 15s, annealing at 58℃for 15s, extension at 72℃for 3.5min, and extension at 72℃for 5min. The ligation product is transformed into competent E.coli Top10 by a calcium chloride method, colony PCR and double enzyme digestion identification are carried out on the selected transformants, 2-3 positive transformants are selected for further verification by sequencing, sequencing results show that the pMA5-Pylb-PGM recombinant co-expression vector is successfully obtained, and a plasmid map is shown in FIG. 2B.
(3) Construction of pMA5-Pylb-IPS
The thermostable inositol-3-phosphate synthase in this example is from Archaeoglobus fulgidus, KEGG accession No. AF1794; archaeoglobus fulgidus is purchased from China general microbiological culture collection center (CGMCC). The thermostable inositol-3-phosphate synthase encoding gene ips was obtained from genomic DNA by PCR using a pair of primers. Primer 323-F was used: 5'-AGAAACAACAAAGGGGGAGATTTGTatgaaagtttggctggttggtgcct-3' and 324-R:5'-agcttgagctcgactctagaggatccTTAtttcaggttgctataccattct-3'; the pMA5-Pylb linear backbone was obtained by PCR using a pair of primers. Primer 325-F was used: 5'-agaatggtatagcaacctgaaaTAAggatcctctagagtcgagctcaagct-3' and 326-R:5'-aggcaccaaccagccaaactttcatACAAATCTCCCCCTTTGTTGTTTCT-3';
all primers were synthesized by su Jin Weizhi biotechnology limited. The PCR conditions of the gene were 94℃for 5min denaturation, and the cycle was 30 times according to the following parameters: denaturation at 94℃for 15s, annealing at 58℃for 15s, extension at 72℃for 1min, and extension at 72℃for 10min. The products obtained by the PCR reaction were analyzed by 0.8% agarose gel electrophoresis, respectively. After the correct size of the fragment is confirmed by imaging of a gel imaging system, a DNA purification recovery kit (Tiangen Biochemical technology Co., china) is adopted to recover the target fragment for constructing the recombinant expression vector.
The thermostable inositol-3-phosphate synthase gene fragment and pMA5-Pylb vector backbone were then assembled using POE-PCR. POE-PCR system is as follows: 200ng of a purified pMA5-Pylb linear framework; 131ng of the purified heat-resistant inositol-3-phosphate synthase gene fragment; 2X PrimeSTAR MAX DNAPolymerase (Dalianbao organism, china), 25. Mu.L, and water in an amount of up to 50. Mu.L. POE-PCR conditions were 98℃for 2min denaturation, 30 cycles according to the following parameters: denaturation at 98℃for 15s, annealing at 58℃for 15s, extension at 72℃for 3.5min, and extension at 72℃for 5min. The ligation product is transformed into competent E.coliTop10 by a calcium chloride method, colony PCR and double enzyme digestion identification are carried out on transformants, 2-3 positive transformants are selected for further verification by sequencing, sequencing results show that pMA5-Pylb-IPS recombinant co-expression vector is successfully obtained, and a plasmid map is shown in figure 2C.
(4) Construction of pMA5-Pylb-IMP
The thermostable inositol monophosphorylase in this example is from Thermotoga maritima, KEGG accession TM1415; thermotoga maritima is purchased from China general microbiological culture collection center (CGMCC). The thermostable inositol monophosphorylase encoding gene ips was obtained from genomic DNA by PCR using a pair of primers. Primer 311-F was used: 5'-AGAAACAACAAAGGGGGAGATTTGTatgctggatcgcctggatttctcta-3' and 312-R:5'-gcttgagctcgactctagaggatccTCAtttaccgccgatttcttcaaca-3'; the pMA5-Pylb linear backbone was obtained by PCR using a pair of primers. Primers are used; 313-F:5'-tgttgaagaaatcggcggtaaaTGAggatcctctagagtcgagctcaagc-3' and 314-R:5'-tagagaaatccaggcgatccagcatACAAATCTCCCCCTTTGTTGTTTCT-3'.
All primers were synthesized by su Jin Weizhi biotechnology limited. The PCR conditions of the gene were 94℃for 5min denaturation, and the cycle was 30 times according to the following parameters: denaturation at 94℃for 15s, annealing at 58℃for 15s, extension at 72℃for 1min, and extension at 72℃for 10min. The products obtained by the PCR reaction were analyzed by 0.8% agarose gel electrophoresis, respectively. After the correct size of the fragment is confirmed by imaging of a gel imaging system, a DNA purification recovery kit (Tiangen Biochemical technology Co., china) is adopted to recover the target fragment for constructing the recombinant expression vector.
The thermostable inositol monophosphorylase gene fragment and pMA5-Pylb vector backbone were then assembled using POE-PCR. POE-PCR system is as follows: 200ng of a purified pMA5-Pylb linear framework; 131ng of the purified heat-resistant inositol monophosphate enzyme gene fragment; 2X PrimeSTAR MAX DNA Polymerase (Dalianbao organism, china), 25. Mu.L, and water in an amount of up to 50. Mu.L. POE-PCR conditions were 98℃for 2min denaturation, 30 cycles according to the following parameters: denaturation at 98℃for 15s, annealing at 58℃for 15s, extension at 72℃for 3.5min, and extension at 72℃for 5min. The ligation product is transformed into competent E.coli Top10 by a calcium chloride method, colony PCR and double enzyme digestion identification are carried out on transformants, 2-3 positive transformants are selected for further verification by sequencing, sequencing results show that pMA5-Pylb-IMP recombinant co-expression vector is successfully obtained, and a plasmid map is shown in figure 2D.
(5) Construction of pMA5-Pylb-aGP-PGM-IPS-IMP
The thermostable α -glucan phosphorylase in this example is from Thermotoga maritima, KEGG accession No. TM1168; thermostable glucose phosphomutase is from Thermococcus kodakarensis, KEGG accession number TK1108; the thermostable inositol-3-phosphate synthase is from Archaeoglobus fulgidus, KEGG accession No. AF1794; and thermotolerant inositol monophosphorylase from Thermotoga maritima, KEGG accession TM1415; thermococcus kodakarensis and Archaeoglobus fulgidus and Thermotoga maritima are purchased from China general microbiological culture collection center (CGMCC). Thermostable a-glucan phosphorylase; the thermostable α -glucan phosphorylase encoding gene agp was obtained by PCR using primer 330-F:5'-AGAAACAACAAAGGGGGAGATTTGTatggtgaacgtttccaatgccgttg-3' and 331-R:5'-cgaaggtaccaaacagtttgcccattcagtcaagtcccttccacttgacc-3'; thermostable a-glucan phosphorylase; the thermostable glucose phosphomutase-encoding gene pgm was obtained by PCR using primer 332-F:5'-ggtcaagtggaagggacttgactgaatgggcaaactgtttggtaccttcg-3' and 333-R:5'-aggcaccaaccagccaaactttcatTTAacctttcagtgcttcttccagc-3'; the thermostable inositol-3-phosphate synthase encoding gene ips was obtained by PCR using primer 334-F:5'-gctggaagaagcactgaaaggtTAAatgaaagtttggctggttggtgcct-3' and 335-R:5'-tagagaaatccaggcgatccagcatTTAtttcaggttgctataccattct-3'; the thermostable inositol monophosphorylase encoding gene imp was obtained by PCR using primer 335-F:5'-agaatggtatagcaacctgaaaTAAatgctggatcgcctggatttctcta-3' and 336-R:5'-gcttgagctcgactctagaggatccTCAtttaccgccgatttcttcaaca-3'; the pMA5-Pylb linear backbone was obtained by PCR using primers; 337-F:5'-tgttgaagaaatcggcggtaaaTGAggatcctctagagtcgagctcaagc-3' and 338-R:5'-caacggcattggaaacgttcaccatACAAATCTCCCCCTTTGTTGTTTCT-3'.
All primers were synthesized by su Jin Weizhi biotechnology limited. The PCR conditions of the gene were 94℃for 5min denaturation, and the cycle was 30 times according to the following parameters: denaturation at 94℃for 15s, annealing at 58℃for 15s, extension at 72℃for 1min, and extension at 72℃for 10min. The products obtained by the PCR reaction were analyzed by 0.8% agarose gel electrophoresis, respectively. After the correct size of the fragment is confirmed by imaging of a gel imaging system, a DNA purification recovery kit (Tiangen Biochemical technology Co., china) is adopted to recover the target fragment for constructing the recombinant expression vector.
Then, a thermostable α -glucan phosphorylase gene fragment, a thermostable glucose phosphomutase gene fragment, a thermostable inositol-3-phosphate synthase gene fragment, a thermostable inositol monophosphorylase gene fragment, and a pMA5-Pylb vector backbone were assembled using POE-PCR. POE-PCR system is as follows: 200ng of a purified pMA5-Pylb linear framework; 131ng of the purified heat-resistant alpha-glucan phosphorylase gene fragment, 131ng of the heat-resistant glucose phosphomutase gene fragment, 131ng of the heat-resistant inositol-3-phosphate synthase gene fragment and 131ng of the heat-resistant inositol monophosphorylase gene fragment; 2X PrimeSTAR MAX DNA Polymerase (Dalianbao organism, china), 25. Mu.L, and water in an amount of up to 50. Mu.L. POE-PCR conditions were 98℃for 2min denaturation, 30 cycles according to the following parameters: denaturation at 98℃for 15s, annealing at 58℃for 15s, extension at 72℃for 3.5min, and extension at 72℃for 5min. The ligation product is transformed into competent E.coli Top10 by a calcium chloride method, colony PCR and double enzyme digestion identification are carried out on transformants, 2-3 positive transformants are selected for further verification by sequencing, sequencing results show that pMA5-Pylb-aGP-PGM-IPS-IMP recombinant co-expression vector is successfully obtained, and a plasmid map is shown in figure 2E.
EXAMPLE 3 construction of recombinant engineering bacteria
The constructed recombinant expression vectors pMA 5-Pyleb-aGP, pMA 5-Pyleb-PGM, pMA 5-Pyleb-IPS, pMA 5-Pyleb-IMP, pMA 5-Pyleb-aGP-PGM-IPS-IMP are respectively transformed into bacillus subtilis host bacteria SCK6/amyE by a calcium chloride method, LB test tubes are cultured overnight, plasmids are extracted by a plasmid extraction kit, and correct clones SCK6/amyE/pMA 5-Pyleb-aGP, SCK6/amyE/pMA 5-Pyleb-PGM, SCK6/amyE/pMA 5-Pyleb-IPS, SCK6/amyE/pMA 5-Pyleb-IMP and SCK6/amyE/pMA 5-Pyleb-aGP-IPS-IMP are preserved.
EXAMPLE 4 preparation of recombinant engineering bacterium Whole cell
Recombinant engineering bacteria SCK6/amyE/pMA5-Pylb-aGP, SCK6/amyE/pMA5-Pylb-PGM, SCK6/amyE/pMA5-Pylb-IPS, SCK6/amyE/pMA5-Pylb-IMP and SCK6/amyE/pMA5-Pylb-aGP-PGM-IPS-IMP are respectively picked and inoculated in LB culture medium containing the Qcomycin, and the culture is carried out at 37 ℃ under shaking overnight. The culture was transferred to fresh LB medium containing Qimycin at an inoculum size of 1%, shake-cultured overnight at 37℃and centrifuged at 5500rpm for 10min, and the supernatant was discarded to obtain whole cells expressing thermostable alpha-glucan phosphorylase, whole cells expressing thermostable glucose phosphomutase, whole cells expressing thermostable inositol-3-phosphate synthase, whole cells expressing thermostable inositol monophosphorylase, and whole cells co-expressing thermostable alpha-glucan phosphorylase, thermostable glucose phosphomutase, thermostable inositol-3-phosphate synthase and thermostable inositol monophosphorylase.
EXAMPLE 5 Whole cell catalyzed starch preparation of inositol
Whole cells of the co-expressed thermostable α -glucan phosphorylase, thermostable glucose phosphomutase, thermostable inositol-3-phosphate synthase and thermostable inositol monophosphorylase prepared in example 4 were washed 1 time with 50mM Tris-HCl buffer (pH 7.5), centrifuged at 5500rpm for 10min, the supernatant was discarded, 50mM Tris-HCl buffer (pH 7.5) was added to the pellet, and the pellet was resuspended to OD 600 =150 or so. The resuspended cells were heat treated at 75℃for 90min.
In a 1L reaction system, 200g/L starch, 50mM sodium phosphate buffer (pH 7.0) and heat-treated whole cells were added to give OD 600 =20 or so. The reaction was performed in a shaking table at 55℃for 63h, and samples were taken for High Performance Liquid Chromatography (HPLC) analysis. The HPLC detection conditions were as follows: the chromatographic column is Bio-Rad HPX-87H; the flow rate is 0.6mL/min; column temperature is 60 ℃; the detector is a differential refraction detector; the sample loading was 20. Mu.L.
Figure 3 shows a graph of inositol yield as a function of reaction time, showing that inositol yields can reach 70%.
EXAMPLE 6 Whole cell catalyzed starch preparation of inositol
The whole cells expressing thermostable α -glucan phosphorylase, whole cells expressing thermostable glucose phosphomutase, whole cells expressing thermostable inositol-3-phosphate synthase, and whole cells expressing thermostable inositol monophosphorylase prepared in example 4 were washed 1 time with 0.9% NaCl, centrifuged at 5500rpm for 10min, the supernatant was discarded, 50mM sodium phosphate buffer (pH 7.0) was added to the pellet, and the pellet was resuspended to OD 600 =150 or so. The resuspended cells were heat treated at 75℃for 90min.
Adding 200g/L starch into 1L reaction system,OD was obtained from whole cells heat-treated with 50mM sodium phosphate buffer (pH 7.0) and four of the above 600 About=20, the ratio of addition of whole cells expressing thermostable α -glucan phosphorylase, whole cells expressing thermostable glucose phosphomutase, whole cells expressing thermostable inositol-3-phosphate synthase, and whole cells expressing thermostable inositol monophosphorylase was 1:1:1:1. The reaction was carried out in a water bath shaker at 55℃for 63h, and samples were taken for HPLC analysis. HPLC detection conditions were the same as in example 5. The results show that inositol yield can reach 73%.
Claims (24)
1. A method for preparing high-concentration inositol by catalyzing high-concentration starch with bacillus subtilis whole cells, which is characterized by comprising the following steps:
(1) Constructing bacillus subtilis host bacteria for knocking out alpha-amylase;
(2) Constructing engineering bacteria for co-expressing heat-resistant alpha-glucan phosphorylase, heat-resistant glucose phosphomutase, heat-resistant inositol-3-phosphate synthase and heat-resistant inositol monophosphate enzyme and/or bacillus subtilis engineering bacteria for respectively expressing heat-resistant alpha-glucan phosphorylase, heat-resistant glucose phosphomutase, heat-resistant inositol-3-phosphate synthase and heat-resistant inositol monophosphate enzyme on the basis of the host bacteria constructed in the step (1), wherein the heat-resistant alpha-glucan phosphorylase is fromThermotoga maritimaThe method comprises the steps of carrying out a first treatment on the surface of the The thermostable glucose phosphomutase is derived fromThermococcus kodakarensisThe method comprises the steps of carrying out a first treatment on the surface of the The thermostable inositol-3-phosphate synthase is derived fromArchaeoglobus fulgidusThe method comprises the steps of carrying out a first treatment on the surface of the The thermostable inositol monophosphorylase is derived fromT h e r m o t o g a m a r i t i m a；
(3) Fermenting the bacillus subtilis engineering bacteria constructed in the step (2) to obtain whole cells;
(4) Performing cell membrane permeability treatment on the bacillus subtilis whole cells obtained in the step (3) to obtain permeable whole cells;
(5) Preparing high concentration inositol by catalyzing high concentration starch with the mixture of the co-expressed thermostable alpha-glucan phosphorylase, the thermostable glucose phosphomutase, the thermostable inositol-3-phosphate synthase and the thermostable inositol monophosphorylase obtained in the step (4), or the permeable whole cell expressing the thermostable alpha-glucan phosphorylase, the permeable whole cell expressing the thermostable glucose phosphomutase, the permeable whole cell expressing the thermostable inositol-3-phosphate synthase and the permeable whole cell expressing the thermostable inositol monophosphorylase.
2. The method of claim 1, wherein the engineered bacterium comprises a vector that co-expresses a thermostable α -glucan phosphorylase, a thermostable glucose phosphomutase, a thermostable inositol-3-phosphate synthase, and a thermostable inositol monophosphate phosphorylase; or a vector expressing a thermostable α -glucan phosphorylase, or a vector expressing a thermostable glucose phosphomutase, or a vector expressing a thermostable inositol-3-phosphate synthase, or a vector expressing a thermostable inositol monophosphate phosphorylase.
3. The method of claim 2, wherein the co-expressed vector comprises a promoter, a thermostable α -glucan phosphorylase gene, a thermostable glucose phosphomutase gene, a thermostable inositol-3-phosphate synthase gene, a thermostable inositol monophosphorylase gene, and a terminator; the vector for expressing the heat-resistant alpha-glucan phosphorylase comprises a promoter, a heat-resistant alpha-glucan phosphorylase gene and a terminator; the vector for expressing the thermostable glucose phosphomutase comprises a promoter, a thermostable glucose phosphomutase gene and a terminator; the vector for expressing the heat-resistant inositol-3-phosphate synthase comprises a promoter, a heat-resistant inositol-3-phosphate synthase gene and a terminator; the vector for expressing the heat-resistant inositol monophosphate enzyme comprises a promoter, a heat-resistant inositol monophosphate enzyme gene and a terminator.
4. A method according to claim 3, wherein the promoter is a P43 promoter, a Pylb promoter, a PamyL promoter, a Plaps promoter, a PhpaII promoter, a pamyle promoter and/or a pgac promoter.
5. The method of claim 4, wherein the promoter is selected from the group consisting of the PhpaII promoter and the Pylb promoter in tandem.
6. The method of claim 1, wherein the cell membrane permeability treatment is a heat treatment, an addition of an organic solvent, and/or an addition of a surfactant.
7. The method of claim 6, wherein the heat treatment temperature is 45-95 ℃.
8. The method of claim 7, wherein the heat treatment temperature is 50-80 ℃.
9. The method of claim 6, wherein the heat treatment time is 1 to 100 minutes.
10. The method of claim 9, wherein the heat treatment time is 10-60 minutes.
11. The method according to claim 6, wherein the cell concentration at the time of the heat treatment is OD 600 = 10-300。
12. The method of claim 11, wherein the cell concentration is OD 600 =30-200。
13. The method of claim 6, wherein the heat treatment is performed in a buffer system, the buffer being HEPES buffer, phosphate buffer, tris buffer, or acetate buffer.
14. The method of claim 13, wherein the phosphate buffer is a sodium phosphate buffer or a potassium phosphate buffer.
15. The method of claim 6, wherein the organic solvent is acetone or acetonitrile.
16. The method of claim 6, wherein the surfactant is cetyltrimethylammonium bromide or Tween-80.
17. The method according to claim 1, wherein the concentration of the substrate starch is 10-300g/L in the reaction system for preparing inositol by catalyzing starch with permeable whole cells or a mixture of permeable whole cells.
18. The method of claim 17, wherein the substrate starch is dosed at a concentration of 20-200g/L.
19. The method of claim 1 or 17, wherein the ratio of permeant whole cells expressing thermostable α -glucan phosphorylase, permeant whole cells expressing thermostable glucose phosphomutase, permeant whole cells expressing thermostable inositol-3-phosphate synthase, permeant whole cells expressing thermostable inositol monophosphorylase is 0.1:1:1:0.1-10:1:1:10 in the mixture of permeant whole cells.
20. The method of claim 19, wherein the ratio is 0.5:1:1:0.5-5:1:1:5.
21. The method according to claim 1 or 17, wherein the conditions of the catalytic reaction are: reacting at pH 6.0-8.0 and 50-80deg.C for 0.5-96 h.
22. The method of claim 21, wherein the conditions of the catalytic reaction are: reacting at pH 6.5-7.5 and 55-75deg.C 12-96h.
23. The method of claim 1 or 17, wherein the catalytic reaction is carried out in a buffer system, the buffer being HEPES buffer, phosphate buffer, tris buffer or acetate buffer.
24. The method of claim 23, wherein the phosphate buffer is a sodium phosphate buffer or a potassium phosphate buffer.
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Inventor after: Zhang Yiheng Inventor after: You Chun Inventor after: Shi Ting Inventor before: You Chun Inventor before: Shi Ting |