CN109913489B - Method for preparing inositol by multi-enzyme reaction system expressed by edible microorganism - Google Patents
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Abstract
The invention discloses a method for preparing inositol by a multi-enzyme reaction system expressed by edible microorganisms. The method uses the strain for food industry as a system for expressing multienzyme to produce isoamylase, glucan phosphorylase, glucose phosphoglucomutase, inositol-3-phosphate synthase and inositol monophosphatase which are required by catalyzing starch and derivatives thereof, thereby fundamentally avoiding the possibility that inositol is polluted by toxic protein, antigenic protein or endotoxin generated by escherichia coli in the production process, avoiding strict and complex purification process and reducing the production cost.
Description
Technical Field
The invention belongs to the field of enzyme catalysis production of inositol, and particularly relates to a method for preparing inositol by a multi-enzyme reaction system expressed by edible microorganisms.
Background
Inositol, also known as inositol, is one of water-soluble vitamin B group, and has a structure shown in formula I. Inositol is an essential substance for growth of human, animals and microorganisms, and is widely applied to industries such as medicine, food, feed and the like. The global demand is about 5,000 tons every year, and the market prospect of inositol is not fully developed due to the high selling price of inositol, for example, the global feed yield is 96 hundred million tons in 2013, and if 0.2 to 0.5 percent of inositol is added, the yield of the inositol required by the feed industry can reach 190 to 480 ten thousand tons. Under the condition, the yield in China and even all over the world is far from meeting the demand at present.
The production of inositol is mainly the traditional high-temperature pressurized hydrolysis of phytic acid (inositol hexaphosphate). The process equipment has strict requirements on materials and large one-time investment; the operation pressure must 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, and the production cost is higher; in addition, the process can generate a large amount of phosphoric acid pollutants, and the pollution to the environment such as water sources is serious. In recent years, atmospheric hydrolysis methods have been developed in order to reduce energy consumption and pollution. At present, the hot point for researching the production of inositol focuses on chemical synthesis and microbial enzymatic hydrolysis, but the problems of high cost, low yield and the like exist to different degrees.
Patent CN106148425A describes a process for the preparation of inositol, and in particular relates to a process for the in vitro multi-enzyme catalysis of the conversion of starch and their derivatives into inositol. The method converts starch into inositol through an in vitro multi-enzyme catalytic system, and the key enzymes and the functions thereof comprise: isoamylase (IA, EC 3.2.1.68), which is used for pruning amylopectin into amylose; glucan phosphorylase (α GP, EC 2.4.1.1), releasing glucose-1-phosphate from starch; phosphoglucomutase (PGM, EC 5.4.2.2), catalyzing the conversion of glucose-1-phosphate to glucose-6-phosphate; inositol-3-phosphate synthase (IPS, EC5.5.1.4), which catalyzes glucose-6-phosphate to inositol-3-phosphate; inositol monophosphatase (IMP, EC 3.1.3.25), which dephosphorylates inositol-3-phosphate into inositol. Since the last two enzymatic reactions are irreversible reactions, the enzymatic system is capable of achieving very high conversions.
Genomic DNA corresponding to these five enzymes is available from the official website (www.atcc.org) of American Type Culture Collection (ATCC). The 5 genes are obtained from corresponding genomic DNA by PCR by using different primers respectively, and are cloned into a prokaryotic expression vector pET20b to obtain corresponding gene expression vectors. And then, transforming the gene expression vectors into escherichia coli expression bacteria BL21(DE3) for protein expression.
Coli has the advantages of simple gene structure, easy gene operation and high growth speed, but the product expressed by the coli may contain toxic protein or antigenic protein and endotoxin generated by the cell wall, so the product produced by the coli needs strict and complex purification process.
Disclosure of Invention
In order to avoid the disadvantages of the enzyme expression system of Escherichia coli, the invention aims to provide a novel method for producing a multienzyme system, and to apply the method to the preparation of inositol with higher safety requirements.
For the above purposes, the present investigators have attempted to screen for suitable species from expression systems that are not harmful to humans and animals. Lactic Acid Bacteria (LAB) are bacteria which can utilize fermentable carbohydrates to produce a large amount of lactic acid, and are widely used in various industries such as light industry, food industry, medicine industry, feed industry and the like at present, no health hazard is caused by thalli or metabolites thereof, and the culture process is mature and suitable for industrial production. Therefore, the inventors first selected lactic acid bacteria as an expression system of a multienzyme system in order to obtain multienzymes having catalytic activity and high safety. Unfortunately, the target enzyme activity detected in the lactic acid bacteria expression product is negative, and the lactic acid bacteria expression system cannot efficiently express the protein multienzyme required for producing inositol.
The inventors have also tried to achieve this in yeasts which are widely used in the food field. The saccharomyces cerevisiae is safe and mature in culture, and has the advantages of fast propagation and short growth cycle; the pichia pastoris has high expression efficiency, the expressed foreign protein can account for more than 90 percent of the total expressed protein, the separation and purification of the target protein are facilitated, the high-density culture can be realized in a simple synthetic culture medium, and if the pichia pastoris can be successfully applied in the invention, the pichia pastoris has huge industrial potential. However, the test results are still unsatisfactory, and the target enzyme activities of saccharomyces cerevisiae and pichia pastoris are also negative.
After a plurality of failures, the inventor surprisingly found that the bacillus subtilis and the corynebacterium glutamicum can effectively express target multienzyme, and provides possibility for producing inositol with high quality requirement, which can be applied to the fields of food, biological medicine and the like. Bacillus subtilis (Bcillus subtilis) is a species of microorganism in the American FDA list of "generally recognized as safe substances" (GRAS). Corynebacterium glutamicum (Corynebacterium glutamicum) is a food-safe microorganism in the appendix XV of the United states Pharmacopeia and American food chemical codex.
Based on the results of the present invention, the present invention firstly provides an expression method of a multi-enzyme system for producing inositol, which is expressed by an edible microorganism.
Further, the edible microorganism is bacillus subtilis and/or corynebacterium glutamicum.
Further, the multienzyme system is one or more of isoamylase, glucan phosphorylase, phosphoglucomutase, inositol-3-phosphate synthase and inositol monophosphatase.
The multi-enzyme system expressed by the food micro-organism can be chosen according to the type of substrate to be catalysed, and the types of enzymes required for starch and different starch derivatives can vary.
Preferably, the proteasome system is glucan phosphorylase, phosphoglucomutase, inositol-3-phosphate synthase and inositol monophosphatase.
More preferably, the proteasome system is glucan phosphorylase, phosphoglucomutase, inositol-3-phosphate synthase, inositol monophosphatase, and isoamylase.
Secondly, the invention also provides a method for preparing inositol by using the edible microorganism expression multienzyme system, the method adopts the multienzyme system expressed by the method, uses starch or starch derivatives as substrates to carry out enzyme catalytic reaction, and then separates and purifies the reaction product to obtain the inositol.
Further, the starch derivative comprises any one or more of partially hydrolyzed starch, starch dextrin and maltodextrin.
In the present invention, isoamylase is derived from Sulfolobus tokodaii, the gene is numbered ST0928 on Kyoto Encyclopedia of Genes and Genomes (hereinafter abbreviated as KEGG), glucan phosphorylase is derived from Thermotoga maritima, the gene is numbered TM1168 on KEGG, phosphoglucomutase is derived from Thermotoga maritima, the gene is numbered TM0769 on KEGG, inositol-3-phosphate synthase is derived from Archaeoglobus fulgidus, the gene is numbered AF1794 on KEGG, inositol monophosphatase is derived from Thermotoga maritima, the gene is numbered TM1415 on KEGG, and these genomic DNAs are all available from official website (www.atcc.org) of American Type Culture Collection (ATCC). The genes corresponding to the 5 enzymes are obtained from the corresponding genome DNA by PCR with different primers respectively, and are cloned into an expression vector to obtain the corresponding gene expression vector. And then transforming the gene expression vectors into bacillus subtilis and/or corynebacterium glutamicum respectively for protein expression.
According to some embodiments of the present invention, when Bacillus subtilis is used as the expression system of the edible microorganism, the vector is pHT 01. When corynebacterium glutamicum is used as an edible microorganism expression system, the vector is pEC-XK 99E.
The present invention refers to the method of PCR, transfection and expression of target genes, which is performed by referring to Simple Cloning (You, C., et al 2012), "Simple Cloning via Direct Transformation of PCR products (DNAmultimer) to Escherichia coli and Bacillus subtilis is./rAppl. environ. Microbiol.78(5): 1593-1595.).
The invention has the beneficial effects that: compared with the CN106148425A patent that uses colon bacillus as an expression system, the method of the invention fundamentally avoids the possibility that the inositol is polluted by toxic protein, antigenic protein or endotoxin generated by the colon bacillus in the production process, avoids strict and complex purification process and reduces the production cost.
Drawings
FIG. 1 is a schematic representation of inositol prepared in example 51H-NMR(D2O, 400MHz) spectrum;
FIG. 2 is a nuclear magnetic resonance analysis chart and a chemical structural diagram of inositol prepared in example 5;
FIG. 3 is a schematic representation of inositol prepared in example 71H-NMR(D2O, 400MHz) spectrum;
FIG. 4 is a nuclear magnetic resonance analysis table and a chemical structural diagram of inositol prepared in example 7.
Detailed Description
The present invention is further illustrated by the following specific examples, but it should not be construed that the scope of the present invention is limited to the following examples, and it will be apparent to those skilled in the art that various technical features in the following examples can be appropriately combined, replaced, adjusted, modified, etc. according to the inventive concept and the entire contents of the present invention, and still fall within the scope of the protection of the present invention.
Main experimental materials
Soluble starch, solubletarch, product of ACROS company, product number 424490020;
expression vectors and host cells (see Table-1), NTCC type culture Collection;
TABLE-1
| Expression system | Carrier | Host cell |
| Lactic acid bacteria | pNZ9530 | NZ9000 |
| Saccharomyces cerevisiae | pYES2 | Y187 |
| Pichia pastoris | pPIC9K | GS115 |
| Bacillus subtilis | pHT01 | BS168 |
| Corynebacterium glutamicum | pEC-XK99E | NTCC910233 |
Example 1: preparation of multienzyme by lactic acid bacteria
Five genes numbered for KEGG, ST0928, TM1168, TM0769, AF1794, TM1415, respectively, were obtained from the ATCC's official website (www.atcc.org). Obtaining different primers from corresponding genomic DNA by PCR, Cloning the primers into pNZ9530 vector by a method of Simple Cloning (You, C., et al. (2012) 'Simple Cloning via direct transformation of PCR products (DNA Multimer) to Escherichia coli and Bacillus subtilis.' appl.Environ.Microbiol.78(5): 1593-1595), obtaining corresponding gene expression vectors, respectively transforming into lactobacillus NZ9000, performing protein expression, and detecting that target enzyme activity is negative.
Example 2: preparation of multienzyme by saccharomyces cerevisiae
5 genes are cloned into a pYES2 vector by adopting the same method as the embodiment 1 to obtain corresponding gene expression vectors, and the corresponding gene expression vectors are respectively transformed into saccharomyces cerevisiae Y187 and carry out protein expression, and the target enzyme activity is negative through detection.
Example 3: preparation of multienzyme from pichia pastoris
5 genes are cloned into a pPIC9K vector by adopting the same method as the embodiment 1 to obtain corresponding gene expression vectors, and the corresponding gene expression vectors are respectively transformed into pichia pastoris GS115 and carry out protein expression, and the target enzyme activity is negative through detection.
Example 4: preparation of multienzyme from Bacillus subtilis
The 5 genes were cloned into pHT01 vector in the same manner as in example 1 to obtain corresponding gene expression vectors, which were then transformed into Bacillus subtilis 168, respectively, and protein expression and purification were carried out.
Example 5: in vitro multienzyme catalytic conversion of starch to inositol
An inositol preparation test was conducted using the multienzyme prepared in example 4. A500-mL reaction system containing 100mM HEPES buffer (pH7.2), 10mM inorganic phosphate, 5mM divalent magnesium ions, 0.5mM zinc ions, 5U/mL dextran phosphorylase, lU/mL phosphoglucomutase, 5U/mL inositol-3-phosphate synthase and 2U/mL inositol monophosphatase, lU/mL isoamylase, and 10g/L soluble starch was subjected to a catalytic reaction at 80 ℃ and, after 40 hours of the reaction, filtered through a microfiltration membrane, the filtrate was concentrated to about 50 mL by a rotary evaporator, cooled, crystallized, filtered and dried to obtain 5.8 g of white powder. The measured melting point is 224.8-225.4 ℃, and the nuclear magnetic resonance1H-NMR(D2O, 400MHz) spectra and analytical tables are shown in fig. 1 and fig. 2.
Example 6: preparation of multienzyme from Corynebacterium glutamicum
5 genes were cloned into pEC-XK99E vector by the same method as in example 1 to obtain corresponding gene expression vectors, which were then transformed into Corynebacterium glutamicum NTCC910233, respectively, and protein expression and purification were performed.
Example 7: in vitro multienzyme catalytic conversion of starch to inositol
An inositol preparation test was conducted using the multienzyme prepared in example 6. A500-mL reaction system containing 100mM HEPES buffer (pH7.2), 10mM inorganic phosphate, 5mM divalent magnesium ions, 0.5mM zinc ions, 5U/mL dextran phosphorylase, lU/mL phosphoglucomutase, 5U/mL inositol-3-phosphate synthase and 2U/mL inositol monophosphatase, lU/mL isoamylase, and 10g/L soluble starch was subjected to a catalytic reaction at 80 ℃ and, after 40 hours of the reaction, filtered through a microfiltration membrane, the filtrate was concentrated to about 50 mL by a rotary evaporator, cooled, crystallized, filtered and dried to obtain 6.6 g of white powder. The measured melting point is 225.2-225.6 ℃, and the nuclear magnetic resonance1H-NMR(D2O, 400MHz) spectra and analytical tables are shown in fig. 3 and fig. 4.
In conclusion, the Corynebacterium glutamicum and the Bacillus subtilis can successfully express a target multienzyme system and catalyze the conversion of starch into inositol.
Claims (3)
1. An expression method of a multi-enzyme system for preparing inositol is characterized in that the multi-enzyme system is expressed by edible microorganisms, the edible microorganisms are corynebacterium glutamicum, or the edible microorganisms are bacillus subtilis and corynebacterium glutamicum, and when the bacillus subtilis is used as an edible microorganism expression system, a carrier is pHT 01; when corynebacterium glutamicum is used as an edible microorganism expression system, a vector of the corynebacterium glutamicum is pEC-XK 99E; the said polyzyme system is glucan phosphorylase, glucose phosphoglucomutase, inositol-3-phosphate synthetase, inositol monophosphatase and isoamylase.
2. A method for preparing inositol by expressing a multienzyme system by edible microorganisms is characterized in that the expression method of claim 1 is applied to express and prepare the multienzyme system, starch or starch derivatives are used as substrates to carry out enzyme catalytic reaction, and then reaction products are separated and purified to obtain the inositol.
3. The method of claim 2, wherein the starch derivative comprises one or more of partially hydrolyzed starch, amylodextrin, and maltodextrin.
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| CN201910266375.5A CN109913489B (en) | 2019-04-03 | 2019-04-03 | Method for preparing inositol by multi-enzyme reaction system expressed by edible microorganism |
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| CN108795837A (en) * | 2018-07-06 | 2018-11-13 | 江南大学 | A kind of bacillus subtilis engineering bacteria of high efficient expression phospholipase D |
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