CN109913489A - The method that inositol is prepared by the multienzymatic reaction system that edible microorganismus is expressed - Google Patents
The method that inositol is prepared by the multienzymatic reaction system that edible microorganismus is expressed Download PDFInfo
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- CN109913489A CN109913489A CN201910266375.5A CN201910266375A CN109913489A CN 109913489 A CN109913489 A CN 109913489A CN 201910266375 A CN201910266375 A CN 201910266375A CN 109913489 A CN109913489 A CN 109913489A
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- inositol
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- multienzyme
- starch
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- 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 46
- SQUHHTBVTRBESD-UHFFFAOYSA-N Hexa-Ac-myo-Inositol Natural products CC(=O)OC1C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C1OC(C)=O SQUHHTBVTRBESD-UHFFFAOYSA-N 0.000 title claims abstract description 45
- 229960000367 inositol Drugs 0.000 title claims abstract description 45
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 title claims abstract description 45
- 238000000034 method Methods 0.000 title claims abstract description 31
- 238000006243 chemical reaction Methods 0.000 title abstract description 12
- 230000014509 gene expression Effects 0.000 claims abstract description 38
- 229920002472 Starch Polymers 0.000 claims abstract description 16
- 235000019698 starch Nutrition 0.000 claims abstract description 16
- 239000008107 starch Substances 0.000 claims abstract description 16
- 108091000115 phosphomannomutase Proteins 0.000 claims abstract description 12
- 108010073135 Phosphorylases Proteins 0.000 claims abstract description 11
- 102000009097 Phosphorylases Human genes 0.000 claims abstract description 11
- 229920001503 Glucan Polymers 0.000 claims abstract description 10
- 101710144867 Inositol monophosphatase Proteins 0.000 claims abstract description 10
- 108010028688 Isoamylase Proteins 0.000 claims abstract description 10
- 102000009569 Phosphoglucomutase Human genes 0.000 claims abstract description 10
- TWNIBLMWSKIRAT-VFUOTHLCSA-N levoglucosan Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@H]2CO[C@@H]1O2 TWNIBLMWSKIRAT-VFUOTHLCSA-N 0.000 claims abstract description 10
- 102000006029 inositol monophosphatase Human genes 0.000 claims abstract description 9
- 108010050335 D-myo-inositol-3-phosphate synthase Proteins 0.000 claims abstract description 8
- 244000063299 Bacillus subtilis Species 0.000 claims description 10
- 235000014469 Bacillus subtilis Nutrition 0.000 claims description 10
- 241000186226 Corynebacterium glutamicum Species 0.000 claims description 10
- 238000006555 catalytic reaction Methods 0.000 claims description 7
- 229920000881 Modified starch Polymers 0.000 claims description 5
- 235000019426 modified starch Nutrition 0.000 claims description 5
- 239000000758 substrate Substances 0.000 claims description 3
- 229920002774 Maltodextrin Polymers 0.000 claims description 2
- 239000005913 Maltodextrin Substances 0.000 claims description 2
- GUBGYTABKSRVRQ-ASMJPISFSA-N alpha-maltose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-ASMJPISFSA-N 0.000 claims description 2
- 238000009835 boiling Methods 0.000 claims description 2
- 229940035034 maltodextrin Drugs 0.000 claims description 2
- 239000007795 chemical reaction product Substances 0.000 claims 1
- 108090000623 proteins and genes Proteins 0.000 abstract description 24
- 102000004169 proteins and genes Human genes 0.000 abstract description 12
- 238000004519 manufacturing process Methods 0.000 abstract description 11
- 241000588724 Escherichia coli Species 0.000 abstract description 9
- 235000013305 food Nutrition 0.000 abstract description 8
- 230000008569 process Effects 0.000 abstract description 6
- 230000003197 catalytic effect Effects 0.000 abstract description 4
- 230000000890 antigenic effect Effects 0.000 abstract description 3
- 239000002158 endotoxin Substances 0.000 abstract description 2
- 238000005516 engineering process Methods 0.000 abstract description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 18
- 102000004190 Enzymes Human genes 0.000 description 15
- 108090000790 Enzymes Proteins 0.000 description 15
- 241000894006 Bacteria Species 0.000 description 11
- 239000004310 lactic acid Substances 0.000 description 9
- 235000014655 lactic acid Nutrition 0.000 description 9
- 239000013604 expression vector Substances 0.000 description 8
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 7
- 239000000047 product Substances 0.000 description 7
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 6
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 6
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 238000005160 1H NMR spectroscopy Methods 0.000 description 4
- 238000005481 NMR spectroscopy Methods 0.000 description 4
- 238000010367 cloning Methods 0.000 description 4
- 239000000706 filtrate Substances 0.000 description 4
- 238000012215 gene cloning Methods 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 241000235058 Komagataella pastoris Species 0.000 description 3
- 241000204666 Thermotoga maritima Species 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 239000007995 HEPES buffer Substances 0.000 description 2
- JLVVSXFLKOJNIY-UHFFFAOYSA-N Magnesium ion Chemical compound [Mg+2] JLVVSXFLKOJNIY-UHFFFAOYSA-N 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- PTFCDOFLOPIGGS-UHFFFAOYSA-N Zinc dication Chemical compound [Zn+2] PTFCDOFLOPIGGS-UHFFFAOYSA-N 0.000 description 2
- HXXFSFRBOHSIMQ-VFUOTHLCSA-N alpha-D-glucose 1-phosphate Chemical compound OC[C@H]1O[C@H](OP(O)(O)=O)[C@H](O)[C@@H](O)[C@@H]1O HXXFSFRBOHSIMQ-VFUOTHLCSA-N 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 235000021472 generally recognized as safe Nutrition 0.000 description 2
- 229910052816 inorganic phosphate Inorganic materials 0.000 description 2
- 229910001425 magnesium ion Inorganic materials 0.000 description 2
- 238000005374 membrane filtration Methods 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 150000004712 monophosphates Chemical class 0.000 description 2
- 235000002949 phytic acid Nutrition 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 238000005185 salting out Methods 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- NDVRKEKNSBMTAX-BTVCFUMJSA-N (2r,3s,4r,5r)-2,3,4,5,6-pentahydroxyhexanal;phosphoric acid Chemical compound OP(O)(O)=O.OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O NDVRKEKNSBMTAX-BTVCFUMJSA-N 0.000 description 1
- 229920000945 Amylopectin Polymers 0.000 description 1
- 229920000856 Amylose Polymers 0.000 description 1
- 241000205042 Archaeoglobus fulgidus Species 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 241000276408 Bacillus subtilis subsp. subtilis str. 168 Species 0.000 description 1
- 241000186216 Corynebacterium Species 0.000 description 1
- 241000588722 Escherichia Species 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 244000286779 Hansenula anomala Species 0.000 description 1
- 235000014683 Hansenula anomala Nutrition 0.000 description 1
- 102100035679 Inositol monophosphatase 1 Human genes 0.000 description 1
- IMQLKJBTEOYOSI-GPIVLXJGSA-N Inositol-hexakisphosphate Chemical compound OP(O)(=O)O[C@H]1[C@H](OP(O)(O)=O)[C@@H](OP(O)(O)=O)[C@H](OP(O)(O)=O)[C@H](OP(O)(O)=O)[C@@H]1OP(O)(O)=O IMQLKJBTEOYOSI-GPIVLXJGSA-N 0.000 description 1
- 241001506991 Komagataella phaffii GS115 Species 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 102100030999 Phosphoglucomutase-1 Human genes 0.000 description 1
- IMQLKJBTEOYOSI-UHFFFAOYSA-N Phytic acid Natural products OP(O)(=O)OC1C(OP(O)(O)=O)C(OP(O)(O)=O)C(OP(O)(O)=O)C(OP(O)(O)=O)C1OP(O)(O)=O IMQLKJBTEOYOSI-UHFFFAOYSA-N 0.000 description 1
- 241000160715 Sulfolobus tokodaii Species 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- PBAYDYUZOSNJGU-UHFFFAOYSA-N chelidonic acid Natural products OC(=O)C1=CC(=O)C=C(C(O)=O)O1 PBAYDYUZOSNJGU-UHFFFAOYSA-N 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 239000012043 crude product Substances 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000005265 energy consumption Methods 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 239000004459 forage Substances 0.000 description 1
- 235000021474 generally recognized As safe (food) Nutrition 0.000 description 1
- 235000021473 generally recognized as safe (food ingredients) Nutrition 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 231100000206 health hazard Toxicity 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 230000002427 irreversible effect Effects 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- -1 phospho phytate Chemical class 0.000 description 1
- 239000000467 phytic acid Substances 0.000 description 1
- 229940068041 phytic acid Drugs 0.000 description 1
- 230000009465 prokaryotic expression Effects 0.000 description 1
- 238000013138 pruning Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000007670 refining Methods 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 0.000 description 1
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention discloses a kind of methods that the multienzymatic reaction system by edible microorganismus expression prepares inositol.This method is the system for expressing multienzyme with food industry with strain, isoamylase, glucosan phosphorylase, phosphoglucomutase, inositol -3- phosphate synthase and inositol monophosphatase needed for producing catalytic starch and its derivative, a possibility that fundamentally avoiding toxalbumin, antigenic protein or contaminated with endotoxins that production technology mysoinositol is generated by Escherichia coli, stringent, complicated purifying process is avoided, production cost is reduced.
Description
Technical field
The invention belongs to the enzymatic production fields of inositol, and in particular to a kind of multienzymatic reaction expressed by edible microorganismus
The method that system prepares inositol.
Background technique
Inositol also known as bios Ⅰ are one of water-soluble (vitamin) B races, and structure is as shown in following formula I.Inositol be people,
The necessary material of animal and microorganism growth, is widely used in the industries such as medicine, food, feed.The demand in the whole world is big at present
Generally at annual 5,000 tons or so, due to the high price of current inositol, so that the market prospects of inositol are not opened adequately also
Hair, such as global forage yield in 2013 is 9,600,000,000 tons, if the inositol of 0.2-0.5% is all added, needed for feed industry
The yield of inositol should reach ten thousand tons of 190-480.In this case, the country or even global yield all far can not at present
Meet demand.
The main or traditional high-temperature pressurizing Hydrolysis of Phytic Acid (phospho phytate) of the production of inositol at present.The process equipment material
Matter requires strictly, and one-time investment is big；Operating pressure must control in a certain range, limit mentioning for utilization rate of raw materials
Height, and crude product process for refining is complicated, loss is more, and production cost is higher；In addition, the technique can generate a large amount of phosphoric acid pollution
Object, to water source etc., environmental pollution is serious.In recent years, in order to reduce energy consumption and pollution, water at atmospheric pressure solution is developed.Research flesh at present
The hot spot of alcohol production concentrates on chemical synthesis and microbion zymolysis, however different degrees of there are at high cost, low yields
The problems such as.
Patent CN106148425A describes a kind of preparation method of inositol, more particularly to external multienzyme catalysis by starch with
And the method that their derivative is converted into inositol.Starch Conversion is flesh by an external multienzyme catalyst system by this method
Alcohol, these key enzymes and its effect include: isoamylase (IA, EC 3.2.1.68), are amylose by amylopectin beta pruning；
Glucosan phosphorylase (α GP, EC 2.4.1.1), releases Cori ester from starch；Phosphoglucomutase
(PGM, EC 5.4.2.2), catalysis Cori ester to glucose _ 6- phosphoric acid；Inositol -3- phosphate synthase (IPS, EC
5.5.1.4), catalysis G-6-P is inositol -3- phosphoric acid；Inositol monophosphatase (IMP, EC 3.1.3.25), will
Inositol -3- phosphoric acid dephosphorization becomes inositol.Since most latter two enzyme reaction is irreversible reaction, so the enzymatic system can obtain
To very high conversion ratio.
This corresponding genomic DNA of five kinds of enzymes all can collect institute (American Type from American Type culture
Culture Collection, hereinafter referred to as ATCC) official website (www.atcc.org) on obtain.By this 5 genes point
It is not obtained, and is cloned into prokaryotic expression carrier pET20b by PCR from corresponding genomic DNA with different primers, obtained
Obtain corresponding expression vector.Expression vector is all converted into Bacillus coli expression bacterium BL21 (DE3) respectively again into
Row protein expression.
Escherichia coli are simple with gene structure, are easy to carry out genetic manipulation, the fast advantage of the speed of growth, but Escherichia coli
It is possible to that proteins toxic or antigenic protein, and the endotoxin generated by its cell wall can be contained in the product of expression, so
The product of Escherichia coli production needs stringent, complicated purifying process.
Summary of the invention
In order to avoid the deficiency of Escherichia coli expression of enzymes system, the purpose of the present invention is to provide a kind of new multi-enzyme systems
Production method, and be applied in the preparation of the higher inositol of safety requirements.
For this purpose, researcher of the invention attempts to screen from the expression system harmless to human body and animal suitable
Suitable strain.Lactic acid bacteria (lactic acid bacteria, LAB) is that one kind can be generated largely using fermentable carbohydrate
The bacterium of lactic acid is widely used in many industries such as light industry, food, medicine and feed industry, whether thallus now
Itself or its metabolite will not all generate health hazard, and culture process is mature, is suitble to industrial production.Therefore, it invents
People chooses lactic acid bacteria as the expression system of multi-enzyme system to obtaining multienzyme with catalytic activity and highly-safe first.But
It is that unfortunately, the target enzyme activity detected in lactic acid bacteria expression product is negative, and lactic acid bacteria expression system can not
Albumen multienzyme needed for effectively expressing produces inositol.
Inventor attempts to realize the purpose in the widely used yeast of field of food again.Wherein, saccharomyces cerevisiae is not only pacified
Entirely, culture is mature, also has breeding fastly, the short advantage of growth cycle；And Pichia anomala expression is high-efficient, the external source egg of expression
Bai Kezhan summary table reaches 90% or more of albumen, is conducive to isolating and purifying for destination protein, and can be in simple synthetic media
Middle realization High Density Cultivation, if it is possible to which application of succeeding in the present invention will have huge industrial potential.But it tries
Test that result is still unsatisfactory, the target enzyme activity of saccharomyces cerevisiae and Pichia pastoris equally shows as feminine gender.
After experience repeatedly failure, inventor surprisingly has found that bacillus subtilis and Corynebacterium glutamicum can be effective
Ground gives expression to target multienzyme, and the inositol that the high-quality that production capacity of making a living enough applies to the fields such as food, biological medicine requires provides
It may.Bacillus subtilis (Bccillus subtilis) is U.S. FDA " generally recognized as safe substance " (generally
Recognized as safe, GRAS) microorganism fungus kind in inventory.Corynebacterium glutamicum (Corynebacterium
It glutamicum) is the food safety level microbe in " United States Pharmacopeia " and " U.S.'s Food Chemical Codex " annex XV.
Based on research achievement of the invention, present invention firstly provides a kind of expression sides of multi-enzyme system for preparing inositol
Method, the multi-enzyme system are expressed by edible microorganismus.
Further, the edible microorganismus is bacillus subtilis and/or Corynebacterium glutamicum.
Further, the multi-enzyme system is isoamylase, glucosan phosphorylase, phosphoglucomutase, inositol-
One of 3- phosphate synthase and inositol monophosphatase are a variety of.
The multi-enzyme system of edible microorganismus expression can be selected according to the substrate type being catalyzed, starch and difference
Starch derivatives needed for enzyme class it is different.
Preferably, the multi-enzyme system is glucosan phosphorylase, phosphoglucomutase, the synthesis of inositol -3- phosphoric acid
Enzyme and inositol monophosphatase.
It is highly preferred that the multi-enzyme system is glucosan phosphorylase, phosphoglucomutase, the conjunction of inositol -3- phosphoric acid
At enzyme, inositol monophosphatase and isoamylase.
Secondly, the present invention also provides a kind of method for preparing inositol by edible microorganismus expression multi-enzyme system, this method
The multi-enzyme system expressed using preceding method carries out enzymic catalytic reaction, then reaction is produced using starch or starch derivatives as substrate
Object is separated, is purified to get inositol.
Further, the starch derivatives include boiling starch, it is amylodextrin, any one in maltodextrin
Kind is a variety of.
In the present invention, isoamylase derives from Sulfolobus tokodaii, and gene is in capital of a country gene and genome encyclopaedia
Number on pandect (Kyoto Encyclopedia of Genes and Genomes, hereinafter referred to as KEGG) is ST0928, Portugal
Phosphorylase derives from Thermotoga maritima, and number of the gene on KEGG is TM1168, and glucose phosphate becomes
For position enzyme source in Thermotoga maritima, number of the gene on KEGG is TM0769, and inositol -3- phosphate synthase comes
Derived from Archaeoglobus fulgidus, number of the gene on KEGG is AF1794, and inositol monophosphatase derives from
Thermotoga maritima, number of the gene on KEGG is TM1415, these genomic DNAs can all be trained from U.S. typical case
Support the official website of object collection institute (American Type Culture Collection, hereinafter referred to as ATCC)
(www.atcc.org) it is obtained on.The corresponding gene of 5 kinds of enzymes is passed through from corresponding genomic DNA with different primers respectively
PCR is obtained, and is cloned into expression vector, and corresponding expression vector is obtained.Expression vector is all converted respectively again
Protein expression is carried out into bacillus subtilis and/or Corynebacterium glutamicum.
Some specific embodiments according to the present invention when using bacillus subtilis as edible microorganismus expression system, carry
Body is pHT01.When using Corynebacterium glutamicum as edible microorganismus expression system, carrier pEC-XK99E.
The present invention is by target gene PCR, reprinting and the method for expression referring to Simple Cloning (You, C., et
al.2012).〃Simple Cloning via Direct Transformation of PCR Product(DNA
Multimer)to Escherichia col i and Bacillus subtil is./
RAppl.Environ.Microbiol.78 (5): 1593-1595.) method carries out.
The beneficial effects of the present invention are: successfully from food industry with filtered out in bacterium can express catalytic starch and its
Derivative prepares the strain of the multi-enzyme system of inositol, compared with patent CN106148425A is expression system with Escherichia coli, this
The method of invention fundamentally avoids toxalbumin, antigenic protein or the endogenous toxic material that production technology mysoinositol is generated by Escherichia coli
A possibility that element pollution, stringent, complicated purifying process is avoided, production cost is reduced.
Detailed description of the invention
Fig. 1 is inositol prepared by embodiment 51H-NMR(D2O, 400MHz) map；
Fig. 2 is the nuclear magnetic resonance resolution table and chemical structural drawing of inositol prepared by embodiment 5；
Fig. 3 is inositol prepared by embodiment 71H-NMR(D2O, 400MHz) map；
Fig. 4 is the nuclear magnetic resonance resolution table and chemical structural drawing of inositol prepared by embodiment 7.
Specific embodiment
It is illustrated below by way of specific embodiment is further to summary of the invention of the invention, but should not be construed as the present invention
Range be only limitted to example below, invention thinking according to the present invention and entire contents, can will be each in following instance
Technical characteristic makes combination/replacement/adjustment/modification appropriate etc., this is will be obvious to those skilled in the art that still
Belong to the scope that the present invention protects.
Main experimental materials
Soluble starch, solublestarch, ACROS Products, product number: 424490020；
Expression vector and host cell (being shown in Table -1), NTCC Type Tissue 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 |
Embodiment 1: lactic acid bacteria prepares multienzyme
Official of five genes of KEGG number respectively ST0928, TM1168, TM0769, AF1794, TM1415 from ATCC
It is obtained on square website (www.atcc.org).It is obtained, is led to by PCR from corresponding genomic DNA with different primers respectively
Cross Simple Cloning (You, C., et al. (2012) " Simple Cloning via Direct
Transformation of PCR Product(DNA Multimer)to Escherichia coli and Bacillus
Subtilis. " (5) Appl.Environ.Microbiol.78: 1593-1595.) method be cloned into pNZ9530 carrier,
Corresponding expression vector is obtained, then is all converted respectively into lactic acid bacteria NZ9000, and carry out protein expression, through detecting,
Target enzyme activity is negative.
Embodiment 2: saccharomyces cerevisiae prepares multienzyme
Using method same as Example 1 by 5 kinds of gene clonings into pYES2 carrier, obtain corresponding gene expression
Carrier, then all convert respectively into saccharomyces cerevisiae Y187, and carry out protein expression, through detecting, target enzyme activity is negative.
Embodiment 3: Pichia pastoris prepares multienzyme
Using method same as Example 1 by 5 kinds of gene clonings into pPIC9K carrier, obtain corresponding gene expression
Carrier, then all convert respectively into Pichia pastoris GS115, and carry out protein expression, through detecting, target enzyme activity is negative.
Embodiment 4: bacillus subtilis prepares multienzyme
Using method same as Example 1 by 5 kinds of gene clonings into pHT01 carrier, obtain corresponding gene expression
Carrier, then converted respectively into paddy bacillus subtilis 168, and carry out protein expression and purifying.
Embodiment 5: starch is catalytically conveted to inositol by external multienzyme
The multienzyme made from embodiment 4 carries out inositol preparation test.Contain in one 500 milliliters of reaction system
The HEPES buffer solution (pH7.2) of 100mM, the inorganic phosphate radical of 10mM, the divalent magnesium ion of 5mM, 0.5mM zinc ion, 5U/mL
Glucosan phosphorylase, the phosphoglucomutase of lU/mL, the inositol of 5U/mL inositol -3- phosphate synthase and 2U/mL
Monophosphate enzyme, the isoamylase of lU/mL, the soluble starch of 10g/L carry out catalysis reaction at 80 DEG C, after reacting 40 hours,
Micro-filtrate membrane filtration, filtrate rotary evaporator are concentrated into about 50 milliliters, and cooling, precipitation crystallization, filtering, drying obtain 5.8 grams of white powder
End.Fusing point is 224.8~225.4 DEG C after measured, nuclear magnetic resonance1H-NMR(D2O, 400MHz) map and resolution table be shown in Fig. 1 and figure
2。
Embodiment 6: Corynebacterium glutamicum prepares multienzyme
Using method same as Example 1 by 5 kinds of gene clonings into pEC-XK99E carrier, obtain corresponding gene
Expression vector, then converted respectively into Corynebacterium glutamicum NTCC910233, and carry out protein expression and purifying.
Embodiment 7: starch is catalytically conveted to inositol by external multienzyme
The multienzyme made from embodiment 6 carries out inositol preparation test.Contain in one 500 milliliters of reaction system
The HEPES buffer solution (pH7.2) of 100mM, the inorganic phosphate radical of 10mM, the divalent magnesium ion of 5mM, 0.5mM zinc ion, 5U/mL
Glucosan phosphorylase, the phosphoglucomutase of lU/mL, the inositol of 5U/mL inositol -3- phosphate synthase and 2U/mL
Monophosphate enzyme, the isoamylase of lU/mL, the soluble starch of 10g/L carry out catalysis reaction at 80 DEG C, after reacting 40 hours,
Micro-filtrate membrane filtration, filtrate rotary evaporator are concentrated into about 50 milliliters, and cooling, precipitation crystallization, filtering, drying obtain 6.6 grams of white powder
End.Fusing point is 225.2~225.6 DEG C after measured, nuclear magnetic resonance1H-NMR(D2O, 400MHz) map and resolution table be shown in Fig. 3 and figure
4。
In conclusion purpose multi-enzyme system can successfully be expressed using Corynebacterium glutamicum and bacillus subtilis, and
Realize that catalytic starch is converted into inositol.
Claims (7)
1. a kind of expression for the multi-enzyme system for preparing inositol, which is characterized in that the multi-enzyme system is by edible microorganismus
Expression.
2. the expression of the multi-enzyme system according to claim 1 for preparing inositol, which is characterized in that it is described eat it is micro-
Biology is bacillus subtilis and/or Corynebacterium glutamicum.
3. the expression of the multi-enzyme system according to claim 1 for preparing inositol, which is characterized in that the multienzyme
System is isoamylase, glucosan phosphorylase, phosphoglucomutase, inositol -3- phosphate synthase and inositol monophosphatase
One of or it is a variety of.
4. the expression of the multi-enzyme system according to claim 3 for preparing inositol, which is characterized in that the multienzyme
System is glucosan phosphorylase, phosphoglucomutase, inositol -3- phosphate synthase and inositol monophosphatase.
5. the expression of the multi-enzyme system according to claim 3 for preparing inositol, which is characterized in that the multienzyme
System is glucosan phosphorylase, phosphoglucomutase, inositol -3- phosphate synthase, inositol monophosphatase and isoamylase.
6. a kind of method for preparing inositol by edible microorganismus expression multi-enzyme system, which is characterized in that this method is wanted using right
The multi-enzyme system for asking 1-5 any one the method to express carries out enzymic catalytic reaction using starch or starch derivatives as substrate,
Reaction product is separated again, is purified to get inositol.
7. the method according to claim 6 for preparing inositol by edible microorganismus expression multi-enzyme system, which is characterized in that institute
The starch derivatives stated includes boiling starch, amylodextrin, any one or more in maltodextrin.
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