CN105838704A - Nanofiber biological membrane immobilized bi-enzyme system and trehalose catalytic synthesis method thereof - Google Patents
Nanofiber biological membrane immobilized bi-enzyme system and trehalose catalytic synthesis method thereof Download PDFInfo
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- CN105838704A CN105838704A CN201610320569.5A CN201610320569A CN105838704A CN 105838704 A CN105838704 A CN 105838704A CN 201610320569 A CN201610320569 A CN 201610320569A CN 105838704 A CN105838704 A CN 105838704A
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- trehalose
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- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 title claims abstract description 63
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 title claims abstract description 63
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 title claims abstract description 53
- 238000000034 method Methods 0.000 title claims abstract description 40
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- 238000007036 catalytic synthesis reaction Methods 0.000 title abstract 2
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- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 4
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 4
- 229910021529 ammonia Inorganic materials 0.000 claims description 4
- QGZKDVFQNNGYKY-UHFFFAOYSA-N ammonia Natural products N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims description 4
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- 238000012163 sequencing technique Methods 0.000 claims description 4
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- 229910019142 PO4 Inorganic materials 0.000 claims description 2
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 2
- 239000000872 buffer Substances 0.000 claims description 2
- 238000004140 cleaning Methods 0.000 claims description 2
- 238000010276 construction Methods 0.000 claims description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 2
- 239000010452 phosphate Substances 0.000 claims description 2
- 238000001338 self-assembly Methods 0.000 claims description 2
- XUWPJKDMEZSVTP-LTYMHZPRSA-N kalafungina Chemical compound O=C1C2=C(O)C=CC=C2C(=O)C2=C1[C@@H](C)O[C@H]1[C@@H]2OC(=O)C1 XUWPJKDMEZSVTP-LTYMHZPRSA-N 0.000 claims 1
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- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 abstract description 13
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 abstract description 13
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- IQFVPQOLBLOTPF-HKXUKFGYSA-L congo red Chemical compound [Na+].[Na+].C1=CC=CC2=C(N)C(/N=N/C3=CC=C(C=C3)C3=CC=C(C=C3)/N=N/C3=C(C4=CC=CC=C4C(=C3)S([O-])(=O)=O)N)=CC(S([O-])(=O)=O)=C21 IQFVPQOLBLOTPF-HKXUKFGYSA-L 0.000 description 7
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- 125000000539 amino acid group Chemical group 0.000 description 4
- 238000004128 high performance liquid chromatography Methods 0.000 description 4
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- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 108020004414 DNA Proteins 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
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- 230000008901 benefit Effects 0.000 description 2
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
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- 239000000126 substance Substances 0.000 description 2
- 238000010189 synthetic method Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 125000000647 trehalose group Chemical group 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 1
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 1
- 241000195493 Cryptophyta Species 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 238000010268 HPLC based assay Methods 0.000 description 1
- 108010093096 Immobilized Enzymes Proteins 0.000 description 1
- 229910017621 MgSO4-7H2O Inorganic materials 0.000 description 1
- 240000000220 Panda oleosa Species 0.000 description 1
- 235000016496 Panda oleosa Nutrition 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 241000235061 Pichia sp. Species 0.000 description 1
- 108010046685 Rho Factor Proteins 0.000 description 1
- 102100029677 Trehalase Human genes 0.000 description 1
- 108010087472 Trehalase Proteins 0.000 description 1
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- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 1
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- AIUDWMLXCFRVDR-UHFFFAOYSA-N dimethyl 2-(3-ethyl-3-methylpentyl)propanedioate Chemical compound CCC(C)(CC)CCC(C(=O)OC)C(=O)OC AIUDWMLXCFRVDR-UHFFFAOYSA-N 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 1
- 235000019797 dipotassium phosphate Nutrition 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 229910000397 disodium phosphate Inorganic materials 0.000 description 1
- 235000019800 disodium phosphate Nutrition 0.000 description 1
- IDAGXRIGDWCIET-SDFKWCIISA-L disodium;(2s,3s,4s,5r)-2,3,4,5-tetrahydroxyhexanedioate Chemical compound [Na+].[Na+].[O-]C(=O)[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C([O-])=O IDAGXRIGDWCIET-SDFKWCIISA-L 0.000 description 1
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- 229930182478 glucoside Natural products 0.000 description 1
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- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
- 229930027917 kanamycin Natural products 0.000 description 1
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 1
- 229960000318 kanamycin Drugs 0.000 description 1
- 229930182823 kanamycin A Natural products 0.000 description 1
- 108010074674 maltooligosyl trehalose trehalohydrolase Proteins 0.000 description 1
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- 238000003199 nucleic acid amplification method Methods 0.000 description 1
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- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 150000002482 oligosaccharides Chemical class 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 230000012743 protein tagging Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
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- 239000002689 soil Substances 0.000 description 1
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- 231100000331 toxic Toxicity 0.000 description 1
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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
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/18—Multi-enzyme systems
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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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- 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
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
- C12N9/2411—Amylases
- C12N9/2425—Beta-amylase (3.2.1.2)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/12—Disaccharides
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y204/00—Glycosyltransferases (2.4)
- C12Y204/01—Hexosyltransferases (2.4.1)
- C12Y204/01245—Alpha,alpha-trehalose synthase (2.4.1.245)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01002—Beta-amylase (3.2.1.2)
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
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Abstract
The invention provides a nanofiber biological membrane immobilized bi-enzyme system and a trehalose catalytic synthesis method thereof. A starchiness nanofiber biological membrane is arranged on the surface of an escherichia coli cell generating high-temperature-resistant trehalose synthase through fermentation, polypeptide tag SpyTag-SpyCatcher capable of gene coding is used for specificity covalent binding and efficient immobilization recombination of beta-amylase, a trehalose synthase-beta amylase bi-enzyme catalytic system is constructed autonomously, and finally extracellular and intracellular two-step catalysis is conducted continuously. The immobilization efficiency of beta-amylase can reach 50-62%, maltose is generated by 20-25wt% of soluble starch under catalysis of beta-amylase on the extracellular biological membrane, enters the cell and reacts with trehalose synthase to generate trehalose, and the conversion rate of starch can reach 45-55% after the immobilized cell is reused for 10-16 times.
Description
Technical field
The invention belongs to biological technical field, relate to using genetic engineering and the technological means of enzyme engineering, utilize escherichia coli to be catalyzed the method that cheap starchy material efficiently prepares trehalose.
Background technology
Trehalose is distributed widely in antibacterial, algae, yeast, rudimentary plant, insecticide and other invertebratess, is one of principal oligosaccharide of recently exploitation in the world.Trehalose can cell be in hunger, be dried, high temperature, freezing, the stressful environmental such as hyperosmosis and toxic reagent time; effectively maintain stablizing of intracellular plasma membrane and protein, obtain stem-winding result at aspects such as Protecting gene engineering enzyme, various virus, vaccine, antibody, rho factor, nucleic acid.The biological function of this uniqueness makes trehalose gather around in fields such as food, cosmetics, molecular biology, medical science, agriculturals to have broad application prospects.
At present trehalose commercial process mostly is enzymatic clarification, but trehalose synthase belongs to endocellular enzyme, it usually needs broken extraction purification trehalase system, causes enzyme in various degree to live loss, and purification process complexity spends huge efficiency low, adds production cost.Either permeability contains enzyme cell or trehalose synthase and substrate maltose catalytic reaction, is restricted by enzyme equilibrium, and the existence of substrate and by-product adds difficulty to the purification of trehalose.
Patent CN105039191A discloses a kind of surface display trehalose synthetase, the method for hydrolysis of trehalose enzyme and application, malt oligosaccharide based mycose synthetase, malt oligosaccharide based mycose hydrolytic enzyme are showed in phage surface with Pir1p protein fusion by the method, realize two kinds of enzymes surface display at Pichia sp., thus realize two enzymes method and produce the efficient application of trehalose.This technology needs substep catalytic reaction trehalose synthesis, malt oligosaccharide based mycose synthetase (MTSase) does not produce conversion to maltose, maltose is caused to accumulate in a large number, the yield finally making trehalose can not reach higher level, cause enzyme utilization rate relatively low, become the limiting factor of double enzyme reaction.
Patent CN103146779A discloses a kind of method utilizing whole-cell catalytic trehalose synthesis, changes permeability of cell membrane by permeabilized treatment and utilizes maltose whole-cell catalytic trehalose synthesis, removes cell breakage operation from.Trehalose synthetase (Trehalose synthase is called for short TreS) can directly act on maltose substrate, and the maltose connected by α, α-Isosorbide-5-Nitrae glycosidic bond turns glucosides one step by intramolecular and is converted into α, α-1, the trehalose that 1 glycosidic bond connects.This approach only needs single step reaction, and technique is simple, but its substrate maltose does not exist competitive advantage compared with starch.
Patent CN104328107A discloses a kind of method of co-immobilization double-enzyme catalysis trehalose synthesis, the method permeabilized treatment engineering bacteria containing trehalose synthase, and fix cell and saccharifying enzyme by preparing chitosan-Sargassum sodium saccharate microsphere, with starch for Material synthesis trehalose, not only increase catalytic efficiency and repeatable usability, and cheaper starting materials, but prepare fixation support by chemical method, immobilization efficiency is the highest, and non-fully green bio catalytic process.
Therefore, the method using method novel, efficient to catalyze and synthesize trehalose still has large development potentiality.
Summary of the invention
The technical problem to be solved is to solve technology path and the efficiency of trehalose synthesis in existing synthetic system；That is, relatively low in existing double-enzyme catalysis system (malt oligosaccharide based mycose synthetase MTSase and malt oligosaccharide based mycose hydrolytic enzyme MTHase) enzyme utilization ratio；And list enzyme catalysis system (trehalose synthetase TreS) immobilization efficiency is the highest, substrate does not possess competitive advantage, and the problem that separation costs is higher.
In order to solve above-mentioned technical problem, the technical scheme is that with starchy material, particularly soluble starch is substrate；Utilization the Polypeptide tags SpyTag-SpyCatcher specific covalent of gene code can combine efficiently fixing recombinant beta amylase, and beta amylase is immobilized onto E. coli cell surface；Soluble starch is catalyzed as maltose at born of the same parents' outer surface, and maltose enters the escherichia coli intracellular that have expressed trehalose synthetase, forms trehalose, and further isolated and purified acquisition trehalose through trehalose synthetase catalysis.
Concrete, the technical scheme is that
A kind of nanofiber biomembrane immobilized bi-enzyme system, it is characterised in that this pair of enzyme system can generate trehalose by catalytic starch in same system, and wherein, this pair of enzyme is respectively beta amylase and trehalose synthetase.
System of the present invention at least includes, expresses the escherichia coli A of trehalose synthetase gene Tre and CsgA gene simultaneously；Express the escherichia coli B of beta amylase gene；Polypeptide tags SpyTag is connected to the fusion protein CsgA-SpyTag that the carbon teminal of CsgA gene is formed；And the fusion protein BA-SpyCatcher that the carbon teminal that Polypeptide tags SpyCatcher is connected to beta amylase gene is formed；And the nanofiber biomembrane covalency being self-assembly of by CsgA-SpyTag high polymeric at escherichia coli A cell surface fixes the immobilization catalytic membrane that BA-SpyCatcher is formed.
Wherein, the gene order of fusion protein CsgA-SpyTag is the CsgA sequence (NCBI announced according to NCBI
Reference Sequence:NC_000913.3) and reported document (Nguyen P Q, Botyanszki Z, Tay P K R, et al.
Programmable biofilm-based materials from engineered curli
Nanofibres [J]. Nature communications, 2014,5.) in SpyTag (peptide sequence AHIVMVDAYKPTK) by six amino acid residue
The flexible linker fusion of GSGGSG is formed by connecting.
Fusion protein BA-SpyCatcher gene order is that the gene order (GenBank:JQ478411.1) of the SpyCatcher of beta amylase BA sequence (GenBank:AJ250858.1) announced according to NCBI and announcement is by six amino acid residue
The flexible linker fusion of GSGGSG is formed by connecting, and beta amylase BA sequence uses full genome synthetic method to obtain.
The method building described system, concrete step is:
(1) build containing trehalose synthetase cell surface formation nanofiber biomembranous escherichia coli A: the TreS gene expanded by carrier PETDuet with PCR double digestion simultaneously is connected acquisition recombiant plasmid PETDuet-TreS, convert after again it being connected with double digestion while of CsgA-SpyTag fusion gene fragment to e. coli bl21 (DE3) competent cell, obtain, through ammonia benzyl antibiotic-screening, bacterium colony PCR checking and gene sequencing, the escherichia coli A that gene order is correct；
(2) the engineering bacteria B containing beta amylase is built: design primer carries out PCR amplification and obtains amylase gene fragment BA and Polypeptide tags SpyCatcher genetic fragment, carries out splicing construction recombination plasmid PET28a-BA-by overlapping PCR method
SC converts to e. coli bl21 (DE3) competent cell, obtains, through kanamycin screening, bacterium colony PCR checking and gene sequencing, the escherichia coli B that gene order is correct
(3) self-induction cultivates escherichia coli A, it is thus achieved that containing the culture fluid of escherichia coli A；
(4) self-induction cultivates escherichia coli B；
(5) by broken for escherichia coli B and isolated and purified acquisition crude enzyme liquid BA-SpyCatcher；
(6) by crude enzyme liquid and culture fluid haptoreaction 48h described in step (3), it is thus achieved that nanofiber biomembrane immobilized bi-enzyme system；
(7) the nanofiber biomembrane immobilized bi-enzyme system also described in cleaning step (6) it is centrifuged, and resuspended to original volume size with phosphate buffer.
Method of the present invention, wherein, in step (6), the amount of crude enzyme liquid is based on the fermentating liquid volume preparing this crude enzyme liquid, and the volume ratio of step (3) culture fluid used is 1:1 with this step.
Method of the present invention, wherein, also includes the engineering bacteria described in step with centrifugal separation (3) and step (4), and by the resuspended step of phosphate buffer.
Method of the present invention, wherein, described in step (5), breaking method is high-pressure breaking, is separated into solid-liquid separation, and sieve aperture is less than 14 mesh；Purification process is membrane filtration, and molecular cut off is 300-400.
A kind of method utilizing nanofiber biomembrane immobilized bi-enzyme to catalyze and synthesize trehalose, utilizes any of the above-described fiber biofilm immobilized bi-enzyme system to contact with soluble starch in phosphoric acid buffer liquid system, and catalysis generates trehalose.Concretely comprising the following steps of the method:
(1) being contacted with nanofiber biomembrane immobilized bi-enzyme system by the soluble starch solution that mass concentration is 20%-25%, condition is 35-60 DEG C, pH
6.0-7.5, rotating speed 100-200rpm, catalytic reaction 20-30 hour；Obtain the solution containing trehalose；
(2) trehalose described in claim 1 is separated；
Wherein, the step reclaiming nanofiber biomembrane immobilized bi-enzyme system is also included.
Soluble starch in the present invention is interpreted as: utilize amyloid raw material or pure starch, through hydro-thermal of the prior art or liquefaction technology, by starch granules swell the starch solution that is dissolved in water, or directly dissolve in the starch material of hot water, as long as wherein meeting its dry matter content at 20-25%.
This catalyzes and synthesizes the method for trehalose, and wherein, the described volume of nanofiber biomembrane immobilized bienzyme system and the volume ratio of soluble starch solution are 1:4.
Any of the above-described described phosphate buffer, its phosphate concentration is 0.04 mol/L, and pH is 7.0.
Pass through the technical program, it is achieved that the process route of trehalose efficiently prepared by cheap raw material soluble starch.
The beneficial effects of the present invention is:
1. the present invention provides functionalization biofilm matrix as beta amylase fixation support by nanofiber biomembrane integration exhibition technology on the colorectal cell surface containing trehalose synthase, construct the double enzyme fixed catalytic system of beta amylase-trehalose synthase, the outer two-step catalysis of intracellular born of the same parents is carried out continuously, and then make enzymic catalytic reaction carry out towards the direction of end-product trehalose all the time, further increase the transformation efficiency of starch.
2. the covalent bond strategy of present invention employing is spontaneous is carried out, without chemical treatment step, simple assembled method specific immobilised enzymes is provided, the complete biosynthesis of fixation support material, binding site dense arrangement produces exhibiting high surface catalysis region, substantially increase enzyme immobilizatio efficiency and utilization ratio, thus improve trehalose production efficiency.
3. whole-cell catalytic and immobilization technology are combined by the present invention, and in reactant liquor system, other compositions are less, decrease the difficulty of later separation Purifing Trehalose, greatly reduce separation costs.
4. the cell containing immobilized enzyme in the present invention can be recycled 10-16 time, and production application is strong.
Accompanying drawing explanation
Fig. 1 is the agarose gel electrophoresis histogram of Tres gene and CsgA-SpyTag fusion gene；
Wherein, Lane
1:DNA Marker 5000；
Lane 2,3:Tres gene PCR amplified production；
Lane 4,5:CsgA-SpyTag fusion gene PCR primer；
Lane 6:DNA
Marker 2000 。
Fig. 2 is β-Amylase-SpyCather fusion gene agarose gel electrophoresis histogram；
Wherein, Lane
1:DNA Marker 5000;
Lane 2,3: β-Amylase-SpyCather fusion gene PCR primer.
Fig. 3 is that E. coli cell surface amyloid fiber is combined full wavelength scanner analysis chart after absorption with Congo red, and illustration is the color contrast before and after Congo red absorption.
Fig. 4 is colibacillary scanning electron microscope (SEM) photograph, amplification be 10000 times (A) be that the escherichia coli (B) expressing CsgA amyloid fiber do not express the escherichia coli of CsgA amyloid fiber.
Detailed description of the invention
Embodiment
1
This example demonstrates that trehalose synthetase gene TreS and the structure of amyloid CsgA-SpyTag fusion gene coexpression engineering bacteria
The gene order of fusion protein CsgA-SpyTag is the CsgA sequence (NCBI announced according to NCBI
Reference Sequence:NC_000913.3) and reported document (Nguyen P Q, Botyanszki Z, Tay P K R, et al.
Programmable biofilm-based materials from engineered curli
Nanofibres [J]. Nature communications, 2014,5.) in SpyTag (peptide sequence AHIVMVDAYKPTK) by six amino acid residue
The flexible linker fusion of GSGGSG is formed by connecting.
Strain that is concrete, that will buildE. coli BL21 (DE3) (pET22b-treS) (Jiang L, Lin M, Zhang Y,
et al. Identification and characterization of a novel trehalose
Synthase gene derived from saline-alkali soil metagenomes [J]. PloS one, 2013,8 (10): e77437.) LB fluid medium (peptone 10 g/L, yeast powder 5 g/L, sodium chloride 10 it are seeded to
G/L) in 37 DEG C, 200rpm incubated overnight, take the method that zymocyte liquid extracts test kit according to TAKARA bacterial plasmid and provided and extract this plasmid pET22b-treS, go forward side by side performing PCR amplification as template design primer T1 and T2.
T1:5'-GGAATTCCATATGCAGATGACCGGGGAC-3'
T2:5'-CCCTCGAGTCACTTCGCCGTCTG -3'
In primer T1 and T2, wherein add NdeI and XhoI restriction enzyme site (line position) respectively.
PCR condition is: 94 DEG C of denaturation 5 min
；94 DEG C of degeneration 30 s, 60 DEG C of annealing 30 s, 72 DEG C extend 1min
50s, circulates 30 times；72 DEG C of supplementary extensions 10
min.The agarose gel electrophoresis detection product of 1% recovery about 1803bp band (such as Fig. 1) of tapping rubber, it is thus achieved that TreS genetic fragment.By its with carrier PETDuet simultaneously through NdeI and XhoI double digestion and be connected acquisition recombiant plasmid PETDuet-TreS, convert to E. coli competentE. coli In BL21 (DE3), screen and check order and obtain, through Amp resistance, bacterium colony PCR, the recombiant plasmid PETDuet-TreS that sequence is correct.
By complete synthesis acquisition CsgA-SpyTag fusion gene.With the puncture bacteria plasmid of complete synthesis CsgA-SpyTag gene as template, design synthesis upstream and downstream primer C1 and C2,
C1:5'-CATGCCATGGATGAAACTTTTAAAAGTA
-3'
C2:5'-CCCAAGCTTTTATTTGGTGGGTTTAT -3'
In primer C1 and C2, wherein add Nco and Hind respectively
Ш restriction enzyme site (line position)
PCR amplification condition is: 94 DEG C of denaturation 5 min
；94 DEG C of degeneration 30 s, 60 DEG C of annealing 30 s, 72 DEG C extend 40 s, circulate 30 times；72 DEG C of supplementary extensions 10
min.The agarose gel electrophoresis detection product of 1% recovery about 525bp band of tapping rubber, it is thus achieved that CsgA-SpyTag gene PCR amplification glue reclaims fragment, and agarose gel electrophoresis band is verified as shown in Figure 1.
After the recovery fragment use of plasmid PETDuet-TreS and CsgA-SpyTag fusion gene PCR glue is cut enzyme Nco and Hind Ш double digestion simultaneously soon, 16 DEG C are overnight connected, obtain heterologous expression vector PETDuet-TreS-CsgA-SpyTag, by recombinant plasmid transformed to expressive host E. coli competentE. coli In BL21 (DE3), through Amp resistance, bacterium colony PCR screening recon obtains clone strainE. coli BL21 (DE3) (PETDuet-TreS-CsgA-SpyTag), preserves after order-checking is correct.
Embodiment
2
This example demonstrates that beta amylase-SpyCatcher fusion gene BA-SC expresses the structure of engineering bacteria.
Fusion protein BA-SpyCatcher gene order is that the gene order (GenBank:JQ478411.1) of the SpyCatcher of beta amylase BA sequence (GenBank:AJ250858.1) announced according to NCBI and announcement is by six amino acid residue
The flexible linker fusion of GSGGSG is formed by connecting, and beta amylase BA sequence uses full genome synthetic method to obtain.
Design synthesis upstream and downstream primers F 1 and R1(according to beta amylase gene order and SpyCatcher gene order and expand beta amylase gene BA), F2 and R2(expand Polypeptide tags SpyCatcher).
F1:5'-CGGGATCCATGTTCATTTTGAGTC-3'
R1:5'-ACTGCCACCGCCACCGCTACCGCCACCGCCCCAATTATCTGTATAA-3'
F2:5'-ACTGCCACCGCCACCGCTACCGCCACCGCCCCAATTATCTGTATAA-3'
R2:5'-CCCTCGAGTTAAATATGAGCGTCACCTTTAGTT-3'
In primers F 1 and R2, wherein add BamH and Xho restriction enzyme site (line position) respectively
PCR amplification is carried out for template with complete synthesis beta amylase genetic fragment.PCR condition is: 94 DEG C of denaturation 5 min
；94 DEG C of degeneration 30 s, 60 DEG C of annealing 30 s, 72 DEG C extend 2 min, circulate 30 times；72 DEG C of supplementary extensions 10
min.The agarose gel electrophoresis detection product of 1% recovery about 1600bp band of tapping rubber, it is thus achieved that BA-linker genetic fragment.
With synthesis SpyCatcher gene as template, with F2 and R2 as primer, carry out PCR amplification.PCR condition is: 94 DEG C of denaturation 5 min
；94 DEG C of degeneration 30 s, 55 DEG C of annealing 30 s, 72 DEG C of extensions
0.5min, circulates 30 times；72 DEG C of supplementary extensions 10
min.The agarose gel electrophoresis detection product of 1% recovery about 450bp band of tapping rubber, it is thus achieved that SpyCatcher-linker genetic fragment.
BA-linker genetic fragment and SpyCatcher-linker genetic fragment are spliced by overlapping PCR method.
PCR condition is: 94 DEG C of denaturation 5 min
；94 DEG C of degeneration 30 s, 55 DEG C of annealing 30 s, 72 DEG C extend 2min, circulate 30 times；72 DEG C of supplementary extensions 10
min.The agarose gel electrophoresis detection product of 1% BA-linker-SpyCatcher genetic fragment (as shown in Figure 2) of recovery about 2000bp band of tapping rubber, by this fusion genes of interest and expression vector pET-28a simultaneously through BamH and Xho double digestion and be connected acquisition recombiant plasmid PET28a-BA-SC, convert to E. coli competentE. coli In BL21 (DE3), obtain clone strain through kana resistance, bacterium colony PCR screening reconE. coliBL21 (DE3) (PET28a-BA-SC), preserves after order-checking.
Embodiment
3
This example demonstrates that containing the trehalose synthetase cell surface biomembranous effect of function of creation nanofiber
From flat board by strainE. coliBL21 (DE3) (PETDuet-TreS-CsgA-ST) is inoculated into the 50ml containing ammonia benzyl resistance
In LB culture medium, 37 DEG C, 200rpm/min
Incubated overnight 12h；Bacterium solution is forwarded to (glycerol 5g/L, Na2HPO4 in the 50 mL self-induction culture medium containing ammonia benzyl resistance by the inoculum concentration by 2%
6g/L、K2HPO4 3g/L、NH4Cl
1g/L、NaCl
0.5g/L, MgSO4-7H2O 0.5g/L, soy peptone 10g/L), 30 DEG C, 180r/min shaken cultivation abduction delivering 10-12
H, collects fermentation liquid, uses pH 7.0 PBS to wash 2 times and resuspended, and the enzyme measuring trehalose synthase cell is lived as 8000U/mL.By Congo red solution, the escherichia coli containing trehalose synthase are combined absorption somatic cells surface be can be observed substantially adsorbed Congo red,
To can produce the escherichia coli of CsgA amyloid fiber by scanning electron microscope analysis (electron-microscope scanning figure refers to accompanying drawing 4) simultaneously, show that surface of E. coli creates nanofiber biomembrane, achieve trehalose synthase TreS and the coexpression of amyloid CsgA, can be as specificity fixation support.
Trehalose synthase enzyme is lived and is defined: at 30 DEG C, and it is 1 enzyme activity unit that every l min generates the amount of the trehalose synthase needed for l nmoL trehalose.
Enzyme activity determination: take 800ul
The maltose solution of PBS preparation and 200ul cell suspending liquid mixing (often group 3 is parallel), 1ml reaction system at 30 DEG C, is reacted 30min, is boiled and boil 10
Min enzyme denaturing.After it cools down, 12000 r/min are centrifuged 2 min, take supernatant, measure the content of trehalose generated.According to standard curve determination content of trehalose and the work of trehalose synthase enzyme is calculated with HPLC after it cools down.
HPLC assay method is: differential refraction detector, and chromatographic column is
Alltima Amino 100A 5u post (4.6mm × 250mm)；Flowing phase: acetonitrile/water=
75/25(v/v)；Column temperature: 35 degree；Sample size 100ul;
Flow velocity: 0.6mL/min;
Quantitatively Congo red combines adsorption analysis: take the somatic cells of 1 abduction delivering CsgA resuspended for mL PBS, add 0.025mmol Congo red solution, mixing vibration 10min, 12000rpm is centrifuged 1min, its supernatant absorbance is surveyed by microplate reader under 490nm, refer to accompanying drawing 3 with the common somatic cells not expressing CsgA for comparison, scanning result and picture, scanning result Congo red has substantially been adsorbed on the somatic cells surface understanding expression CsgA albumen.
Implement
Example
4
This example demonstrates that and utilize nanofiber biomembrane to fix the beta amylase effect for double-enzyme catalysis trehalose synthesis.
The strain that will buildE. coli BL21 (DE3) (PET28a-BA-SC) is seeded in 50ml LB fluid medium, 37 degree of incubated overnight, transfers in LB culture medium by 2% inoculum concentration, 37 degree of shaken cultivation 2.5h, the IPTG of interpolation final concentration 0.2mmol/L, 25 DEG C
180r/min continues to cultivate 10h, centrifugal collection thalline, and use pH 7.0 PBS washs 2 times and 1/2 volume is resuspended, uses hyperpressure to crush and carries out cell breakage acquisition beta amylase enzyme liquid.Take 0.9 mL PBS (pH
The concentration 2% 7.0) prepared (W/V) soluble starch solution, 40 DEG C of preheating 5 min, add 0.1
40 DEG C of insulation reaction 30 min after mL crude enzyme liquid, boiling water bath heats 5 min, and centrifuging and taking supernatant carries out 0.22 μm membrane filtration, and the supernatant after filtration carries out high performance liquid chromatography (HPLC) detection maltose concentration and calculates enzyme work.Measure beta amylase enzyme alive 25
U/mL。
Enzyme activity is defined as: under these conditions, and it is 1 enzyme activity unit (U) that l ml enzyme liquid catalysis per minute 2% soluble starch generates the enzyme amount of 1 μm ol maltose.
Biomembranous for surface display nanofiber Bacillus coli cells suspension is mixed shaken overnight with beta amylase enzyme liquid according to equal-volume ratio, it is centrifuged and abandons residual enzyme liquid, and with unadsorbed fixing beta amylase content in Coomassie Brilliant Blue detection residual liquid, clean somatic cells twice with phosphate buffer and 1/4 volume is resuspended, obtaining surface fixing Beta-diastatic escherichia coli, beta amylase immobilization efficiency on nanofiber biomembrane can reach 50%-62% after measured.
Soluble starch solution and 500mL surface fixing Beta-diastatic Bacillus coli cells suspension stirring at low speed reaction that 2L mass concentration is 20%-25% is added in 5L reactor, catalytic reaction 20-30 hour, reaction condition is 35-60 DEG C, pH 6.0-7.5, rotating speed 100-200rpm；Use 14 mesh sieve hole Filter Press separating reaction liquid, filtrate is trehalose mixed liquor, filtering residue is the escherichia coli that double enzyme is fixing, HPLC is used to detect its content of trehalose, and calculate the conversion ratio of trehalose, evaluating the catalysis activity of cell with this, converting soluble starch solution with this understanding through test is that the first transfonning rate of trehalose is up to 55%.
Implement
Example
5
This example demonstrates that engineered biological film fixes the recycling situation of double-enzyme catalysis system
Surface fixing Beta-diastatic escherichia coli re-suspension liquid is mixed according to volume ratio 1: 4 with the soluble starch solution that mass concentration is 20%-25%.Adding soluble starch solution and 500mL surface fixing Beta-diastatic Bacillus coli cells suspension stirring at low speed reaction that 2L mass concentration is 20%-25% in i.e. 5L reactor, catalytic reaction 24 hours, reaction condition is 40 DEG C, pH 7.0, rotating speed 100rpm；Use 14 mesh sieve hole Filter Press separating reaction liquid, filtrate is trehalose mixed liquor, HPLC is used to detect its content of trehalose, and calculate the conversion ratio of trehalose, filtering residue is the escherichia coli that double enzyme is fixing, with the resuspended rear recycling of PBS, it is 50% that biomembrane fixes conversion ratio after double-enzyme catalysis system reuses 10 times, and after reusing 16 times, conversion ratio is 45%.Result above can be seen that, utilize nanofiber biofilm surface displaying beta amylase is that full cell double-enzyme catalysis system converting soluble starch solution can recycle containing trehalose synthase escherichia coli, have enzyme loss alive every time after converting, but after reusing 10-16 time, conversion ratio is more than 45%.
Claims (10)
1. a nanofiber biomembrane immobilized bi-enzyme system, it is characterised in that this pair of enzyme system can generate trehalose by catalytic starch in same system, and wherein, this pair of enzyme is respectively beta amylase and trehalose synthetase.
2., according to the system described in right 1, it is characterised in that this system at least includes, express the escherichia coli A of trehalose synthetase gene Tre and CsgA gene simultaneously；Express the escherichia coli B of beta amylase gene；Polypeptide tags SpyTag is connected to the fusion protein CsgA-SpyTag that the carbon teminal of CsgA gene is formed；And the fusion protein BA-SpyCatcher that the carbon teminal that Polypeptide tags SpyCatcher is connected to beta amylase gene is formed；And the nanofiber biomembrane covalency being self-assembly of by CsgA-SpyTag high polymeric at escherichia coli A cell surface fixes the immobilization catalytic membrane that BA-SpyCatcher is formed.
3. build the method for system described in claim 1, it is characterised in that
The step that the method is concrete is:
(1) build containing trehalose synthetase cell surface formation nanofiber biomembranous escherichia coli A: after the TreS genetic fragment expanded by carrier PETDuet with PCR double digestion simultaneously, be connected acquisition recombiant plasmid PETDuet-TreS, convert after again it being connected with double digestion while of CsgA-SpyTag fusion gene fragment to e. coli bl21 (DE3) competent cell, obtain, through ammonia benzyl antibiotic-screening, bacterium colony PCR checking and gene sequencing, the escherichia coli A that gene order is correct；
(2) the escherichia coli B containing beta amylase is built: design primer carries out PCR amplification and obtains amylase gene fragment BA and Polypeptide tags SpyCatcher genetic fragment, carry out splicing construction recombination plasmid PET28a-BA-SC by overlapping PCR method to convert to e. coli bl21 (DE3) competent cell, obtain, through kalamycin resistance screening, bacterium colony PCR checking and gene sequencing, the escherichia coli B that gene order is correct
(3) self-induction cultivates escherichia coli A, it is thus achieved that containing the culture fluid of escherichia coli A；
(4) self-induction cultivates escherichia coli B；
(5) by broken for escherichia coli B and isolated and purified acquisition crude enzyme liquid BA-SpyCatcher；
(6) by crude enzyme liquid and culture fluid haptoreaction 48h described in step (3), it is thus achieved that nanofiber biomembrane immobilized bi-enzyme system；
(7) the nanofiber biomembrane immobilized bi-enzyme system also described in cleaning step (6) it is centrifuged, and resuspended to original volume size with phosphate buffer.
Method the most according to claim 3, it is characterised in that in described step (6), the amount of crude enzyme liquid is based on the fermentating liquid volume preparing this crude enzyme liquid, and the volume ratio of step (3) culture fluid used is 1:1 with this step.
Method the most according to claim 3, it is characterised in that also include the engineering bacteria described in step with centrifugal separation (3) and step (4), and by the resuspended step of phosphate buffer.
Method the most according to claim 3, it is characterised in that described in step (5), breaking method is high-pressure breaking, is separated into solid-liquid separation, sieve aperture is less than 14 mesh；Purification process is membrane filtration, and molecular cut off is 300-400.
7. one kind utilizes the method that nanofiber biomembrane immobilized bi-enzyme catalyzes and synthesizes trehalose, it is characterised in that utilize the system described in any of the above-described claim to contact with soluble starch in phosphoric acid buffer liquid system, and catalysis generates trehalose.
Method the most according to claim 7, it is characterised in that concretely comprising the following steps of the method:
(1) being contacted with nanofiber biomembrane immobilized bi-enzyme system by the soluble starch solution that mass concentration is 20%-25%, condition is 35-60 DEG C, pH 6.0-7.5, rotating speed 100-200rpm, catalytic reaction 20-30 hour；Obtain the solution containing trehalose；
(2) trehalose described in claim 1 is separated；
Wherein, the step reclaiming nanofiber biomembrane immobilized bi-enzyme system is also included.
The most in accordance with the method for claim 8, it is characterised in that the described volume of nanofiber biomembrane immobilized bienzyme system and the volume ratio of soluble starch solution are 1:4.
Phosphate buffer described in the most any of the above-described claim, it is characterised in that its phosphate concentration is 0.04 mol/L, pH is 7.0.
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| CN109148924A (en) * | 2018-08-19 | 2019-01-04 | 南京工业大学 | A kind of enzyme thermistor devices producing trehalose power generation using Escherichia coli in sewage |
| CN109148924B (en) * | 2018-08-19 | 2021-08-03 | 南京工业大学 | Enzyme biofuel cell for generating electricity by utilizing trehalose produced by escherichia coli in sewage |
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| CN114891776A (en) * | 2022-05-24 | 2022-08-12 | 天津大学 | Construction of double-enzyme layered cascade nanocrystal and method for degrading PET (polyethylene terephthalate) plastic by using double-enzyme layered cascade nanocrystal |
| CN114891776B (en) * | 2022-05-24 | 2024-03-12 | 天津大学 | Construction of double-enzyme layered cascade nanocrystals and method for degrading PET plastic by using same |
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