WO2023004770A1 - Pichia pastoris expression vector of fructosamine deglycase, genetically engineered bacterium and construction method, and protein expression method - Google Patents

Pichia pastoris expression vector of fructosamine deglycase, genetically engineered bacterium and construction method, and protein expression method Download PDF

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WO2023004770A1
WO2023004770A1 PCT/CN2021/109677 CN2021109677W WO2023004770A1 WO 2023004770 A1 WO2023004770 A1 WO 2023004770A1 CN 2021109677 W CN2021109677 W CN 2021109677W WO 2023004770 A1 WO2023004770 A1 WO 2023004770A1
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fructosamine
desugarase
pichia pastoris
expression vector
genetically engineered
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PCT/CN2021/109677
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French (fr)
Chinese (zh)
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王怀英
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武汉百氨汇基生物技术有限公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • 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)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/84—Pichia

Definitions

  • the invention relates to the technical field of protein expression, in particular to an expression vector of fructosamine desugarase Pichia pastoris, a genetic engineering bacterium, a construction method and a protein expression method.
  • Glucosamine Glucosamine, GlcN
  • 2-amino-2-deoxy-D-glucose also known as glucosamine, glucosamine or simply glucosamine, ammonia sugar
  • Glucosamine GlcN
  • 2-amino-2-deoxy-D-glucose also known as glucosamine, glucosamine or simply glucosamine, ammonia sugar
  • glucosamine has some special physiologically active functions, such as anti-inflammatory and analgesic, repairing cartilage damage, treating rheumatoid arthritis; strengthening white blood cell proliferation and differentiation, promoting cytokine secretion, improving immune regulation, anti-tumor; restoring mitochondrial enzymes Expression, improve mitochondrial glutathione antioxidant capacity, enhance immune function; promote endoplasmic reticulum-related protein degradation, strengthen protease activity, improve protein homeostasis, prolong cell life, etc.
  • fructosamine desugarase can use alanine or glutamic acid as an amino donor to convert fructose-6-phosphate into glucosamine, and simultaneously produce pyruvate or ⁇ -ketopentyl Diacid. Therefore, fructosamine desugarase plays a key role in the co-production technology of enzymatically preparing glucosamine and pyruvate or ⁇ -ketoglutarate.
  • the fructosamine desugarase is currently constructed in Escherichia coli, which is not conducive to the large-scale industrial production of the enzyme in the field of food and medicine.
  • the purpose of the present invention is to provide an expression vector of fructosamine desugarase Pichia pastoris, genetic engineering bacteria and its construction method and protein expression method.
  • the carrier of the invention is beneficial to the industrialized production of the enzyme, and the production method is green, safe, environment-friendly, pollution-free and controllable, is beneficial to the large-scale preparation of glucosamine, and can generate greater social value and economic benefits.
  • the present invention provides a Pichia pastoris expression vector for fructosamine desugarase, including a skeleton carrier and a gene encoding fructosamine desugarase; the nucleotide of the gene encoding fructosamine desugarase is as shown in SEQ ID NO.1 shown.
  • the backbone vector comprises pPIC9K.
  • the present invention also provides a method for constructing an expression vector of fructosamine desugarase Pichia pastoris described in the above technical scheme, comprising the following steps:
  • the gene encoding fructosamine desugarase is connected to the backbone vector by double enzyme digestion to obtain an expression vector.
  • each 40-60 ⁇ L double enzyme digestion system includes: 10 ⁇ quitcut buffer 4-6 ⁇ L, pPIC9K or nucleic acid containing a gene encoding fructosamine desugarase 28-32 ⁇ L, 4-6 U 0.8-1.5 ⁇ L each of SnaBI and NotI per ⁇ L and the rest of water.
  • the double enzyme digestion condition of the double enzyme digestion method is 36-38° C. for 1-3 hours.
  • the present invention also provides a genetically engineered bacterium containing the expression vector described in the above technical solution, and the host bacterium of the genetically engineered bacterium includes Pichia pastoris.
  • the Pichia pastoris comprises Pichia pastoris GS115.
  • the present invention also provides a method for constructing genetically engineered bacteria described in the above technical solution, comprising the following steps:
  • the fructosamine desugarase Pichia pastoris expression vector is linearized, and the competent cells of the Pichia pastoris are electrotransformed to obtain genetically engineered bacteria.
  • the present invention also provides a method for expressing fructosamine desugarase based on the genetically engineered bacteria described in the above technical scheme, comprising the following steps:
  • the methanol is 100% methanol or an aqueous methanol solution with a methanol content of more than 80% by volume.
  • the invention provides a Pichia pastoris expression vector of fructosamine desugarase.
  • the present invention provides the Pichia pastoris expression vector for the first time, and constructs it in the eukaryotic Pichia pastoris to realize the successful expression of the fructosamine desugarase.
  • the invention has the advantages of simple operation, low cost, short period and easy realization.
  • the expression vector of the invention can successfully express the fructosamine desugarase with good thermostability.
  • the source of raw materials used in the technical solution of the invention is abundant and easy to obtain, the cost is low, and there is no environmental pollution.
  • the equipment and reagents used are cheap, and are convenient for large-scale production.
  • the present invention uses Pichia pastoris GS115 to successfully express highly soluble fructosamine desugarase, and the expressed biological enzyme protein is purified.
  • the present invention has verified the biological activity of the obtained recombinant fructosamine desugarase, and found that through the above-mentioned fructosamine desugarase in sequence, starch, glucose, fructose and fructose-6-phosphate can be synthesized into glucosamine, and at the same time, alanine can be used Acid produces pyruvate, glutamic acid produces ketoglutaric acid, aspartic acid produces carbonyl succinate, and glutamine produces glutamic acid and ketoglutaric acid.
  • the data show that the above-mentioned recombinant biological enzyme has the important function of well synthesizing glucosamine, pyruvate, ketoglutarate and carbonylsuccinate, and has great application potential.
  • Fig. 1 is the plate screening result figure provided by the present invention
  • Fig. 2 is a graph of SDS-PAGE electrophoresis results of the fructosamine desugarase FrIB provided by the present invention.
  • the present invention provides a kind of fructosamine desugarase (Fructosamine deglycase, FrIB) Pichia pastoris expression vector, comprising a backbone vector and a gene encoding fructosamine deglyase; the nucleotide of the gene encoding fructosamine deglyase As shown in SEQ ID NO.1:
  • the method for obtaining the fructosamine desugarase is not particularly limited in the present invention, and artificial synthesis methods known to those skilled in the art can be used.
  • the present invention preferably builds the gene into plasmid pCold II, and transforms it into Escherichia coli JM109 for preservation.
  • the present invention has no special limitations on the methods of construction and transformation, and conventional methods can be used.
  • the backbone vector preferably includes pPIC9K.
  • the source of the skeleton carrier is not particularly limited, and conventional commercially available skeleton carriers known to those skilled in the art can be used.
  • the present invention also provides a method for constructing an expression vector of fructosamine desugarase Pichia pastoris described in the above technical scheme, comprising the following steps:
  • the gene encoding fructosamine desugarase is connected to the backbone vector by double enzyme digestion to obtain an expression vector.
  • each 40-60 ⁇ L double enzyme digestion system preferably includes: 10 ⁇ quitcut buffer 4-6 ⁇ L, pPIC9K or 28-32 ⁇ L of nucleic acid containing a gene encoding fructosamine desugarase, 4 0.8-1.5 ⁇ L each of ⁇ 6U/ ⁇ L of SnaBI and NotI and the rest in water.
  • the present invention has no special limitation on the sources of 10 ⁇ quitcut buffer, pPIC9K, SnaBI and NotI, and conventional commercially available products well known to those skilled in the art can be used.
  • the double enzyme digestion condition of the double enzyme digestion method is preferably 36-38° C. for 1-3 hours, more preferably 37° C. for 3 hours.
  • Escherichia coli JM109 containing the plasmid pCold II of the gene encoding fructosamine desugarase as a template for PCR amplification
  • the amplification primers preferably include forward primers: 5 '- TACGTA AATATATTGTAATATCAGATTACGT-3' (SEQ ID NO.3) and reverse primer: 5'- GCGGCCG CGTTATATAACATTATAGTCTAATGCA-3' (SEQ ID NO.4), where the underline is the restriction site, and the enzymes used are SnaBI and NotI .
  • the sources of the primers are not particularly limited, and conventional artificial synthesis methods well known to those skilled in the art can be used.
  • the method of linking the gene encoding fructosamine desugarase to the backbone carrier is not particularly limited, and conventional linking systems can be used for linking.
  • the present invention also provides a genetically engineered bacterium containing the expression vector described in the above technical solution, and the host bacterium of the genetically engineered bacterium includes Pichia pastoris.
  • the Pichia pastoris preferably includes Pichia pastoris GS115.
  • the source of Pichia pastoris is not particularly limited, and conventional commercially available Pichia pastoris GS115 well known to those skilled in the art can be used.
  • the present invention also provides a method for constructing genetically engineered bacteria described in the above technical solution, comprising the following steps:
  • the fructosamine desugarase Pichia pastoris expression vector is linearized, and the competent cells of the Pichia pastoris are electrotransformed to obtain genetically engineered bacteria.
  • the present invention it is preferred to transform the expression vector of fructosamine desugarase Pichia pastoris into Escherichia coli competent cells, culture and screen single clones, pick single clones to expand culture, extract plasmids, and obtain recombinant plasmid pPIC9K-FrIB; then The recombinant plasmid pPIC9K-FrIB was linearized with Sacl, and the competent cells of Pichia pastoris were electrotransformed to obtain genetically engineered bacteria.
  • the present invention has no special limitations on the methods of transformation, screening, expanded culture, plasmid extraction and linearization, and conventional methods can be used.
  • the present invention also provides a method for expressing fructosamine desugarase based on the genetically engineered bacteria described in the above technical scheme, comprising the following steps:
  • the genetically engineered bacteria are inoculated into the BMGY liquid medium, the first shaking culture is carried out until the OD 600 is between 2 and 6, the cultured bacteria are obtained by centrifuging and discarding the supernatant.
  • the centrifugation condition is preferably 2000-4000 r/min for 3-8 minutes.
  • the time of the first shaking culture is preferably 12-20 h, and the temperature is preferably 26-30°C.
  • the present invention uses BMGY liquid medium to resuspend the cultured thalli, and carries out the second shaking culture at 100-300r/min for 84-108h; during the second shaking culture, every 10-14h to culture Methanol is added to the base until the final volume percentage of methanol is 0.5% to 1.0%.
  • the temperature of the second shaking culture is preferably 26-30°C.
  • the methanol is preferably 100% methanol or an aqueous methanol solution with a final volume percentage of methanol of more than 80%.
  • the fructosamine desugarase of the present invention can be used to synthesize or convert glucosamine.
  • Escherichia coli can produce endotoxin etc. in the fermentation process, and the present invention adopts Pichia pastoris to be able to realize food grade production of glucosamine.
  • the synthetic or converted substrate preferably includes one or more of starch, glucose, fructose, and fructose-6-phosphate.
  • Amino donors are alanine, glutamic acid, aspartic acid or glutamine.
  • the synthesis or conversion method is to control the pH to 6-8 and the temperature to be 30-50° C. to carry out the synthesis or conversion in the presence of substrates and amino donors.
  • the pH is more preferably 6.7-7.5, and the temperature is more preferably 40°C-50°C.
  • the synthesis and/or conversion process of the present invention can also obtain ketoacid products at the same time, such as when alanine is used as a substrate, pyruvate can be obtained, and when glutamic acid is used as a substrate, ⁇ -ketoglutadiene can be obtained acid.
  • the synthesis or conversion reaction in the present invention is a reversible reaction, and the present invention can also use ketoacid and glucosamine as reaction raw materials to prepare amino acids and sugars.
  • fructosamine desugarase when ketoacid and glucosamine are used as reaction raw materials, fructosamine desugarase can catalyze the amino acid corresponding to ketoacid and fructose-6-phosphate. Utilizing fructose-6-phosphate, fructose, glucose and starch can be synthesized by conventional methods to obtain other sugars.
  • fructosamine desugarase Pichia pastoris expression vector A kind of fructosamine desugarase Pichia pastoris expression vector, genetic engineering bacteria and construction method and protein expression method of the present invention will be described in further detail below in conjunction with specific examples.
  • the technical scheme of the present invention includes but is not limited to the following implementation example.
  • Primer design design primers from the mature peptide sequence after the signal peptide (as shown in SEQ ID NO.3 and SEQ ID NO.4);
  • the linearized plasmid was subjected to electrophoresis and gel recovery, and after the corresponding fragments were recovered, the competent Pichia pastoris GS115 was electrotransformed. Take 2-3 ⁇ L linearized plasmid with a concentration of 5 ⁇ g/ ⁇ L and mix it with 80 ⁇ L competent cells evenly, transfer it to a 0.2 cm pre-cooled electroporation cuvette, place it on ice for 5 min, and perform electric shock with a voltage of 1500 V, a capacitance of 25 ⁇ F, and a resistance of 200 ⁇ . Immediately after the electric shock Add 1mL 1mol/L pitol to revive, and incubate at 30°C for 1h. The transformation liquid was centrifugally spread on the MD plate, cultured at 30°C for 3-4 days, and a single colony (transformant) visible to the naked eye grew.
  • the single colonies of the four transformed transformants were picked and inoculated into 50mL BMGY liquid medium respectively, cultured with shaking at 28°C for 16h, and the OD 600 was determined to be between 2 and 6. Centrifuge at 3000r/min for 5min, discard the supernatant. The bacteria were resuspended in 20 mL of BMGY liquid medium, and cultured with shaking at 28°C and 200 r/min for 96 hours. During this period, 100% methanol was added to the medium every 12 hours until the final volume percentage was 0.5%-1.0%.
  • sample buffer solution [0.1mol/L Tris-HCl, pH 6.8; 2% SDS (weight: volume), 10% glycerol ( volume: volume), 0.01% bromophenol blue (weight: volume)] placed in a water bath at 37°C for 5-10 min, and then separated by electrophoresis.
  • sample buffer solution [0.1mol/L Tris-HCl, pH 6.8; 2% SDS (weight: volume), 10% glycerol ( volume: volume), 0.01% bromophenol blue (weight: volume)] placed in a water bath at 37°C for 5-10 min, and then separated by electrophoresis.
  • sample buffer solution [0.1mol/L Tris-HCl, pH 6.8; 2% SDS (weight: volume), 10% glycerol ( volume: volume), 0.01% bromophenol blue (weight: volume)] placed in a water bath at 37°C for 5-10 min, and then separated by electrophoresis.
  • ⁇ -Mercaptoethanol Used to open disulfide bonds,
  • Gel preparation and electrophoresis add 0.2% (volume percentage) gelatin in the process of preparing the separating gel, mix well and then pour the gel, and it will be Gelatin-SDS-PAGE (substrate gel) after solidification.
  • the density of polyacrylamide in the "stacking gel” is 5%
  • the density of polyacrylamide in the "separating gel” is 12%
  • the thickness is 1 mm 3 .
  • E. Staining and decolorization stain with Coomassie Brilliant Blue for 30 minutes, and then change the decolorization solution (5% (volume percentage) acetic acid + 10% (volume percentage) methanol) every few hours until the background is clear. Note: The background color of the stained and decolorized gel is blue-black, and the color of the protease reaction site becomes lighter. The size of the area in the gel where the protease reaction is present and the light transmittance of that site are directly proportional to the protease activity.
  • lane 1 is the protein marker
  • lane 2 is the SDS-PAGE electrophoresis of fructosamine desugarase FrIB.
  • the protein has an obvious band around 39.7kD, which is consistent with the theoretical value, which proves that the protein can be successfully expressed and purified in yeast.
  • 1mM reaction system contains 15mM alanine, 20mM fructose-6-phosphate, 0.2mL fructosamine desugarase, 2.5mM EDTA and 100mM different pH buffers (Na 2 HPO 4 -NaH 2 PO 4 , pH2.0 ⁇ 10.0), put the above reaction system in a 1.5mL centrifuge tube and mix well, then put it in a PCR instrument for 20min at 37°C, heat at 95°C for 5min to terminate the reaction, centrifuge and take the supernatant through High-performance liquid chromatography (HPLC) was used to detect the pyruvate production, thereby calculating the enzyme activity of fructosamine desugarase.
  • HPLC High-performance liquid chromatography
  • Pyruvate was quantitatively analyzed by high performance liquid chromatography (HPLC).
  • HPLC high performance liquid chromatography
  • the chromatographic column used is a C18 column
  • the mobile phase is an aqueous solution of acetonitrile (containing 0.1% (volume percent) dicyclohexylamine, 0.1% (volume percent) formic acid) (volume ratio 5:95)
  • the flow rate is 0.6mL /min
  • the column temperature is 40°C
  • the detection wavelength is 230nm.
  • sodium pyruvate is used as a reference substance, and the content of pyruvate in the reaction solution is calculated by the peak area according to the external standard method.
  • the amount of enzyme required to catalyze the conversion of 1 ⁇ mol of alanine into pyruvate per minute is defined as an enzyme activity unit, ie 1U.

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Abstract

Provided are a Pichia pastoris expression vector of fructosamine deglycase, a genetically engineered bacterium and a construction method therefor, and a protein expression method. The Pichia pastoris expression vector of fructosamine deglycase comprises a skeleton vector and a gene encoding fructosamine deglycase, wherein the nucleotide sequence of the gene encoding fructosamine deglycase is as shown in SEQ ID NO: 1.

Description

一种果糖胺脱糖酶毕赤酵母表达载体、基因工程菌及构建方法和蛋白表达方法A kind of fructosamine desugarase Pichia pastoris expression vector, genetic engineering bacteria and its construction method and protein expression method 技术领域technical field
本发明涉及蛋白表达技术领域，具体涉及一种果糖胺脱糖酶毕赤酵母表达载体、基因工程菌及构建方法和蛋白表达方法。The invention relates to the technical field of protein expression, in particular to an expression vector of fructosamine desugarase Pichia pastoris, a genetic engineering bacterium, a construction method and a protein expression method.
背景技术Background technique
氨基葡萄糖(Glucosamine，GlcN)，即2-氨基-2-脱氧-D-葡萄糖，又称葡萄糖胺、氨基葡糖或简称为葡糖胺、氨糖，是葡萄糖的一个羟基被氨基取代后的化合物。研究表明，氨基葡萄糖具有一些特殊的生理活性功能，如消炎镇痛，修复软骨损伤，治疗风湿性关节炎；强化白细胞增殖和分化，促进细胞因子分泌，改善免疫调节、抗肿瘤；恢复线粒体内酶表达，提高线粒体谷胱甘肽抗氧化能力，增强免疫功能；促进内质网相关蛋白降解，强化蛋白酶活性，改善蛋白稳态水平，延长细胞寿命等。Glucosamine (Glucosamine, GlcN), namely 2-amino-2-deoxy-D-glucose, also known as glucosamine, glucosamine or simply glucosamine, ammonia sugar, is a compound in which a hydroxyl group of glucose is replaced by an amino group . Studies have shown that glucosamine has some special physiologically active functions, such as anti-inflammatory and analgesic, repairing cartilage damage, treating rheumatoid arthritis; strengthening white blood cell proliferation and differentiation, promoting cytokine secretion, improving immune regulation, anti-tumor; restoring mitochondrial enzymes Expression, improve mitochondrial glutathione antioxidant capacity, enhance immune function; promote endoplasmic reticulum-related protein degradation, strengthen protease activity, improve protein homeostasis, prolong cell life, etc.
果糖胺脱糖酶作为一种新发现的生物酶类，它可以利用丙氨酸或谷氨酸作为氨基供体，将果糖-6-磷酸转化为氨基葡萄糖，同时产生丙酮酸或α-酮戊二酸。因此，果糖胺脱糖酶在酶法制备氨基葡萄糖及丙酮酸或α-酮戊二酸的联产技术中发挥着关键的作用。As a newly discovered biological enzyme, fructosamine desugarase can use alanine or glutamic acid as an amino donor to convert fructose-6-phosphate into glucosamine, and simultaneously produce pyruvate or α-ketopentyl Diacid. Therefore, fructosamine desugarase plays a key role in the co-production technology of enzymatically preparing glucosamine and pyruvate or α-ketoglutarate.
果糖胺脱糖酶目前构建于大肠杆菌中，不利于该酶在食品药品领域的大规模工业生产。The fructosamine desugarase is currently constructed in Escherichia coli, which is not conducive to the large-scale industrial production of the enzyme in the field of food and medicine.
发明内容Contents of the invention
本发明的目的在于提供一种果糖胺脱糖酶毕赤酵母表达载体、基因工程菌及构建方法和蛋白表达方法。本发明所述载体有利于该酶的工业化生产，生产方法绿色，安全，环保，无污染，可控，对氨基葡萄糖的大规模制备有益，会产生较大的社会价值和经济效益。The purpose of the present invention is to provide an expression vector of fructosamine desugarase Pichia pastoris, genetic engineering bacteria and its construction method and protein expression method. The carrier of the invention is beneficial to the industrialized production of the enzyme, and the production method is green, safe, environment-friendly, pollution-free and controllable, is beneficial to the large-scale preparation of glucosamine, and can generate greater social value and economic benefits.
本发明提供了一种果糖胺脱糖酶毕赤酵母表达载体，包括骨架载体和编码果糖胺脱糖酶的基因；所述编码果糖胺脱糖酶的基因的核苷酸如SEQ ID NO.1所示。The present invention provides a Pichia pastoris expression vector for fructosamine desugarase, including a skeleton carrier and a gene encoding fructosamine desugarase; the nucleotide of the gene encoding fructosamine desugarase is as shown in SEQ ID NO.1 shown.
优选的是，所述骨架载体包括pPIC9K。Preferably, the backbone vector comprises pPIC9K.
本发明还提供了上述技术方案所述果糖胺脱糖酶毕赤酵母表达载体的构建方法，包括以下步骤：The present invention also provides a method for constructing an expression vector of fructosamine desugarase Pichia pastoris described in the above technical scheme, comprising the following steps:
使用双酶切法将编码果糖胺脱糖酶的基因与骨架载体相连接，得到表达载体。The gene encoding fructosamine desugarase is connected to the backbone vector by double enzyme digestion to obtain an expression vector.
优选的是，所述双酶切法，每40～60μL的双酶切体系包括：10×quitcut buffer 4～6μL、pPIC9K或含编码果糖胺脱糖酶的基因的核酸28～32μL，4～6U/μL的SnaBI和NotI各0.8～1.5μL和余量的水。Preferably, in the double enzyme digestion method, each 40-60 μL double enzyme digestion system includes: 10×quitcut buffer 4-6 μL, pPIC9K or nucleic acid containing a gene encoding fructosamine desugarase 28-32 μL, 4-6 U 0.8-1.5 μL each of SnaBI and NotI per μL and the rest of water.
优选的是，所述双酶切法的双酶切条件为36～38℃，1～3h。Preferably, the double enzyme digestion condition of the double enzyme digestion method is 36-38° C. for 1-3 hours.
本发明还提供了一种含上述技术方案所述表达载体的基因工程菌，所述基因工程菌的宿主菌包括毕赤酵母。The present invention also provides a genetically engineered bacterium containing the expression vector described in the above technical solution, and the host bacterium of the genetically engineered bacterium includes Pichia pastoris.
优选的是，所述毕赤酵母包括毕赤酵母GS115。Preferably, the Pichia pastoris comprises Pichia pastoris GS115.
本发明还提供了上述技术方案所述基因工程菌的构建方法，包括以下步骤：The present invention also provides a method for constructing genetically engineered bacteria described in the above technical solution, comprising the following steps:
将果糖胺脱糖酶毕赤酵母表达载体线性化，电转化毕赤酵母的感受态细胞，得到基因工程菌。The fructosamine desugarase Pichia pastoris expression vector is linearized, and the competent cells of the Pichia pastoris are electrotransformed to obtain genetically engineered bacteria.
本发明还提供了基于上述技术方案所述基因工程菌的果糖胺脱糖酶表达方法，包括以下步骤：The present invention also provides a method for expressing fructosamine desugarase based on the genetically engineered bacteria described in the above technical scheme, comprising the following steps:
将所述基因工程菌接种到BMGY液体培养基中，进行第一振荡培养至OD 600在2～6之间，离心，弃上清，得到培养菌体； Inoculate the genetically engineered bacteria into the BMGY liquid medium, perform the first shaking culture until the OD600 is between 2 and 6, centrifuge, discard the supernatant, and obtain the cultured bacteria;
用BMGY液体培养基重悬所述培养菌体，在100～300r/min进行第二振荡培养84～108h；所述第二振荡培养的期间，每10～14h向培养基中加甲醇至甲醇的终体积百分含量为0.5％～1.0％。Resuspend the cultured bacteria with BMGY liquid medium, and carry out the second shaking culture at 100-300r/min for 84-108h; during the second shaking culture, add methanol to the medium every 10-14h The final volume percentage is 0.5%-1.0%.
优选的是，所述甲醇为100％的甲醇或甲醇的体积百分含量为80％以上的甲醇水溶液。Preferably, the methanol is 100% methanol or an aqueous methanol solution with a methanol content of more than 80% by volume.
本发明提供了一种果糖胺脱糖酶毕赤酵母表达载体。本发明首次提供果糖胺脱糖酶毕赤酵母表达载体，并将其构建于真核生物毕赤酵母菌中，实现果糖胺脱糖酶的成功表达。本发明具有操作简单，成本低，周期短和 易于实现的优点。本发明所述表达载体能够成功表达具有良好的热稳定性的果糖胺脱糖酶。本发明技术方案使用的原料来源丰富易取，成本低，无环境污染，所用设备、试剂价格便宜，便于规模化生产。The invention provides a Pichia pastoris expression vector of fructosamine desugarase. The present invention provides the Pichia pastoris expression vector for the first time, and constructs it in the eukaryotic Pichia pastoris to realize the successful expression of the fructosamine desugarase. The invention has the advantages of simple operation, low cost, short period and easy realization. The expression vector of the invention can successfully express the fructosamine desugarase with good thermostability. The source of raw materials used in the technical solution of the invention is abundant and easy to obtain, the cost is low, and there is no environmental pollution. The equipment and reagents used are cheap, and are convenient for large-scale production.
进一步的，本发明利用毕赤酵母GS115成功表达了高可溶性的果糖胺脱糖酶，并对表达的生物酶蛋白进行了纯化。Furthermore, the present invention uses Pichia pastoris GS115 to successfully express highly soluble fructosamine desugarase, and the expressed biological enzyme protein is purified.
本发明对所获得的重组果糖胺脱糖酶进行了生物活性验证，发现依次通过上述果糖胺脱糖酶，可以将淀粉，葡萄糖，果糖和果糖-6-磷酸合成氨基葡萄糖，并且同时利用丙氨酸生成丙酮酸，谷氨酸生成酮戊二酸，天门冬氨酸生成羰基丁二酸，谷氨酰胺生产谷氨酸、酮戊二酸。通过数据表明上述重组生物酶具有很好地合成氨基葡萄糖和丙酮酸，酮戊二酸，羰基丁二酸的重要功能，具有较大应用潜力。The present invention has verified the biological activity of the obtained recombinant fructosamine desugarase, and found that through the above-mentioned fructosamine desugarase in sequence, starch, glucose, fructose and fructose-6-phosphate can be synthesized into glucosamine, and at the same time, alanine can be used Acid produces pyruvate, glutamic acid produces ketoglutaric acid, aspartic acid produces carbonyl succinate, and glutamine produces glutamic acid and ketoglutaric acid. The data show that the above-mentioned recombinant biological enzyme has the important function of well synthesizing glucosamine, pyruvate, ketoglutarate and carbonylsuccinate, and has great application potential.
说明书附图Instructions attached
图1为本发明提供的平板筛选结果图；Fig. 1 is the plate screening result figure provided by the present invention;
图2为本发明提供的果糖胺脱糖酶FrIB的SDS-PAGE电泳结果图。Fig. 2 is a graph of SDS-PAGE electrophoresis results of the fructosamine desugarase FrIB provided by the present invention.
具体实施方式Detailed ways
本发明提供了一种果糖胺脱糖酶(Fructosamine deglycase，FrIB)毕赤酵母表达载体，包括骨架载体和编码果糖胺脱糖酶的基因；所述编码果糖胺脱糖酶的基因的核苷酸如SEQ ID NO.1所示：The present invention provides a kind of fructosamine desugarase (Fructosamine deglycase, FrIB) Pichia pastoris expression vector, comprising a backbone vector and a gene encoding fructosamine deglyase; the nucleotide of the gene encoding fructosamine deglyase As shown in SEQ ID NO.1:
Figure PCTCN2021109677-appb-000001
Figure PCTCN2021109677-appb-000001
Figure PCTCN2021109677-appb-000002
Figure PCTCN2021109677-appb-000002
在本发明中，所述果糖胺脱糖酶的氨基酸序列如SEQ ID NO.2所示：MSQATAKVNREVQAFLQDLKGKTIDHVFFVACGGSSAIMYPSKYVFDRESKSINSDLYSANEFIQRNPVQLGEKSLVILCSHSGNTPETVKAAAFARGKGALTIAMTFKPESPLAQEAQYVAQYDWGDEALAINTNYGVLYQIVFGTLQVLENNTKFQQAIEGLDQLQAVYEKALKQEADNAKQFAKAHEKESIIYTMASGANYGVAYSYSICILMEMQWIHSHAIHAGEYFHGPFEIIDESVPFIILLGLDETRPLEERALTFSKKYGKKLTVLDAASYDFTAIDDSVKGYLAPLVLNRVLRSYADELAEERNHPLSHRRYMWKVEY。本发明所述果糖胺脱糖酶的原始序列来自NCBI(National Center for Biotechnology Information)数据库，所表达的氨基酸序列编号为GenBank:AOR99552.1，来源为枯草芽孢杆菌。本发明对果糖胺脱糖酶的获得方法没有特殊限定，采用本领域技术人员公知的人工合成方法即可。获得果糖胺脱糖酶的编码基因后，本发明优选将基因建于质粒pCold Ⅱ中，转化入大肠杆菌JM109中进行保存。本发明对所述构建和转化的方法没有特殊限定，采用常规方 法即可。在本发明中，所述骨架载体优选包括pPIC9K。本发明对所述骨架载体的来源没有特殊限定，采用本领域技术人员公知的常规市售骨架载体即可。在本发明中，所述果糖胺脱糖酶的氨基酸序列如SEQ ID NO.2所示：MSQATAKVNREVQAFLQDLKGKTIDHVFFVACGGSSAIMYPSKYVFDRESKSINSDLYSANEFIQRNPVQLGEKSLVILCSHSGNTPETVKAAAFARGKGALTIAMTFKPESPLAQEAQYVAQYDWGDEALAINTNYGVLYQIVFGTLQVLENNTKFQQAIEGLDQLQAVYEKALKQEADNAKQFAKAHEKESIIYTMASGANYGVAYSYSICILMEMQWIHSHAIHAGEYFHGPFEIIDESVPFIILLGLDETRPLEERALTFSKKYGKKLTVLDAASYDFTAIDDSVKGYLAPLVLNRVLRSYADELAEERNHPLSHRRYMWKVEY。 The original sequence of the fructosamine desugarase of the present invention comes from the NCBI (National Center for Biotechnology Information) database, the expressed amino acid sequence number is GenBank: AOR99552.1, and the source is Bacillus subtilis. The method for obtaining the fructosamine desugarase is not particularly limited in the present invention, and artificial synthesis methods known to those skilled in the art can be used. After obtaining the coding gene of fructosamine desugarase, the present invention preferably builds the gene into plasmid pCold II, and transforms it into Escherichia coli JM109 for preservation. The present invention has no special limitations on the methods of construction and transformation, and conventional methods can be used. In the present invention, the backbone vector preferably includes pPIC9K. In the present invention, the source of the skeleton carrier is not particularly limited, and conventional commercially available skeleton carriers known to those skilled in the art can be used.
本发明还提供了上述技术方案所述果糖胺脱糖酶毕赤酵母表达载体的构建方法，包括以下步骤：The present invention also provides a method for constructing an expression vector of fructosamine desugarase Pichia pastoris described in the above technical scheme, comprising the following steps:
使用双酶切法将编码果糖胺脱糖酶的基因与骨架载体相连接，得到表达载体。The gene encoding fructosamine desugarase is connected to the backbone vector by double enzyme digestion to obtain an expression vector.
在本发明中，所述双酶切法，每40～60μL的双酶切体系优选包括：10×quitcut buffer 4～6μL、pPIC9K或含编码果糖胺脱糖酶的基因的核酸28～32μL，4～6U/μL的SnaBI和NotI各0.8～1.5μL和余量的水。本发明对10×quitcut buffer、pPIC9K、SnaBI和NotI的来源没有特殊限定，采用本领域技术人员熟知的常规市售产品即可。In the present invention, in the double enzyme digestion method, each 40-60 μL double enzyme digestion system preferably includes: 10×quitcut buffer 4-6 μL, pPIC9K or 28-32 μL of nucleic acid containing a gene encoding fructosamine desugarase, 4 0.8-1.5 μL each of ~6U/μL of SnaBI and NotI and the rest in water. The present invention has no special limitation on the sources of 10×quitcut buffer, pPIC9K, SnaBI and NotI, and conventional commercially available products well known to those skilled in the art can be used.
在本发明中，所述双酶切法的双酶切条件优选为36～38℃，1～3h，更优选为37℃，3h。In the present invention, the double enzyme digestion condition of the double enzyme digestion method is preferably 36-38° C. for 1-3 hours, more preferably 37° C. for 3 hours.
在本发明中，所述双酶切前，优选以含编码果糖胺脱糖酶的基因的质粒pCold Ⅱ的大肠杆菌JM109为模板，进行PCR扩增，扩增用引物优选包括正向引物：5’- TACGTAAATATATTGTAATATCAGATTACGT-3’(SEQ ID NO.3)和反向引物：5’- GCGGCCGCGTTATATAACATTATAGTCTAATGCA-3’(SEQ ID NO.4)，其中，下划线为酶切位点，所用酶为SnaBⅠ和NotⅠ。本发明对所述引物的来源没有特殊限定，采用本领域技术人员熟知的常规人工合成方法即可。 In the present invention, before the double enzyme digestion, it is preferred to use Escherichia coli JM109 containing the plasmid pCold II of the gene encoding fructosamine desugarase as a template for PCR amplification, and the amplification primers preferably include forward primers: 5 '- TACGTA AATATATTGTAATATCAGATTACGT-3' (SEQ ID NO.3) and reverse primer: 5'- GCGGCCG CGTTATATAACATTATAGTCTAATGCA-3' (SEQ ID NO.4), where the underline is the restriction site, and the enzymes used are SnaBI and NotⅠ . In the present invention, the sources of the primers are not particularly limited, and conventional artificial synthesis methods well known to those skilled in the art can be used.
本发明对编码果糖胺脱糖酶的基因与骨架载体相连接的方法没有特殊限定，采用常规连接体系进行连接即可。In the present invention, the method of linking the gene encoding fructosamine desugarase to the backbone carrier is not particularly limited, and conventional linking systems can be used for linking.
本发明还提供了一种含上述技术方案所述表达载体的基因工程菌，所述基因工程菌的宿主菌包括毕赤酵母。在本发明中，所述毕赤酵母优选包括毕赤酵母GS115。本发明对毕赤酵母的来源没有特殊限定，采用本领域技术人员熟知的常规市售毕赤酵母GS115即可。The present invention also provides a genetically engineered bacterium containing the expression vector described in the above technical solution, and the host bacterium of the genetically engineered bacterium includes Pichia pastoris. In the present invention, the Pichia pastoris preferably includes Pichia pastoris GS115. In the present invention, the source of Pichia pastoris is not particularly limited, and conventional commercially available Pichia pastoris GS115 well known to those skilled in the art can be used.
本发明还提供了上述技术方案所述基因工程菌的构建方法，包括以下步骤：The present invention also provides a method for constructing genetically engineered bacteria described in the above technical solution, comprising the following steps:
将果糖胺脱糖酶毕赤酵母表达载体线性化，电转化毕赤酵母的感受态细胞，得到基因工程菌。The fructosamine desugarase Pichia pastoris expression vector is linearized, and the competent cells of the Pichia pastoris are electrotransformed to obtain genetically engineered bacteria.
具体的，本发明优选先将所述果糖胺脱糖酶毕赤酵母表达载体转化大肠杆菌感受态细胞，培养筛选单克隆，挑取单克隆扩大培养，提取质粒，得到重组质粒pPIC9K-FrIB；然后用Sacl线性化重组质粒pPIC9K-FrIB，电转化毕赤酵母的感受态细胞，得到基因工程菌。本发明对所述转化、筛选、扩大培养、提取质粒和线性化的方法没有特殊限定，采用常规方法即可。Specifically, in the present invention, it is preferred to transform the expression vector of fructosamine desugarase Pichia pastoris into Escherichia coli competent cells, culture and screen single clones, pick single clones to expand culture, extract plasmids, and obtain recombinant plasmid pPIC9K-FrIB; then The recombinant plasmid pPIC9K-FrIB was linearized with Sacl, and the competent cells of Pichia pastoris were electrotransformed to obtain genetically engineered bacteria. The present invention has no special limitations on the methods of transformation, screening, expanded culture, plasmid extraction and linearization, and conventional methods can be used.
本发明还提供了基于上述技术方案所述基因工程菌的果糖胺脱糖酶表达方法，包括以下步骤：The present invention also provides a method for expressing fructosamine desugarase based on the genetically engineered bacteria described in the above technical scheme, comprising the following steps:
将所述基因工程菌接种到BMGY液体培养基中，进行第一振荡培养至OD 600在2～6之间，离心，弃上清，得到培养菌体； Inoculate the genetically engineered bacteria into the BMGY liquid medium, perform the first shaking culture until the OD600 is between 2 and 6, centrifuge, discard the supernatant, and obtain the cultured bacteria;
用BMGY液体培养基重悬所述培养菌体，在100～300r/min进行第二振荡培养84～108h；所述第二振荡培养的期间，每10～14h向培养基中加甲醇至甲醇的终体积百分含量为0.5％～1.0％。Resuspend the cultured bacteria with BMGY liquid medium, and carry out the second shaking culture at 100-300r/min for 84-108h; during the second shaking culture, add methanol to the medium every 10-14h The final volume percentage is 0.5%-1.0%.
本发明将所述基因工程菌接种到BMGY液体培养基中，进行第一振荡培养至OD 600在2～6之间，离心，弃上清，得到培养菌体。在本发明中，所述离心的条件优选为2000～4000r/min离心3～8min。在本发明中，所述第一振荡培养的时间优选为12～20h，温度优选为26～30℃。 In the present invention, the genetically engineered bacteria are inoculated into the BMGY liquid medium, the first shaking culture is carried out until the OD 600 is between 2 and 6, the cultured bacteria are obtained by centrifuging and discarding the supernatant. In the present invention, the centrifugation condition is preferably 2000-4000 r/min for 3-8 minutes. In the present invention, the time of the first shaking culture is preferably 12-20 h, and the temperature is preferably 26-30°C.
弃上清后，本发明用BMGY液体培养基重悬所述培养菌体，在100～300r/min进行第二振荡培养84～108h；所述第二振荡培养的期间，每10～14h向培养基中加甲醇至甲醇的终体积百分含量为0.5％～1.0％。在本发明中，所述第二振荡培养的温度优选为26～30℃。在本发明中，所述甲醇优选为100％的甲醇或甲醇的终体积百分含量为80％以上的甲醇水溶液。After discarding the supernatant, the present invention uses BMGY liquid medium to resuspend the cultured thalli, and carries out the second shaking culture at 100-300r/min for 84-108h; during the second shaking culture, every 10-14h to culture Methanol is added to the base until the final volume percentage of methanol is 0.5% to 1.0%. In the present invention, the temperature of the second shaking culture is preferably 26-30°C. In the present invention, the methanol is preferably 100% methanol or an aqueous methanol solution with a final volume percentage of methanol of more than 80%.
本发明所述果糖胺脱糖酶能够用于合成或转化氨基葡萄糖。大肠杆菌在发酵过程会产生内毒素等，本发明采用毕赤酵母能够实现氨基葡萄糖食 品级生产。在本发明中，所述合成或转化的底物优选包括淀粉，葡萄糖，果糖，果糖-6-磷酸中的一种或两种以上。氨基供体为丙氨酸，谷氨酸，天门冬氨酸或谷氨酰胺。在本发明中，所述合成或转化的方法为在底物和氨基供体存在的环境下，控制pH为6～8，温度为30～50℃进行合成或转化。在本发明中，所述pH更优选为6.7～7.5，温度更优选为40℃～50℃。本发明所述合成和/或转化过程，还能同时得到酮酸产物，如以丙氨酸为底物时，可以获得丙酮酸，以谷氨酸为底物时，能够获得α-酮戊二酸。本发明所述合成或转化的反应为可逆反应，本发明也可以利用酮酸和氨基葡萄糖作为反应原料，制备氨基酸和糖类。在本发明中，利用酮酸和氨基葡萄糖为反应原料时，果糖胺脱糖酶能够催化得到酮酸对应的氨基酸，以及果糖-6-磷酸。利用果糖-6-磷酸可以采用常规方法合成果糖、葡萄糖和淀粉，实现其它糖类的获得。The fructosamine desugarase of the present invention can be used to synthesize or convert glucosamine. Escherichia coli can produce endotoxin etc. in the fermentation process, and the present invention adopts Pichia pastoris to be able to realize food grade production of glucosamine. In the present invention, the synthetic or converted substrate preferably includes one or more of starch, glucose, fructose, and fructose-6-phosphate. Amino donors are alanine, glutamic acid, aspartic acid or glutamine. In the present invention, the synthesis or conversion method is to control the pH to 6-8 and the temperature to be 30-50° C. to carry out the synthesis or conversion in the presence of substrates and amino donors. In the present invention, the pH is more preferably 6.7-7.5, and the temperature is more preferably 40°C-50°C. The synthesis and/or conversion process of the present invention can also obtain ketoacid products at the same time, such as when alanine is used as a substrate, pyruvate can be obtained, and when glutamic acid is used as a substrate, α-ketoglutadiene can be obtained acid. The synthesis or conversion reaction in the present invention is a reversible reaction, and the present invention can also use ketoacid and glucosamine as reaction raw materials to prepare amino acids and sugars. In the present invention, when ketoacid and glucosamine are used as reaction raw materials, fructosamine desugarase can catalyze the amino acid corresponding to ketoacid and fructose-6-phosphate. Utilizing fructose-6-phosphate, fructose, glucose and starch can be synthesized by conventional methods to obtain other sugars.
下面结合具体实施例对本发明所述的一种果糖胺脱糖酶毕赤酵母表达载体、基因工程菌及构建方法和蛋白表达方法做进一步详细的介绍，本发明的技术方案包括但不限于以下实施例。A kind of fructosamine desugarase Pichia pastoris expression vector, genetic engineering bacteria and construction method and protein expression method of the present invention will be described in further detail below in conjunction with specific examples. The technical scheme of the present invention includes but is not limited to the following implementation example.
实施例1Example 1
果糖胺脱糖酶基因的酵母表达载体的构建Construction of yeast expression vector of fructosamine desugarase gene
(1)引物设计：从信号肽之后的成熟肽序列开始起设计引物(如SEQ ID NO.3和SEQ ID NO.4所示)；(1) Primer design: design primers from the mature peptide sequence after the signal peptide (as shown in SEQ ID NO.3 and SEQ ID NO.4);
(2)PCR反应，以克隆载体pCold Ⅱ-FrIB为模板(本发明对pCold Ⅱ-FrIB的构建方法没有限定，采用常规构建方法将FrIB构建于pCold Ⅱ-FrIB中即可)，62℃退火，35cycles。(2) PCR reaction, using the cloning vector pCold Ⅱ-FrIB as a template (the present invention has no limitation on the construction method of pCold Ⅱ-FrIB, just use conventional construction methods to construct FrIB in pCold Ⅱ-FrIB), anneal at 62°C, 35cycles.
(3)SnaBI或NotI双酶切的相应生物酶基因的PCR产物和质粒pPIC9K。(3) The PCR product of the corresponding biological enzyme gene digested by SnaBI or NotI and the plasmid pPIC9K.
表1 双酶切体系Table 1 Double enzyme digestion system
成分Element 使用量Usage amount
纯化PCR产物/质粒Purification of PCR products/plasmids 30μL30μL
10*quitcut buffer10*quitcut buffer 5μL5μL
QuitCut SnaBIQuitCut SnaBI 1μL1μL
QuitCut NotIQuit Cut NotI 1μL1μL
ddH 2O ddH 2 O 13μL13μL
总体积total capacity 50μL50μL
37℃酶切2h。Enzyme digestion at 37°C for 2h.
(4)如常规方法连接，转化，并进行双酶切鉴定。(4) Ligate, transform, and carry out double-enzyme digestion identification according to conventional methods.
对这个克隆抽提质粒，从AOX3和AOX5两端进行全长测序，进一步验证插入的目的基因的正确性。Extract the plasmid from this clone, and perform full-length sequencing from both ends of AOX3 and AOX5 to further verify the correctness of the inserted target gene.
实施例2Example 2
含有果糖胺脱糖酶基因的重组酵母菌的构建和诱导表达Construction and Induced Expression of Recombinant Yeast Containing Fructosamine Desugarase Gene
1、毕赤酵母电转化感受态的制备1. Preparation of Pichia pastoris electroporation competent
向25mLYPD培养基中接种毕赤酵母GS115单菌落，30℃，250r/min过夜培养至OD 600约为2～6。转接1mL培养物至100mLYPD培养基中，继续培养至OD 600约为1.4。6,000r/min，离心10min后，弃掉上清液。将细胞轻轻重悬于50mL SolutionI溶液中，室温静止30min后，6,000r/min,4℃离心10min。弃掉上清后，将细胞轻轻重悬于10mL 1mo1/L山梨醇溶液中，将细胞洗涤4次后，再次将细胞轻轻重悬于0.5mL 1mo1/L山梨醇溶液中，每管80μL分装，-80℃冰箱冻存备用。 Inoculate a single colony of Pichia pastoris GS115 into 25mL of YPD medium, and culture overnight at 30°C and 250r/min until the OD600 is about 2-6. Transfer 1 mL of the culture to 100 mL of YPD medium, and continue to culture until the OD 600 is about 1.4. After centrifugation at 6,000 r/min for 10 min, discard the supernatant. The cells were gently resuspended in 50mL Solution I solution, and after standing at room temperature for 30min, centrifuged at 6,000r/min at 4°C for 10min. After discarding the supernatant, gently resuspend the cells in 10mL 1mol/L sorbitol solution, wash the cells 4 times, and gently resuspend the cells in 0.5mL 1mol/L sorbitol solution again, aliquot 80μL per tube , and stored in a -80°C refrigerator for later use.
2、重组质粒pPIC9K-FrIB线性化2. Linearization of recombinant plasmid pPIC9K-FrIB
用SacⅠ线性化重组质粒pPIC9K-FrIB，体系50μL：10×Cut smart Buffer 5μL，重组质粒pPIC9K-FrIB 30μg，5U/μL的Q.cut SacⅠ2μL，补水到50μL。37℃金属浴消化5min，65℃灭活10min。加入体积量10％的3M pH 5.2乙酸钠水溶液，1倍体积量的100％乙醇沉淀，-20℃静置30min，离心弃上清，再用75％乙醇洗1次，离心弃上清，干燥，加灭菌水溶解即可。Linearize recombinant plasmid pPIC9K-FrIB with SacⅠ, system 50 μL: 10×Cut smart Buffer 5 μL, recombinant plasmid pPIC9K-FrIB 30 μg, 5 U/μL Q.cut SacⅠ 2 μL, replenish water to 50 μL. Digest in a metal bath at 37°C for 5 minutes, and inactivate at 65°C for 10 minutes. Add 10% volume of 3M pH 5.2 sodium acetate aqueous solution, 1 volume volume of 100% ethanol for precipitation, stand at -20°C for 30 minutes, centrifuge to discard the supernatant, then wash once with 75% ethanol, centrifuge to discard the supernatant, and dry , add sterile water to dissolve.
3、电转化毕赤酵母3. Electrotransformation of Pichia pastoris
将线性化质粒进行电泳以及胶回收，回收得到相应的片段后，电转化毕赤酵母GS115感受态。取2～3μL浓度为5μg/μL的线性化质粒与80μL感受态细胞混合均匀，转移至0.2cm预冷的电转杯，冰上放置5min，电压1500V，电容25μF，电阻200Ω进行电击，电击后立刻加入1mL 1mo1/L梨醇复苏，30℃培养1h。将转化液离心涂布于MD平板，30℃培养3～4d，长出肉眼可见的单菌落(转化子)。The linearized plasmid was subjected to electrophoresis and gel recovery, and after the corresponding fragments were recovered, the competent Pichia pastoris GS115 was electrotransformed. Take 2-3 μL linearized plasmid with a concentration of 5 μg/μL and mix it with 80 μL competent cells evenly, transfer it to a 0.2 cm pre-cooled electroporation cuvette, place it on ice for 5 min, and perform electric shock with a voltage of 1500 V, a capacitance of 25 μF, and a resistance of 200 Ω. Immediately after the electric shock Add 1mL 1mol/L pitol to revive, and incubate at 30°C for 1h. The transformation liquid was centrifugally spread on the MD plate, cultured at 30°C for 3-4 days, and a single colony (transformant) visible to the naked eye grew.
4、重组质粒在酵母中的诱导表达4. Induced expression of recombinant plasmids in yeast
选择长出可分的单菌落的MD平板，并将MD平板上长出的单菌落编号，灭菌牙签挑取单菌落，复制到YPD平板上的划线格中，29℃培养1～2d。利用含遗传霉素G418的YPD平板筛选，获得抗2.0mg/ml遗传霉素G418的转化子，如图1所示，由图1可知，于平板中长出阳性克隆子，说明该重组质粒已经成功转入宿主酵母中。Select the MD plate with separable single colony, and number the single colony grown on the MD plate, pick a single colony with a sterilized toothpick, copy it to the streaked grid on the YPD plate, and incubate at 29°C for 1-2 days. Utilize the YPD plate screening that contains Geneticin G418, obtain the transformant of resistance 2.0mg/ml Geneticin G418, as shown in Figure 1, as can be seen from Figure 1, grow positive clone in plate, illustrate that this recombinant plasmid has successfully transferred into the host yeast.
挑取筛选出的四株转化子单菌落，分别接种到50mLBMGY液体培养基中，28℃振荡培养16h，测定其OD 600在2～6之间。3000r/min离心5min，弃上清。用20mLBMGY液体培养基重悬菌体，28℃、200r/min振荡培养96h。期间每12h向培养基中加100％甲醇至终体积百分含量为0.5％～1.0％。 The single colonies of the four transformed transformants were picked and inoculated into 50mL BMGY liquid medium respectively, cultured with shaking at 28°C for 16h, and the OD 600 was determined to be between 2 and 6. Centrifuge at 3000r/min for 5min, discard the supernatant. The bacteria were resuspended in 20 mL of BMGY liquid medium, and cultured with shaking at 28°C and 200 r/min for 96 hours. During this period, 100% methanol was added to the medium every 12 hours until the final volume percentage was 0.5%-1.0%.
实施例3Example 3
果糖胺脱糖酶蛋白的纯化Purification of fructosamine desugarase protein
重组酶的纯化及其SDS-PAGE凝胶电泳分析Purification of recombinant enzyme and its analysis by SDS-PAGE gel electrophoresis
蛋白质纯化参考GE Healthcare指南，SDS-PAGE分析按照《分子克隆实验指南》(第三版)，使用的凝胶浓度为12.5％，上样量5～25μL。蛋白质用考马斯亮蓝R-250染色。Refer to the GE Healthcare guidelines for protein purification, and follow the "Molecular Cloning Experiment Guide" (Third Edition) for SDS-PAGE analysis. The gel concentration used is 12.5%, and the loading volume is 5-25 μL. Proteins were stained with Coomassie brilliant blue R-250.
其中native-SDS-PAGE实验步骤：Among them, native-SDS-PAGE experimental steps:
A、在5～10μL酶液中加入5～10μL样品缓冲液[0.1mol/L三羟甲基氨基甲烷盐酸(Tris-HC1)，pH 6.8；2％SDS(重量︰体积)，10％甘油(体积︰体积)，0.01％溴酚蓝(重量︰体积)]在37℃水浴中放置5～10min，再进行上样电泳分离。注：在提取样品时，样品提取液中不加巯基乙醇是为了在电泳过程中使蛋白酶适度变性，以便在电泳结束后能恢复这些蛋白酶的活性。β-巯基乙醇：用于打开二硫键的，使蛋白质的四级或三级结构被破坏。是一种具有特殊臭味的无色透明液体，易燃、易溶于水和醇、醚等多种有机溶剂。A. Add 5-10 μL sample buffer solution [0.1mol/L Tris-HCl, pH 6.8; 2% SDS (weight: volume), 10% glycerol ( volume: volume), 0.01% bromophenol blue (weight: volume)] placed in a water bath at 37°C for 5-10 min, and then separated by electrophoresis. Note: When extracting samples, the purpose of not adding mercaptoethanol to the sample extract is to moderately denature proteases during electrophoresis, so that the activity of these proteases can be restored after electrophoresis. β-Mercaptoethanol: Used to open disulfide bonds, destroying the quaternary or tertiary structure of proteins. It is a colorless transparent liquid with a special odor, flammable and soluble in water, alcohol, ether and other organic solvents.
B、制胶与电泳：在制备分离胶的过程中加入0.2％(体积百分含量)的明胶，混合均匀后再进行灌胶，凝固后即为Gelatin-SDS-PAGE(底物胶)。“浓缩胶”的聚丙烯酰胺密度为5％，“分离胶”中聚丙烯酰胺密度为 12％，厚度为1mm 3。加样跑电泳。注：明胶因为是在制备凝胶时加入，所以已经交联在凝胶中，在电泳中不会在电场的作用下泳动。 B. Gel preparation and electrophoresis: add 0.2% (volume percentage) gelatin in the process of preparing the separating gel, mix well and then pour the gel, and it will be Gelatin-SDS-PAGE (substrate gel) after solidification. The density of polyacrylamide in the "stacking gel" is 5%, the density of polyacrylamide in the "separating gel" is 12%, and the thickness is 1 mm 3 . Add sample and run electrophoresis. Note: Because gelatin is added during the preparation of the gel, it has been cross-linked in the gel and will not swim under the action of an electric field during electrophoresis.
C、去SDS：电泳完成后，将分离胶在复性缓冲液[2％(体积百分含量)TritonX-100，50mmol/L Tris-HC1，pH 7.5]中浸洗2～3次，每次5～10min。C. Remove SDS: After the electrophoresis is completed, soak the separating gel in the refolding buffer [2% (volume percentage) TritonX-100, 50mmol/L Tris-HC1, pH 7.5] for 2 to 3 times, each time 5~10min.
D、复性：将分离胶置于缓冲液[50mmol/L Tris-HC1，pH7.5]中在37℃下放置进行酶反应3h。D. Renaturation: Put the separating gel in buffer [50mmol/L Tris-HC1, pH7.5] and place it at 37°C for 3h.
E、染色与脱色：用考马斯亮蓝染色30min，然后数小时换一次脱色液(5％(体积百分含量)乙酸+10％(体积百分含量)甲醇)，直至背景清晰。注：经染色和脱色处理的凝胶背景颜色为蓝黑色，蛋白酶反应部位颜色变浅。凝胶中呈现蛋白酶反应的区域大小和该部位的透光率与蛋白酶活性成正比。E. Staining and decolorization: stain with Coomassie Brilliant Blue for 30 minutes, and then change the decolorization solution (5% (volume percentage) acetic acid + 10% (volume percentage) methanol) every few hours until the background is clear. Note: The background color of the stained and decolorized gel is blue-black, and the color of the protease reaction site becomes lighter. The size of the area in the gel where the protease reaction is present and the light transmittance of that site are directly proportional to the protease activity.
由图2所示，泳道1为蛋白Marker；泳道2为果糖胺脱糖酶FrIB的SDS-PAGE电泳图。蛋白在39.7kD左右有明显条带，与理论值相符，证明该蛋白可以在酵母菌中成功表达并被纯化出来。As shown in Figure 2, lane 1 is the protein marker; lane 2 is the SDS-PAGE electrophoresis of fructosamine desugarase FrIB. The protein has an obvious band around 39.7kD, which is consistent with the theoretical value, which proves that the protein can be successfully expressed and purified in yeast.
实施例4Example 4
纯化后的果糖胺脱糖酶的活性检测Activity detection of purified fructosamine desugarase
反应体系：reaction system:
1mL反应体系中含有15mM的丙氨酸，20mM的果糖-6-磷酸，0.2mL的果糖胺脱糖酶，2.5mM的EDTA及100mM的不同pH值缓冲液(Na 2HPO 4-NaH 2PO 4，pH2.0～10.0)，将以上反应体系放在1.5mL离心管中混匀，然后放入PCR仪中37℃下反应20min，反应结束后95℃下加热5min终止反应，离心取上清通过使用高效液相色谱法(HPLC)检测丙酮酸产量，从而计算出果糖胺脱糖酶酶活。 1mM reaction system contains 15mM alanine, 20mM fructose-6-phosphate, 0.2mL fructosamine desugarase, 2.5mM EDTA and 100mM different pH buffers (Na 2 HPO 4 -NaH 2 PO 4 , pH2.0～10.0), put the above reaction system in a 1.5mL centrifuge tube and mix well, then put it in a PCR instrument for 20min at 37℃, heat at 95℃ for 5min to terminate the reaction, centrifuge and take the supernatant through High-performance liquid chromatography (HPLC) was used to detect the pyruvate production, thereby calculating the enzyme activity of fructosamine desugarase.
检测条件：Detection conditions:
采用高效液相色谱法(HPLC)定量分析丙酮酸。所用色谱柱为C18柱，流动相为乙腈(含0.1％(体积百分含量)二环已胺、0.1％(体积百分含量)甲酸)的水溶液(体积比5：95)，流速为0.6mL/min，柱温40℃，检测波长为230nm。此法以丙酮酸钠作对照品，按外标法以峰面积计算 反应液中丙酮酸的含量。Pyruvate was quantitatively analyzed by high performance liquid chromatography (HPLC). The chromatographic column used is a C18 column, the mobile phase is an aqueous solution of acetonitrile (containing 0.1% (volume percent) dicyclohexylamine, 0.1% (volume percent) formic acid) (volume ratio 5:95), and the flow rate is 0.6mL /min, the column temperature is 40°C, and the detection wavelength is 230nm. In this method, sodium pyruvate is used as a reference substance, and the content of pyruvate in the reaction solution is calculated by the peak area according to the external standard method.
酶活力定义：Enzyme activity definition:
在最适反应温度37℃条件下，每分钟催化1μmol丙氨酸转化为丙酮酸所需的酶量定义为一个酶活单位，即1U。Under the optimal reaction temperature of 37°C, the amount of enzyme required to catalyze the conversion of 1 μmol of alanine into pyruvate per minute is defined as an enzyme activity unit, ie 1U.
经过测试，发现酶活为2.49U/mg。上述数据表明该酵母表达系统的重组果糖胺脱糖酶具有很好合成氨基葡萄糖和丙酮酸的功能，具有较大应用潜力。After testing, it was found that the enzyme activity was 2.49U/mg. The above data show that the recombinant fructosamine desugarase in the yeast expression system has a good function of synthesizing glucosamine and pyruvate, and has great application potential.
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。The above is only a preferred embodiment of the present invention, it should be pointed out that, for those of ordinary skill in the art, without departing from the principle of the present invention, some improvements and modifications can also be made, and these improvements and modifications can also be made. It should be regarded as the protection scope of the present invention.

Claims (10)

  1. 一种果糖胺脱糖酶毕赤酵母表达载体，其特征在于，包括骨架载体和编码果糖胺脱糖酶的基因；所述编码果糖胺脱糖酶的基因的核苷酸如SEQ ID NO.1所示。A kind of expression vector of fructosamine desugarase Pichia pastoris, is characterized in that, comprises skeleton carrier and the gene of coding fructosamine desugarase; The nucleotide of the gene of described coding fructosamine desugarase is as SEQ ID NO.1 shown.
  2. 根据权利要求1所述的果糖胺脱糖酶毕赤酵母表达载体，其特征在于，所述骨架载体包括pPIC9K。The expression vector of fructosamine desugarase Pichia pastoris according to claim 1, wherein the backbone vector comprises pPIC9K.
  3. 权利要求1或2所述果糖胺脱糖酶毕赤酵母表达载体的构建方法，包括以下步骤：The construction method of the fructosamine desugarase Pichia pastoris expression vector described in claim 1 or 2, comprising the following steps:
    使用双酶切法将编码果糖胺脱糖酶的基因与骨架载体相连接，得到表达载体。The gene encoding fructosamine desugarase is connected to the backbone vector by double enzyme digestion to obtain an expression vector.
  4. 根据权利要求3所述的构建方法，其特征在于，所述双酶切法，每40～60μL的双酶切体系包括：10×quitcut buffer 4～6μL、pPIC9K或含编码果糖胺脱糖酶的基因的核酸28～32μL，4～6U/μL的SnaBI和NotI各0.8～1.5μL和余量的水。The construction method according to claim 3, characterized in that, in the double enzyme digestion method, each 40-60 μL double enzyme digestion system includes: 10×quitcut buffer 4-6 μL, pPIC9K or the enzyme containing the encoding fructosamine desugarase 28-32 μL of nucleic acid of the gene, 0.8-1.5 μL of 4-6 U/μL of SnaBI and NotI and the rest of water.
  5. 根据权利要求4所述的构建方法，其特征在于，所述双酶切法的双酶切条件为36～38℃，1～3h。The construction method according to claim 4, characterized in that, the double enzyme digestion condition of the double enzyme digestion method is 36-38°C, 1-3h.
  6. 一种含权利要求1或2所述表达载体的基因工程菌，其特征在于，所述基因工程菌的宿主菌包括毕赤酵母。A genetically engineered bacterium containing the expression vector according to claim 1 or 2, characterized in that the host bacterium of the genetically engineered bacterium comprises Pichia pastoris.
  7. 根据权利要求6所述的基因工程菌，其特征在于，所述毕赤酵母包括毕赤酵母GS115。The genetically engineered bacterium according to claim 6, wherein the Pichia pastoris comprises Pichia pastoris GS115.
  8. 权利要求6或7所述基因工程菌的构建方法，包括以下步骤：The construction method of genetic engineering bacterium described in claim 6 or 7, comprises the following steps:
    将果糖胺脱糖酶毕赤酵母表达载体线性化，电转化毕赤酵母的感受态细胞，得到基因工程菌。The fructosamine desugarase Pichia pastoris expression vector is linearized, and the competent cells of the Pichia pastoris are electrotransformed to obtain genetically engineered bacteria.
  9. 基于权利要求6或7所述基因工程菌的果糖胺脱糖酶表达方法，包括以下步骤：The fructosamine desugarase expression method based on the genetically engineered bacterium described in claim 6 or 7 comprises the following steps:
    将所述基因工程菌接种到BMGY液体培养基中，进行第一振荡培养至OD 600在2～6之间，离心，弃上清，得到培养菌体； Inoculate the genetically engineered bacteria into the BMGY liquid medium, perform the first shaking culture until the OD600 is between 2 and 6, centrifuge, discard the supernatant, and obtain the cultured bacteria;
    用BMGY液体培养基重悬所述培养菌体，在100～300r/min进行第二振荡培养84～108h；所述第二振荡培养的期间，每10～14h向培养基中加甲醇至甲醇的终体积百分含量为0.5％～1.0％。Resuspend the cultured bacteria with BMGY liquid medium, and carry out the second shaking culture at 100-300r/min for 84-108h; during the second shaking culture, add methanol to the medium every 10-14h The final volume percentage is 0.5%-1.0%.
  10. 根据权利要求9所述的表达方法，其特征在于，所述甲醇为100％的甲醇或甲醇的体积百分含量为80％以上的甲醇水溶液。The expression method according to claim 9, characterized in that the methanol is 100% methanol or an aqueous methanol solution with a volume percentage of methanol of more than 80%.
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