WO2022213477A1 - 一种从葡萄糖到氨基葡萄糖的酶法制备方法与酶用途 - Google Patents

一种从葡萄糖到氨基葡萄糖的酶法制备方法与酶用途 Download PDF

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WO2022213477A1
WO2022213477A1 PCT/CN2021/099567 CN2021099567W WO2022213477A1 WO 2022213477 A1 WO2022213477 A1 WO 2022213477A1 CN 2021099567 W CN2021099567 W CN 2021099567W WO 2022213477 A1 WO2022213477 A1 WO 2022213477A1
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glucosamine
phosphate
glucose
seq
amino
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French (fr)
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陈延静
詹金明
王松叶
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江苏澳新生物工程有限公司
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    • C—CHEMISTRY; METALLURGY
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    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates

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  • the invention relates to an enzymatic preparation method from glucose to glucosamine and the types of biological enzymes used in the preparation method, belonging to the technical field of bioengineering; the invention also relates to the use of the enzyme.
  • Glucosamine namely 2-amino-2-deoxy-D-glucose, also known as glucosamine, glucosamine or simply glucosamine, glucosamine, is a compound in which a hydroxyl group of glucose is replaced by an amino group , is another important functional monosaccharide.
  • glucosamine is abundant in prokaryotic, eukaryotic, plant cell walls and animal bones.
  • Glucosamine is needle-like crystal, safe and non-toxic, easily soluble in water, and has no special odor. It is a component of various antibiotics and can be synthesized in the human body, usually in the form of N-acetyl derivatives or muramic acid in animals.
  • glucosamine The stability of glucosamine is related to pH. At room temperature, it can exist stably at pH lower than 4.8, and the stability of glucosamine decreases with the increase of pH.
  • glucosamine on the market mainly exists in the form of glucosamine hydrochloride, glucosamine sulfate and compound salt, which can exist stably at room temperature, and is beneficial to the absorption of the human body and the storage and transportation of medicines.
  • Glucosamine is a natural water-soluble monosaccharide and one of the most abundant monosaccharides in the human body. Studies have shown that glucosamine has some special physiological active functions, such as anti-inflammatory and analgesic, repairing cartilage damage, and treating rheumatoid arthritis; strengthening leukocyte proliferation and differentiation, promoting cytokine secretion, improving immune regulation, and anti-tumor; restoring mitochondrial enzymes Expression, improve mitochondrial glutathione antioxidant capacity, enhance immune function; promote endoplasmic reticulum-related protein interpretation, strengthen protease activity, improve protein homeostasis, prolong cell life, etc. Therefore, it is widely used in the fields of medicine, food nutrition, health care and cosmetics.
  • glucosamine on the market are mainly chemical hydrolysis method and microbial fermentation method.
  • the production of glucosamine by chemical hydrolysis is based on shrimp and crab shells or fungal cell walls as raw materials for acid hydrolysis with strong acid, using chitin in shrimp and crab shells as substrates, and using concentrated hydrochloric acid for hydrolysis under high temperature conditions. After the hydrolysis of concentrated hydrochloric acid The glycosidic bond in chitin is broken, and the amide bond is hydrolyzed.
  • activated carbon needs to be added for decolorization, and the steps of heating and dissolving, vacuum distillation, crystallization and recrystallization, pulverization, drying, etc.
  • glucosamine double salt was prepared by using sodium sulfate.
  • the production of glucosamine by chemical hydrolysis requires the use of a large amount of strong acid, which is easy to cause environmental pollution, the reaction needs to be carried out under high temperature conditions, the reaction conditions are harsh, the steps in the production process are complex, the technical requirements are high, and the production efficiency is low.
  • the material source of the method is shrimp and crab shells, and the source of the raw material is limited, and the glucosamine produced by this method is not suitable for people allergic to seafood, so the scope of use is limited.
  • Glucosamine can also be produced by microbial fermentation.
  • Escherichia coli, Bacillus subtilis and fungi that have undergone metabolic engineering have been developed for the production of glucosamine.
  • the fermentation process is optimized and strictly controlled, and key issues such as temperature, dissolved oxygen, pH, and feeding are optimized to achieve efficient production of glucosamine.
  • the microbial fermentation method has a simple process compared with the chemical method. It is a renewable production method, with mild conditions and unlimited sources of raw materials.
  • the fermentation period is long, the fermentation process is strictly controlled, and the concentration of glucosamine in the final product is low.
  • the solution method converts N-acetylglucosamine to produce glucosamine, but there are still problems of low production efficiency and environmental pollution.
  • the purpose of the present invention is to aim at the deficiencies of the prior art, and provide a kind of enzymatic preparation method from glucose to glucosamine, which is a method for combining multiple in vitro biological enzymes for preparing glucosamine with glucose as the starting substrate,
  • the method has the advantages of cheap raw materials, abundant sources, low production cost, environmental friendliness and safety to human body.
  • Another object of the present invention is to provide uses involving biological enzymes used in the above-mentioned methods.
  • the present invention is an enzymatic preparation method from glucose to glucosamine, comprising a combination of biological enzymes, or an expression vector or cloning vector expressing the combined biological enzymes, or a transgenic cell line expressing the combined biological enzymes, Or express the genetically engineered bacteria of the combined biological enzyme; the amino acid sequence of the combined biological enzyme contains:
  • amino acid sequence of an enzyme formed by deletion, substitution, insertion or mutation of bases based on the amino acid sequence defined in 1) and having the activity of catalyzing the transfer of an amino group from an amino-donor compound to an amino-acceptor compound.
  • the enzymes are hexokinase, 6-phosphate glucose isomerase, glucosamine synthase and glucosamine-6-phosphate dephosphorase.
  • nucleotide sequences of the enzyme are respectively SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6 and SEQ ID NO.8. the sequence shown.
  • the genetically engineered bacteria are recombinant Escherichia coli genetically engineered bacteria.
  • the method for catalyzing the transfer of amino groups from an amino-donor compound to an amino-acceptor compound includes a method for preparing glucosamine or a method for preparing glucose 6-phosphate, ammonia A method of sugar-6-phosphate or fructose-6-phosphate.
  • the synthetic reaction method of preferred glucosamine is:
  • the invention also discloses a method for synthesizing or converting glucosamine from glucose: the substrate for synthesizing or converting glucosamine is glucose; the method comprises using any one or more groups of biological enzymes in the following (1) to (6) :
  • transgenic cell line containing the genes encoding hexokinase, 6-phosphate glucose isomerase, glucosamine synthase and glucosamine-6-phosphate dephosphorase;
  • the amino acid sequence of the biological enzyme contains:
  • amino acid sequence of an enzyme formed by deletion, substitution, insertion or mutation of bases based on the amino acid sequence defined in 1) and having the activity of catalyzing the transfer of an amino group from an amino-donor compound to an amino-acceptor compound.
  • the synthesis/conversion is carried out in an environment containing the substrate glucose, at a pH of 6-8 and a temperature of 30-50°C.
  • the optimum pH of the synthesis/transformation environment is 7.0 and the temperature is 45°C.
  • the present invention also discloses the use of the combined biological enzyme involved in the technical solution of the above preparation method:
  • the application is an enzyme that uses the combined biological enzyme to catalyze the transfer of an amino group from an amino-donor compound to an amino-acceptor compound.
  • the amino acid sequence of the biological enzyme contains:
  • amino acid sequence of an enzyme formed by deletion, substitution, insertion or mutation of bases based on the amino acid sequence defined in 1) and having the activity of catalyzing the transfer of an amino group from an amino-donor compound to an amino-acceptor compound.
  • nucleotide sequences of the combined biological enzymes are the sequences shown in SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6 and SEQ ID NO.8, respectively.
  • the method for catalyzing the transfer of an amino group from an amino-donor compound to an amino-acceptor compound includes a method for preparing glucosamine or for preparing glucosamine 6-phosphate, glucosamine-6-phosphate or 6-phosphate The fructose method.
  • the combined biological enzyme is selected from any one or more groups of biological enzymes in the following (1) to (6):
  • transgenic cell line containing the genes encoding hexokinase, 6-phosphate glucose isomerase, glucosamine synthase and glucosamine-6-phosphate dephosphorase;
  • the recombinant Escherichia coli genetically engineered bacteria are preferably constructed by using Escherichia coli BL21, DH5 ⁇ or Top10 as a host, and more preferably, the host is Escherichia coli BL21.
  • the expression vector is preferably pCold II, pCold I or pUC19.
  • the recombinant Escherichia coli genetically engineered bacteria are obtained by constructing a recombinant expression vector with pCold II and using Escherichia coli BL21 as a host.
  • the construction method of recombinant Escherichia coli genetically engineered bacteria includes:
  • IPTG isopropyl ⁇ -D-1-thiogalactopyranoside
  • the present invention has the following beneficial effects:
  • the method of the present invention utilizes the preparation method of the biological enzyme method to prepare glucosamine with glucose as the substrate.
  • the combination of four biological enzymes including hexokinase, 6-phosphate glucose isomerase, glucosamine synthase and glucosamine 6-phosphate dephosphorase, is involved for the first time.
  • the technical route is concise, the substrate sources involved in the method are sufficient, not limited by raw materials, the preparation conditions are mild, the environmental pollution is small, and the reaction specificity is high, the impurities in the system are few, and the downstream separation and purification is easy, and the production cost is low.
  • the above-mentioned four biological enzymes are synthesized by gene sequence synthesis, and are constructed in plasmid pCold II and stored in Escherichia coli JM109.
  • the above four biological enzyme proteins were obtained by successful high soluble expression in Escherichia coli BL21, and the expressed four biological enzyme proteins were purified.
  • the biological activity of the obtained four recombinant enzymes was verified, and it was found that glucose can be gradually synthesized into glucosamine through the above four biological enzymes. And the data show that the above four recombinant biological enzymes have a good function of synthesizing glucosamine and have great application potential.
  • Example 1 Acquisition of hexokinase, 6-phosphate glucose isomerase, glucosamine synthase and glucosamine-6-phosphate dephosphorase genes
  • the synthesized gene sequence was transformed, that is, 10 ⁇ l of the ligation product was added to the competent cell JM109, placed on ice for 30 min, immediately placed on ice after heat shock at 42°C for 90 s, placed on ice for 2 min, and 1 ml of LB without antibiotics was added to culture
  • the cells were cultured on a shaker at 37°C for 1 h, and the transformed bacterial solution was spread on LB (Amp) blue and white plates, and cultured at 37°C overnight. Multiple colonies were picked on the plate for PCR identification.
  • plasmid extraction was used, and the cloned plasmid identified as positive by PCR was extracted with the plasmid extraction kit of omega company.
  • Example 2 Construction, recombinant expression and protein expression of prokaryotic expression vector for four enzyme genes of hexokinase, 6-phosphate glucose isomerase, glucosamine synthase and glucosamine 6-phosphate dephosphorase
  • Primer design primers are designed starting from the mature peptide sequence after the signal peptide (as shown in SEQ ID NO: 4 and SEQ ID NO: 5)
  • the plasmid was extracted from this clone, and full-length sequencing was performed on both ends of AOX3 and AOX5 to further verify the correctness of the inserted target gene.
  • Recombinant E. coli was cultured in 25-50mL LB medium (250mL Erlenmeyer flask) to an OD of 0.4-0.6, then transferred to 15°C for culture and added with a final concentration of 0.4mM isopropyl ⁇ -D-1-thiogalactopyranoside (IPTG) to induce Expression 24h, speed 200rpm. Then ultrasonically disrupt the cells and extract the recombinase. At the same time, the SDS-PAGE of the culture medium and the determination of the enzyme activity are carried out.
  • IPTG isopropyl ⁇ -D-1-thiogalactopyranoside
  • sample buffer [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 5-10min, and then subjected to electrophoresis separation.
  • sample buffer [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 5-10min, and then subjected to electrophoresis separation.
  • mercaptoethanol is not added to the sample extract in order to moderately denature proteases during electrophoresis, so that the activities of these proteases can be restored after electrophoresis.
  • ⁇ -Mercaptoethanol Used to open disulfide bonds and disrupt the quaternary or tertiary structure of proteins. It is a colorless and transparent liquid with
  • E. Staining and destaining stain with Coomassie brilliant blue for 30 minutes, and then change the destaining solution (5% acetic acid + 10% methanol) every few hours until the background is clear. Note: The background color of the stained and destained gel is blue-black, and the color of the protease reaction site becomes lighter. The size of the region in the gel showing the protease reaction and the light transmittance of this site are proportional to the protease activity.
  • Example 4 Activity detection of four kinds of biological enzymes after purification
  • bacterial supernatant or purified diluted enzyme solution
  • 500 ⁇ L of sodium dihydrogen phosphate/disodium hydrogen phosphate buffer 100 mM, pH 7.0
  • the substrate is glucose
  • the generated product is glucose 6-phosphate
  • the hexokinase activity is measured according to the instructions of the hexokinase activity detection kit. The hexokinase activity was calculated using the formula.
  • glucose 6-phosphate isomerase Since the reaction catalyzed by glucose 6-phosphate isomerase is a reversible reaction, that is, it can not only catalyze glucose 6-phosphate to generate fructose 6-phosphate, but also catalyze fructose 6-phosphate to generate glucose 6-phosphate. Moreover, glucose 6-phosphate can generate NADPH and lactone under the action of NADP and gluconate dehydrogenase. At the same time, the specific absorption wavelength of NADPH is 340nm. Therefore, by measuring the change of the absorbance value of NADPH at 340nm with a UV spectrophotometer, the enzyme activity of glucose-6-phosphate isomerase can be measured correspondingly, which is expressed as OD 340nm .
  • the amount of enzyme required to raise the OD 340nm reading by 1 per minute is one unit of enzyme activity.
  • 1mL reaction system contains 15mM glutamine, 20mM fructose-6-phosphate, 0.2mL glucosamine synthase, 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 into PCR at 37°C for 20min, after the reaction is completed, heat at 95°C for 5min to terminate the reaction, centrifuge to take the supernatant by using SBA Glutamate production was detected to calculate glucosamine synthase activity.
  • pH buffers Na 2 HPO 4 -NaH 2 PO 4 , pH2.0-10.0
  • V enzyme volume of enzyme solution added to the reaction ( ⁇ L)
  • Total V the final volume of the reaction system for the determination of glucosamine synthase activity ( ⁇ L)
  • the amount of enzyme required to catalyze the conversion of 1 ⁇ mol of glutamine to glutamate per minute was defined as one unit of enzyme activity, ie 1U.
  • Glucosamine was quantitatively analyzed by high performance liquid chromatography (HPLC).
  • HPLC high performance liquid chromatography
  • the chromatographic column used is an amino column
  • the mobile phase is 80% acetonitrile aqueous solution
  • the flow rate is 0.6 mL/min
  • the column temperature is 40° C.
  • the detector used is a refractive index detector.
  • Glucosamine retention time is approximately 12.7 minutes.
  • the glucosamine concentration is proportional to the response intensity of the HPLC characteristic peak of glucosamine.
  • Embodiment 5 the synthetic reaction system of glucosamine
  • Glucosamine was quantitatively analyzed by high performance liquid chromatography (HPLC).
  • HPLC high performance liquid chromatography
  • the chromatographic column used is an amino column
  • the mobile phase is 80% acetonitrile aqueous solution
  • the flow rate is 0.6 mL/min
  • the column temperature is 40° C.
  • the detector used is a refractive index detector.
  • Glucosamine retention time is approximately 12.7 minutes.
  • the glucosamine concentration is proportional to the response intensity of the HPLC characteristic peak of glucosamine.
  • the reactor add glucose, HEPES buffer (pH 7.0), magnesium chloride, glutamine, EDTA so that the glucose concentration is 1 mM; the HEPES buffer (pH 7.0) concentration is 100 mM; the magnesium chloride concentration is 10 mM; The amide concentration was 15 mM; the EDTA concentration was 2.5 mM.
  • the reactor was heated, and when the internal temperature of the reactor reached 40 °C, the heating rate was controlled so that the internal temperature of the reactor was between 40 °C and 50 °C, and 10U of hexokinase was added. During the reaction, glucose was added to control the reaction.
  • the glucose concentration in the liquid is about 1mM, until the glucose concentration does not change; then add 25U 6-phosphate glucose isomerase; 16U glucosamine synthase; 10U glucosamine-6-phosphate dephosphorase. Until there is no change in the glucosamine generated in the reaction solution.
  • Crude product glucosamine hydrochloride is again processed, recrystallized, and dried to obtain finished product glucosamine hydrochloride, and the yield of the finished product is 80% (calculated according to the weight of glucose feeding), and the obtained glucosamine hydrochloride is detected according to the USP method, and the content reaches 98%, which fully meets the USP quality requirements. .
  • Embodiment 6 the application of this technical route in other product synthesis
  • the present invention not only relates to the biological enzymatic technical route for synthesizing glucosamine by using glucose, but also relates to all biological enzymes on the route, so it has various applications. These include:
  • Glucose 6-phosphate is not only used as an important intermediate product to synthesize various compounds, but also often used as a biological agent in enzymatic research, and also plays a certain role in pathological research.
  • the efficient synthesis of NADPH is obtained by using glucose-6-phosphate dehydrogenase to catalyze glucose 6-phosphate and NADP, so glucose 6-phosphate also has an important application in the efficient synthesis of NADPH.
  • the glucosamine synthesized from glucose through this technical route can further synthesize N-acetylglucosamine, that is, glucosamine can be the synthetic precursor of N-acetylglucosamine.
  • N-acetylglucosamine is one of the important monomers composed of polysaccharides hyaluronic acid, heparin, keratan sulfate, etc. normal victory function of the body.
  • N-acetylglucosamine is widely used in food, medical care, cosmetics and other fields, and the demand is increasing year by year, and the market application prospect is broad. Even in developed countries, N-acetylglucosamine is widely used as an important component of health care products, providing necessary biological active substances for the human body.
  • the technical route and the four biological enzymes included in the technical route have become important synthetic routes and key enzymes for the preparation of glucosamine and related intermediates.
  • the method uses glucose as a substrate to prepare glucosamine, including combining biological enzymes, or expression vectors or cloning vectors expressing the combined biological enzymes, or transgenic cell lines expressing the combined biological enzymes, or expressing the combined biological enzymes.
  • Genetically engineered bacteria that use biological enzymes in combination; preferably hexokinase, 6-phosphate glucose isomerase, glucosamine synthase and glucosamine 6-phosphate dephosphorase in combination with four biological enzymes; the technical route of the method is simple and the method is simple.
  • the involved substrate has sufficient sources, is not limited by raw materials, mild preparation conditions, low environmental pollution, high reaction specificity, less impurities in the system, easy downstream separation and purification, and low production cost.
  • Glucose can be gradually synthesized into glucosamine, which has great potential for industrial application in the preparation of glucosamine.

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Abstract

提供了一种从葡萄糖到氨基葡萄糖的酶法制备方法，包括以葡萄糖为底物，联用生物酶制备氨基葡萄糖，优选己糖激酶、6-磷酸葡萄糖异构酶、氨基葡萄糖合酶和氨糖6-磷酸脱磷酶联用。该方法技术路线简洁，底物来源充足，制备条件温和，环境污染小，反应专一性高，体系中杂质少易于下游分离纯化，生产成本低。

Description

一种从葡萄糖到氨基葡萄糖的酶法制备方法与酶用途 技术领域
本发明涉及一种从葡萄糖到氨基葡萄糖的酶法制备方法及该制备方法中所用生物酶的种类，属于生物工程技术领域；本发明还涉及酶的用途。
背景技术
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，又是一种重要的功能单糖。在自然界中，氨基葡萄糖大量存在于原核、真核、植物的细胞壁以及动物的骨骼之中。氨基葡萄糖为针状晶体，安全无毒，易溶于水，无特殊气味，是多种抗生素的组分，可在人体中合成，通常以N-乙酰基衍生物或胞壁酸的形式存在动物及微生物来源的多糖中。氨基葡萄糖的稳定性与pH有关，在室温环境下，其可在pH低于4.8的条件下稳定存在，随着pH的升高氨基葡萄糖的稳定性下降。目前市场上氨基葡萄糖主要以氨基葡萄糖盐酸盐、氨基葡萄糖硫酸盐及复合盐的形式存在，在室温下可稳定存在，且利于人体的吸收及药品的储存运输。
氨基葡萄糖是一种天然的水溶性单糖，是在人体中含量最丰富的单糖之一。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；强化白细胞增值和分化，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 关蛋白讲解，强化蛋白酶活性，改善蛋白稳态水平，延长细胞寿命等。因此，其被广泛应用于医药、食品营养、健康保健及化妆品等领域。据报道，2019年全球氨基葡萄糖市场总额约为50亿美元。随着生活质量的进一步提高以及人口老龄化问题的加剧，氨基葡萄糖类的医药、食品营养保健品的全球需求会持续增加。预计在未来五年内，氨基葡萄糖的全球市场规模会以5％左右的复合年均增长率(Compound annual growth rate,CAGR)上升。
目前市场上氨基葡萄糖的生产方法主要是化学水解法和微生物发酵法。化学水解法生产氨基葡萄糖是以虾蟹壳或真菌细胞壁为原料利用强酸进行酸水解反应，以虾蟹壳中的甲壳素为底物，利用浓盐酸在高温条件下进行水解，经过浓盐酸的水解作用，甲壳素中的糖苷键断裂，酰胺键水解，反应过程中需加入活性炭进行脱色，多次加热溶解与减压蒸馏、结晶与重结晶、粉碎、烘干等步骤，制成成品氨基葡萄糖盐酸盐。在此基础上，利用硫酸钠制备氨基葡萄糖复盐。但利用化学水解法生产氨基葡萄糖需要使用大量强酸，易造成环境污染，反应需高温条件下进行，反应条件苛刻，生产过程中步骤复杂，技术要求高，生产效率低。且该方法的材料来源是虾蟹壳类物质，原料来源受限，用该方法生产出的氨基葡萄糖不适合对海鲜过敏的人群，因此使用范围受限制。
氨基葡萄糖还可以通过微生物发酵法生产，近年来，已经开发出利用经过代谢工程改造过的大肠杆菌、枯草芽孢杆菌及真菌进行氨基葡萄糖的生产。通过菌种的构建改良，对发酵过程进行优化与严格控 制，对温度、溶氧、pH、补料等关键问题进行优化，来实现氨基葡萄糖的高效生产。目前，微生物发酵法与化学法相比过程简单，为可再生的生产方法，条件温和，原料来源丰富不受限制，但是发酵周期长，发酵过程控制严苛，终产物氨基葡萄糖浓度低，需结合酸解法转化N-乙酰氨基葡萄糖生产氨基葡萄糖，仍然存在生产效率低和环境污染的问题。
因此，急需开发一种低成本，低污染的生产氨基葡萄糖的新方法。
发明内容
本发明的目的是针对现有技术的不足，提供了一种从葡萄糖到氨基葡萄糖的酶法制备方法，它是以葡萄糖为起始底物制备氨基葡萄糖的多个体外生物酶联用的方法，该方法具有原料廉价、来源丰富、生产成本低、环境友好和对人体安全等优点。
本发明的另一个目的是提供了涉及上述方法中所用生物酶的用途。
实现本发明的目的技术方案如下：
本发明是一种从葡萄糖到氨基葡萄糖的酶法制备方法，包括联用生物酶，或者表达所述联用生物酶的表达载体或克隆载体、或者表达所述联用生物酶的转基因细胞系、或者表达所述联用生物酶的基因工程菌；所述联用生物酶的氨基酸序列含有：
1)SEQ ID NO.1、SEQ ID NO.3、SEQ ID NO.5和SEQ ID NO.7所示的氨基酸序列；或者
2)在1)限定的氨基酸序列基础上经碱基的缺失、取代、插入或突变而成且具有催化氨基由氨基供体化合物转移至氨基受体化合物的活性的酶的氨基酸序列。
以上所述的本发明方法，其进一步优选的技术方案是：所述酶为己糖激酶、6-磷酸葡萄糖异构酶、氨基葡萄糖合酶和氨糖-6-磷酸脱磷酶。
以上所述的本发明方法，其进一步优选的技术方案是：所述酶的核苷酸序列分别为SEQ ID NO.2、SEQ ID NO.4、SEQ ID NO.6和SEQ ID NO.8所示的序列。
以上所述的本发明方法，其进一步优选的技术方案是：所述基因工程菌为重组大肠杆菌基因工程菌。
以上所述的本发明方法，其进一步优选的技术方案是：所述催化氨基由氨基供体化合物转移至氨基受体化合物的方法，包括制备氨基葡萄糖的方法或者用于制备6-磷酸葡萄糖、氨糖-6-磷酸或者6-磷酸果糖的方法。
本发明所述的方法中，优选的氨基葡萄糖的合成反应方法是：
在反应器中，加入葡萄糖，pH7.0HEPES缓冲液，氯化镁，谷氨酰胺，EDTA，各原料的摩尔浓度比为，葡萄糖：HEPES缓冲液：氯化镁：谷氨酰胺：EDTA＝1：90－110：9－11：13－17：2－3；对反应器进行加热，当反应器内温达到40℃时，控制加热速率，使得反应器内温在40℃-50℃之间，加入的适量的己糖激酶，反应过程中，补加葡萄糖，控制反应液中葡萄糖浓度，直到葡萄糖浓度不变为止； 再加入适理的6-磷酸葡萄糖异构酶、氨基葡萄糖合酶和氨糖-6-磷酸脱磷酶；直到反应液中产生的氨基葡萄糖没有变化为止；所加入的酶的单位比为：己糖激酶：6-磷酸葡萄糖异构酶：氨基葡萄糖合酶：氨糖-6-磷酸脱磷酶＝10：20－30：10－20：8－12。
各原料的摩尔浓度比为进一步成选为，葡萄糖：HEPES缓冲液：氯化镁：谷氨酰胺：EDTA＝1：100：10：15：2.5；所加入的酶的单位比为：己糖激酶：6-磷酸葡萄糖异构酶：氨基葡萄糖合酶：氨糖-6-磷酸脱磷酶＝10：25：16：10。
本发明还公开了一种葡萄糖合成或转化氨基葡萄糖的方法：合成或转化氨基葡萄糖的底物为葡萄糖；所述方法包括利用如下(1)至(6)中任意一组或多组的生物酶：
(1)己糖激酶、6-磷酸葡萄糖异构酶、氨基葡萄糖合酶和氨糖-6-磷酸脱磷酶；
(2)编码己糖激酶、6-磷酸葡萄糖异构酶、氨基葡萄糖合酶和氨糖-6-磷酸脱磷酶的基因；
(3)含有所述己糖激酶、6-磷酸葡萄糖异构酶、氨基葡萄糖合酶和氨糖-6-磷酸脱磷酶的编码基因的蛋白；(4)含有所述己糖激酶、6-磷酸葡萄糖异构酶、氨基葡萄糖合酶和氨糖-6-磷酸脱磷酶的编码基因的表达载体或克隆载体；
(5)含有所述己糖激酶、6-磷酸葡萄糖异构酶、氨基葡萄糖合酶和氨糖-6-磷酸脱磷酶的编码基因的转基因细胞系；
(6)含有所述己糖激酶、6-磷酸葡萄糖异构酶、氨基葡萄糖合酶和氨糖-6-磷酸脱磷酶的编码基因的基因工程菌；
所述生物酶的氨基酸序列含有：
1)SEQ ID NO.1、SEQ ID NO.3、SEQ ID NO.5和SEQ ID NO.7所示的氨基酸序列；或者
2)在1)限定的氨基酸序列基础上经碱基的缺失、取代、插入或突变而成且具有催化氨基由氨基供体化合物转移至氨基受体化合物的活性的酶的氨基酸序列。
在一种优选的实施方式中，所述合成/转化，是在含有底物葡萄糖的环境下，在pH为6-8，温度为30-50℃的条件下进行合成/转化。所述合成/转化环境的最优pH为7.0，温度为45℃。
本发明还公开了如以上制备方法技术方案中涉及的联用生物酶的用途：该用途为将联用生物酶用于催化氨基由氨基供体化合物转移至氨基受体化合物的酶，所述联用生物酶的氨基酸序列含有：
1)SEQ ID NO.1、SEQ ID NO.3、SEQ ID NO.5和SEQ ID NO.7所示的氨基酸序列；或者
2)在1)限定的氨基酸序列基础上经碱基的缺失、取代、插入或突变而成且具有催化氨基由氨基供体化合物转移至氨基受体化合物的活性的酶的氨基酸序列。
优选的本发明用途中：联用生物酶的核苷酸序列分别为SEQ ID NO.2、SEQ ID NO.4、SEQ ID NO.6和SEQ ID NO.8所示的序列。
优选的本发明用途中：所述催化氨基由氨基供体化合物转移至氨基受体化合物的方法，包括制备氨基葡萄糖的方法或者用于制备6-磷酸葡萄糖、氨糖-6-磷酸或者6-磷酸果糖的方法。
优选的本发明用途中：所述的联用生物酶选自下述(1)至(6)中任意一组或多组的生物酶：
(1)己糖激酶、6-磷酸葡萄糖异构酶、氨基葡萄糖合酶和氨糖-6-磷酸脱磷酶；
(2)编码己糖激酶、6-磷酸葡萄糖异构酶、氨基葡萄糖合酶和氨糖-6-磷酸脱磷酶的基因；
(3)含有所述己糖激酶、6-磷酸葡萄糖异构酶、氨基葡萄糖合酶和氨糖-6-磷酸脱磷酶的编码基因的蛋白；(4)含有所述己糖激酶、6-磷酸葡萄糖异构酶、氨基葡萄糖合酶和氨糖-6-磷酸脱磷酶的编码基因的表达载体或克隆载体；
(5)含有所述己糖激酶、6-磷酸葡萄糖异构酶、氨基葡萄糖合酶和氨糖-6-磷酸脱磷酶的编码基因的转基因细胞系；
(6)含有所述己糖激酶、6-磷酸葡萄糖异构酶、氨基葡萄糖合酶和氨糖-6-磷酸脱磷酶的编码基因的基因工程菌。
本发明制备方法中，所述重组大肠杆菌基因工程菌，优选是以大肠杆菌BL21，DH5α或Top10为宿主构建得到的，进一步优选地，宿主为大肠杆菌BL21。表达载体优选pCold Ⅱ、pCold Ⅰ或pUC19。最优选的方案是：所述重组大肠杆菌基因工程菌，是以pCold Ⅱ构建 重组表达载体、以大肠杆菌BL21为宿主而构建得到的。重组大肠杆菌基因工程菌的构建方法包括：
(1)通过NCBI(National Center for Biotechnology Information)数据库寻找己糖激酶(Hexokinase)、6-磷酸葡萄糖异构酶(6-phosphate glucose isomerase)、氨基葡萄糖合酶(Glucosamine synthase)和氨糖-6-磷酸脱磷酶(Glucosamine 6-phosphate dephosphorase)基因。找到符合相对应底物的生物酶的基因后，交由生物公司进行基因合成，从而得到上述四种酶基因；
(2)选取大肠杆菌表达载体pCold Ⅱ，将扩增得到的上述四种基因分别连接到表达载体上，构建重组表达载体pCold Ⅱ-ota3，将重组表达质粒pCold Ⅱ-ota3转化大肠杆菌BL21，筛选阳性转化子并测序验证。
优选的一种利用重组菌生产酶的方法包括：将电转化线性化重组表达质粒pCold Ⅱ-ota3的阳性重组大肠杆菌BL21/pCold Ⅱ-ota3在37℃、200rpm条件下培养至OD 600＝0.4-0.6，随后转入15℃培养并加入终浓度为0.4mM isopropyl β-D-1-thiogalactopyranoside(IPTG)，诱导表达24h，转速200rpm。
与现有技术相比，本发明具有以下有益效果：
1、本发明方法利用生物酶法的制备方法，以葡萄糖为底物制备氨基葡萄糖。其中首次涉及到己糖激酶、6-磷酸葡萄糖异构酶、氨基葡萄糖合酶和氨糖6-磷酸脱磷酶共四种生物酶联用。该技术路线简洁，方法所涉及的底物来源充足，不受原材料限制，制备条件温和， 环境污染小，且反应专一性高，体系中杂质少易于下游分离纯化，生产成本低。
2、本发明中，通过基因合成的方法对上述四种生物酶进行了基因序列合成，并构建于质粒pCold Ⅱ中，储存于大肠杆菌JM109中。利用大肠杆菌BL21成功高可溶性表达分别获得了上述四种生物酶蛋白，并对表达的四种生物酶蛋白进行了纯化。对所获得的四种重组酶进行了生物活性验证，发现依次通过上述四种生物酶，可以将葡萄糖逐步合成氨基葡萄糖。并通过数据表明上述四种重组生物酶联用具有很好地合成氨基葡萄糖的功能，并具有较大应用潜力。
具体实施方式
虽然本发明已以较佳实施例公开如下，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。
在本发明中所使用的术语，除非有另外说明，一般具有本领域普通技术人员通常理解的含义。
下面结合具体的制备实施例和应用实施例，并参照数据进一步详细地描述本发明。应理解，这些实施例只是为了举例说明本发明，而非以任何方式限制本发明的范围。
在以下的实施例中，未详细描述的各种过程和方法是本领域中公知的常规方法。
实施例1 己糖激酶、6-磷酸葡萄糖异构酶、氨基葡萄糖合酶和 氨糖-6-磷酸脱磷酶基因的获得
首先通过NCBI(National Center for Biotechnology Information)数据库寻找己糖激酶(Hexokinase)、6-磷酸葡萄糖异构酶(6-phosphate glucose isomerase)、氨基葡萄糖合酶(Glucosamine synthase)和氨糖-6-磷酸脱磷酶(Glucosamine 6-phosphate dephosphorase)基因。找到符合相对应底物的生物酶的基因后，交由生物公司进行基因合成。其中合成后的基因序列引入XhoI和PstI限制性酶切位点(不带信号肽)，酶切位点为保护碱基，有效序列为酶切位点后序列。
合成后的基因序列进行转化，即取10μl连接产物加入感受态细胞JM109中，置于冰上30min，42℃热激90s后立即放在冰上，放置2min后，加入1ml不含抗生素的LB培养基，37℃摇床培养1h，将转化的菌液涂于LB(Amp)蓝白板上，37℃培养过夜。在平板上分别挑取多个菌落做PCR鉴定。
同时用到质粒提取，用omega公司的质粒抽提试剂盒提取PCR鉴定为阳性的克隆质粒。
实施例2 己糖激酶、6-磷酸葡萄糖异构酶、氨基葡萄糖合酶和氨糖6-磷酸脱磷酶四种酶基因的原核表达载体的构建，重组表达及其蛋白表达
1、原核表达载体的构建
(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:4和SEQ ID NO:5所示)
(2)PCR反应，以克隆载体pMD-19T为模板，62℃退火，35cycles。
(3)XhoI和PstI双酶切的相应生物酶基因的PCR产物和质粒pCold Ⅱ。
表1 双酶切体系
成分 使用量
纯化PCR产物/质粒 30μl
10*quitcut buffer 5μl
QuitCut SnaBI 1μl
QuitCut NotI 1μl
ddH 2O 13μl
总体积 50μl
37℃酶切2hr
(4)如常规方法连接，转化，并进行双酶切鉴定。
对这个克隆抽提质粒，从AOX3和AOX5两端进行全长测序，进一步验证了插入的目的基因的正确性。
2、重组质粒在大肠杆菌BL21中的表达
1)转化大肠杆菌及阳性转化子的筛选
据大肠杆菌表达系统操作手册，取10μl连接产物加入感受态细胞DE3中，置于冰上30min，42℃热激90s后立即放在冰上，放置2min后，加入1ml不含抗生素的LB培养基，37℃摇床培养1h，将转化的菌液涂于LB(Amp)蓝白板上，37℃培养过夜。在平板上分别挑取多个菌落做PCR鉴定。以空载体转化菌作对照，进一步验证了目的基因已经转入大肠杆菌BL21。
2)重组质粒在大肠杆菌中的诱导表达
重组大肠杆菌在25-50mL LB培养基(250mL三角瓶)中培养至OD为0.4-0.6，随后转入15℃培养并加入终浓度为0.4mM isopropyl β-D-1-thiogalactopyranoside(IPTG)，诱导表达24h，转速200rpm。然后进行超声波破碎菌体并提取重组酶，同时进行培养液的SDS-PAGE和酶活测定。
实施例3：上述四种生物酶蛋白的纯化
重组酶的纯化及其SDS-PAGE凝胶电泳分析
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。
其中native-SDS-PAGE实验步骤：
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。
B、制胶与电泳：在制备分离胶的过程中加入0.2％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。
C、去SDS：电泳完成后，将分离胶在复性缓冲液[2％TritonX-100，50mmol/LTris-HC1，pH 7.5]中浸洗2-3次，每次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。
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％乙酸+10％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。
实施例4：分别纯化后的四种生物酶的活性检测
1、己糖激酶的活性检测
取适量菌体上清液(或者纯化稀释酶液)，加入500μL磷酸二氢钠/磷酸氢二钠缓冲液(100mM，pH7.0)。其中底物为葡萄糖，生成产物为6-磷酸葡萄糖，己糖激酶活性测定根据己糖激酶活性检测试剂盒说明书。利用公式计算己糖激酶活性。
己糖激酶活性(mU/mg)＝B/(△T×m)×D
B:根据NADPH标曲计算△T内产生的NADPH；m:蛋白质含量；D:稀释倍数。
2、6-磷酸葡萄糖异构酶的活性检测
6-磷酸葡萄糖异构酶活力的测定原理：
由于6-磷酸葡萄糖异构酶催化的反应为可逆反应，即它不仅可以将6-磷酸葡萄糖催化生成6-磷酸果糖，也可以催化6-磷酸果糖生成6-磷酸葡萄糖。而且6-磷酸葡萄糖在NADP和葡萄糖酸脱氢酶的作用下可生成NADPH和內酯。同时，NADPH的特定吸收波长为340nm，因此，利用紫外分光光度计在340nm处测定NADPH的吸光度值的变化，即可相应测定6-磷酸葡萄糖异构酶的酶活力，以OD 340nm表示。
反应体系：
将33μL 20mmol/L的6-磷酸果糖，20μL 10mmol/L的NADP，35μL100mmol/L的MgCl 2及15μL 0.1654mg/mL的磷酸葡萄糖脱氢酶溶解于375μL 0.1mol/L的Tris-Hcl(pH8.0)，混匀后转移至0.5mL比色皿中，立即加入25μL酶液开始反应，在紫外分光光度计上每隔30s读取吸光度值，直至数值趋近于恒定。以时间为横坐标，OD 340nm为纵坐标，绘制曲线。
酶活力定义：
每分钟使OD 340nm读数升高1所需要的酶量为一个酶活力单位。
3、氨基葡萄糖合酶的活性检测
反应体系：
1mL反应体系中含有15mM的谷氨酰胺，20mM的果糖-6-磷酸，0.2mL的氨基葡萄糖合酶，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SBA检测谷氨酸产量，从而计 算出氨基葡萄糖合酶酶活。
氨基葡萄糖合酶酶活计算公式：
U/L＝(C 谷氨酸×10 4÷147×V 总÷T)/V 酶
V 酶：反应添加的酶液体积(μL)
C 谷氨酸：谷氨酸浓度(mg/dL)
V 总：氨基葡萄糖合酶酶活测定反应体系的终体积(μL)
T：反应时间(min)
酶活力定义：
在最适反应温度37℃条件下，每分钟催化1μmol谷氨酰胺转化为谷氨酸所需的酶量定义为一个酶活单位，即1U。
4、氨糖6-磷酸脱磷酶的活性检测
在含有10mM氯化镁的100mM HEPES缓冲液(pH7.0)中，能够脱磷酸基团的酶，即氨糖-6-磷酸脱磷酶对底物氨糖6-磷酸的脱磷活性，从而生成最终底物氨基葡萄糖。
检测条件：
采用高效液相色谱法(HPLC)定量分析氨基葡萄糖。所用色谱柱为氨基柱，流动相为80％乙腈水溶液，流速0.6mL/min，柱温40℃，所用检测器为示差折光检测器。氨基葡萄糖保留时间约为12.7分钟。氨基葡萄糖浓度与氨基葡萄糖的HPLC特征峰的响应强度成正比。
实施例5：氨基葡萄糖的合成反应体系
反应体系：
1mM葡萄糖；100mM HEPES缓冲液(pH7.0)；10mM氯化镁； 15mM谷氨酰胺；2.5mM EDTA；10U己糖激酶；25U 6-磷酸葡萄糖异构酶；16U氨基葡萄糖合酶；10U氨糖-6-磷酸脱磷酶。利用这个反应体系总共反应1小时后检测。
检测条件：
采用高效液相色谱法(HPLC)定量分析氨基葡萄糖。所用色谱柱为氨基柱，流动相为80％乙腈水溶液，流速0.6mL/min，柱温40℃，所用检测器为示差折光检测器。氨基葡萄糖保留时间约为12.7分钟。氨基葡萄糖浓度与氨基葡萄糖的HPLC特征峰的响应强度成正比。
合成反应：
在反应器中，加入葡萄糖，HEPES缓冲液(pH7.0)，氯化镁，谷氨酰胺，EDTA，使得葡萄糖浓度为1mM；HEPES缓冲液(pH7.0)浓度为100mM；氯化镁浓度为10mM；谷氨酰胺浓度为15mM；EDTA浓度为2.5mM。对反应器进行加热，当反应器内温达到40℃时，控制加热速率，使得反应器内温在40℃-50℃之间，加入10U己糖激酶，反应过程中，补加葡萄糖，控制反应液中葡萄糖浓度在1mM左右，直到葡萄糖浓度不变为止；再加入25U 6-磷酸葡萄糖异构酶；16U氨基葡萄糖合酶；10U氨糖-6-磷酸脱磷酶。直到反应液中产生的氨基葡萄糖没有变化为止。
反应液中加入盐酸使得反应液中氨基葡萄糖完全成盐，溶液过滤，除去不溶物，滤液减压浓缩，除去大部分的水，至溶液中出现大量结晶，冷却结晶。结晶完全后，过滤，固体为粗品盐酸氨基葡萄糖。
粗品盐酸氨基葡萄糖再经过，重结晶，干燥，得到成品盐酸氨基 葡萄糖，成品收率80％(按照葡萄糖投料重量计算)，所得盐酸氨基葡萄糖按照USP方法检测，含量达到98％，完全符合USP质量要求。
实施例6：该技术路线在其他产物合成中的应用
本发明不仅涉及利用葡萄糖合成氨基葡萄糖的生物酶法技术路线，还涉及该路线上的所有生物酶，因此具有多种应用。其中包括：
(1)6-磷酸葡萄糖的合成
利用该技术路线上的生物酶，不仅可以制备氨基葡萄糖，还可以制备6-磷酸葡萄糖。6-磷酸葡萄糖不仅作为重要的中间产物用来合成多种化合物，而且常作为生物制剂应用于酶学研究中，也在病理学研究中发挥着一定的作用。同时，NADPH的有效合成是利用葡萄糖-6-磷酸脱氢酶催化6-磷酸葡萄糖和NADP而来，因此6-磷酸葡萄糖在NADPH的有效合成中也有着重要的应用。
(2)氨糖-6-磷酸和6-磷酸果糖的合成
利用该技术路线上的生物酶，不仅可以制备氨基葡萄糖，还可以制备6-磷酸果糖和氨糖-6-磷酸。这些都是其他重要化合物的关键中间体。
(3)N-乙酰氨基葡萄糖的合成
葡萄糖通过该技术路线所合成的氨基葡萄糖可以进一步合成N-乙酰氨基葡萄糖，即氨基葡萄糖可以是N-乙酰氨基葡萄糖的合成前体。而N-乙酰氨基葡萄糖是生物体内多糖透明质酸、肝素、硫酸角质素等组成的重要单体之一，同时也是母乳寡糖、神经氨酸、壳寡糖合成的前体物质，可以维持生物体正常的胜利功能。N-乙酰氨基葡萄 糖广泛应用于食品、医疗保健、化妆品等领域，并且需求量逐年增加，市场应用前景广阔。更是在发达国家，N-乙酰氨基葡萄糖作为保健产品的重要组成成分，为人体提供必需的生物活性物质，因而被广泛应用。
该技术路线和技术路线中包含的四种生物酶成为了制备氨基葡萄糖和相关中间产物的重要合成路线和关键酶。该方法以葡萄糖为底物来制备氨基葡萄糖，包括联用生物酶，或者表达所述联用生物酶的表达载体或克隆载体、或者表达所述联用生物酶的转基因细胞系、或者表达所述联用生物酶的基因工程菌；优选己糖激酶、6-磷酸葡萄糖异构酶、氨基葡萄糖合酶和氨糖6-磷酸脱磷酶共四种生物酶联用；方法技术路线简洁，方法所涉及的底物来源充足，不受原材料限制，制备条件温和，环境污染小，且反应专一性高，体系中杂质少易于下游分离纯化，生产成本低。可以将葡萄糖逐步合成氨基葡萄糖，在氨基葡萄糖的制备中具有较大的工业应用潜力。

Claims (10)

  1. 一种从葡萄糖到氨基葡萄糖的酶法制备方法，其特征在于：包括联用生物酶，或者表达所述联用生物酶的表达载体或克隆载体、或者表达所述联用生物酶的转基因细胞系、或者表达所述联用生物酶的基因工程菌；所述联用生物酶的氨基酸序列含有：
    1)SEQ ID NO.1、SEQ ID NO.3、SEQ ID NO.5和SEQ ID NO.7所示的氨基酸序列；或者
    2)在1)限定的氨基酸序列基础上经碱基的缺失、取代、插入或突变而成且具有催化氨基由氨基供体化合物转移至氨基受体化合物的活性的酶的氨基酸序列。
  2. 根据权利要求1所述的方法，其特征在于：联用的生物酶为己糖激酶、6-磷酸葡萄糖异构酶、氨基葡萄糖合酶和氨糖-6-磷酸脱磷酶；其核苷酸序列分别为SEQ ID NO.2、SEQ ID NO.4、SEQ ID NO.6和SEQ ID NO.8所示的序列。
  3. 根据权利要求1所述的方法，其特征在于，所述基因工程菌为重组大肠杆菌基因工程菌。
  4. 根据权利要求1所述的方法，其特征在于，所述催化氨基由氨基供体化合物转移至氨基受体化合物的方法，包括制备氨基葡萄糖的方法或者用于制备6-磷酸葡萄糖、氨糖-6-磷酸或者6-磷酸果糖的方法。
  5. 根据权利要求1所述的方法，其特征在于，氨基葡萄糖的合成反应方法是：
    在反应器中，加入葡萄糖，pH7.0HEPES缓冲液，氯化镁，谷氨 酰胺，EDTA，各原料的摩尔浓度比为，葡萄糖：HEPES缓冲液：氯化镁：谷氨酰胺：EDTA＝1：90－110：9－11：13－17：2－3；对反应器进行加热，当反应器内温达到40℃时，控制加热速率，使得反应器内温在40℃-50℃之间，加入的适量的己糖激酶，反应过程中，补加葡萄糖，控制反应液中葡萄糖浓度，直到葡萄糖浓度不变为止；再加入适理的6-磷酸葡萄糖异构酶、氨基葡萄糖合酶和氨糖-6-磷酸脱磷酶；直到反应液中产生的氨基葡萄糖没有变化为止；所加入的酶的单位比为：己糖激酶：6-磷酸葡萄糖异构酶：氨基葡萄糖合酶：氨糖-6-磷酸脱磷酶＝10：20－30：10－20：8－12。
  6. 一种葡萄糖合成或转化氨基葡萄糖的方法，其特征在于，合成或转化氨基葡萄糖的底物为葡萄糖；所述方法包括利用如下(1)至(6)中任意一组或多组的生物酶：
    (1)己糖激酶、6-磷酸葡萄糖异构酶、氨基葡萄糖合酶和氨糖-6-磷酸脱磷酶；
    (2)编码己糖激酶、6-磷酸葡萄糖异构酶、氨基葡萄糖合酶和氨糖-6-磷酸脱磷酶的基因；
    (3)含有所述己糖激酶、6-磷酸葡萄糖异构酶、氨基葡萄糖合酶和氨糖-6-磷酸脱磷酶的编码基因的蛋白；(4)含有所述己糖激酶、6-磷酸葡萄糖异构酶、氨基葡萄糖合酶和氨糖-6-磷酸脱磷酶的编码基因的表达载体或克隆载体；
    (5)含有所述己糖激酶、6-磷酸葡萄糖异构酶、氨基葡萄糖合酶和氨糖-6-磷酸脱磷酶的编码基因的转基因细胞系；
    (6)含有所述己糖激酶、6-磷酸葡萄糖异构酶、氨基葡萄糖合酶和氨糖-6-磷酸脱磷酶的编码基因的基因工程菌；
    所述生物酶的氨基酸序列含有：
    1)SEQ ID NO.1、SEQ ID NO.3、SEQ ID NO.5和SEQ ID NO.7所示的氨基酸序列；或者
    2)在1)限定的氨基酸序列基础上经碱基的缺失、取代、插入或突变而成且具有催化氨基由氨基供体化合物转移至氨基受体化合物的活性的酶的氨基酸序列。
  7. 一种如权利要求1－5中所述的制备方法中涉及的联用生物酶的用途，其特征在于，该用途为将联用生物酶用于催化氨基由氨基供体化合物转移至氨基受体化合物的酶，所述联用生物酶的氨基酸序列含有：
    1)SEQ ID NO.1、SEQ ID NO.3、SEQ ID NO.5和SEQ ID NO.7所示的氨基酸序列；或者
    2)在1)限定的氨基酸序列基础上经碱基的缺失、取代、插入或突变而成且具有催化氨基由氨基供体化合物转移至氨基受体化合物的活性的酶的氨基酸序列。
  8. 根据权利要求7所述的用途，其特征在于：联用生物酶的核苷酸序列分别为SEQ ID NO.2、SEQ ID NO.4、SEQ ID NO.6和SEQ ID NO.8所示的序列。
  9. 根据权利要求7所述的用途，其特征在于，所述催化氨基由氨基供体化合物转移至氨基受体化合物的方法，包括制备氨基葡萄 糖的方法或者用于制备6-磷酸葡萄糖、氨糖-6-磷酸或者6-磷酸果糖的方法。
  10. 根据权利要求7所述的用途，其特征在于，所述的联用生物酶选自下述(1)至(6)中任意一组或多组的生物酶：
    (1)己糖激酶、6-磷酸葡萄糖异构酶、氨基葡萄糖合酶和氨糖-6-磷酸脱磷酶；
    (2)编码己糖激酶、6-磷酸葡萄糖异构酶、氨基葡萄糖合酶和氨糖-6-磷酸脱磷酶的基因；
    (3)含有所述己糖激酶、6-磷酸葡萄糖异构酶、氨基葡萄糖合酶和氨糖-6-磷酸脱磷酶的编码基因的蛋白；(4)含有所述己糖激酶、6-磷酸葡萄糖异构酶、氨基葡萄糖合酶和氨糖-6-磷酸脱磷酶的编码基因的表达载体或克隆载体；
    (5)含有所述己糖激酶、6-磷酸葡萄糖异构酶、氨基葡萄糖合酶和氨糖-6-磷酸脱磷酶的编码基因的转基因细胞系；
    (6)含有所述己糖激酶、6-磷酸葡萄糖异构酶、氨基葡萄糖合酶和氨糖-6-磷酸脱磷酶的编码基因的基因工程菌。
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FANG JIAJU: "Glucokinase is an Isoenzyme of Hexokinase D", CHEMISTRY OF LIFE : COMMUNICATIONS OF THE CHINESE BIOCHEMICAL SOCIETY, SHANGHAI : ZHONGGUO SHENGWU HUAXUEHUI, CN, vol. 12, no. 5, 15 October 1992 (1992-10-15), CN , pages 35 - 35, XP055975283, ISSN: 1000-1336 *
LIU DIANLEI, LI PIWU, LI RUIRUI, WANG SHENG, WANG RUIMING: "Research advance of the construction of gene engineering strains for glucosamine", BIOTECHNOLOGY BULLETIN, no. 3, 31 December 2014 (2014-12-31), pages 36 - 41, XP009525425, ISSN: 1002-5464 *
YOU CHUN: "In Vitro Multi-enzyme System Catalyzed Synthesis of Glucosamine", BAIDU, 29 November 2020 (2020-11-29), pages 270 - 270, XP055975284 *

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