CN113584007B

CN113584007B - Fructosamine dehydrase vector, transgenic cell line expressing fructosamine dehydrase, genetically engineered bacterium and application of fructosamine dehydrase

Info

Publication number: CN113584007B
Application number: CN202110872810.6A
Authority: CN
Inventors: 王怀英
Original assignee: Wuhan Baiammonia Huiji Biotechnology Co ltd
Current assignee: Wuhan Baiammonia Huiji Biotechnology Co ltd
Priority date: 2021-07-30
Filing date: 2021-07-30
Publication date: 2024-08-23
Anticipated expiration: 2041-07-30
Also published as: CN113584007A

Abstract

The invention relates to a fructosamine dehydrase vector, a transgenic cell line expressing fructosamine dehydrase, genetically engineered bacteria and application of fructosamine dehydrase, and belongs to the technical field of biological enzyme production. The present invention provides a method of catalyzing the transfer of an amino group from an amino donor compound to an amino acceptor compound, the method comprising: the transfer of amino groups from an amino donor compound to an amino acceptor compound is catalyzed by an enzyme (fructosamine dehydrase), or an expression or cloning vector expressing the enzyme, or a transgenic cell line expressing the enzyme, or a genetically engineered bacterium expressing the enzyme. The method disclosed by the invention has the advantages of sufficient substrate source, no limitation of raw materials, mild preparation conditions, small environmental pollution, high reaction specificity, few impurities in a system, easiness in downstream separation and purification and low production cost.

Description

Fructosamine dehydrase vector, transgenic cell line expressing fructosamine dehydrase, genetically engineered bacterium and application of fructosamine dehydrase

Technical Field

The invention relates to the technical field of biological enzyme method production, in particular to a fructosamine-removing enzyme vector, a transgenic cell line for expressing fructosamine-removing enzyme, genetically engineered bacteria and application of fructosamine-removing enzyme.

Background

Glucosamine (GlcN), i.e. 2-amino-2-deoxy-D-glucose. The glucosamine has important physiological functions on human bodies, is widely applied to the fields of foods, health products, medicines and the like, can be used as a substance for treating bone joint diseases in medical clinic, can also be used as a medicine for treating rheumatic arthritis, and can be used as an additive in foods for infant foods. With further improvement of life quality and aggravation of population aging, global demands for medicines, foods, nutritional and health products of glucosamine are continuously increasing.

The traditional glucosamine preparation method has two types, namely, the glucosamine is prepared by hydrolyzing chitin, but the technology has the limitations of seasonal influence on raw materials, complex hydrolysis technology, large acid and alkali consumption, environmental pollution and other factors. Secondly, N-acetylglucosamine can be produced by a microbial fermentation method, and then the glucosamine is prepared by hydrolysis. The microbial fermentation process has been widely used, and in recent years, the production of glucosamine by using metabolic engineering-modified escherichia coli, bacillus subtilis and fungi has been developed. But has low yield, high cost and complex refining process.

Disclosure of Invention

The invention aims to provide a fructosamine dehydrase vector, a transgenic cell line expressing fructosamine dehydrase, genetically engineered bacteria and application of fructosamine dehydrase. The method disclosed by the invention has the advantages of sufficient substrate source, no limitation of raw materials, mild preparation conditions, small environmental pollution, high reaction specificity, few impurities in a system, easiness in downstream separation and purification, low production cost and high glucosamine yield.

The present invention provides a method of catalyzing the transfer of an amino group from an amino donor compound to an amino acceptor compound, the method comprising: catalyzing transfer of an amino group from an amino donor compound to an amino acceptor compound using an enzyme, or an expression vector or cloning vector expressing the enzyme, or a transgenic cell line expressing the enzyme, or a genetically engineered bacterium expressing the enzyme; the enzyme comprises:

1) An amino acid sequence as shown in SEQ ID NO. 1; or alternatively

2) An amino acid sequence of an enzyme which is obtained by deletion, substitution, insertion or mutation of amino acids based on the amino acid sequence shown in SEQ ID NO.1 and has an activity of catalyzing the transfer of an amino group from an amino donor compound to an amino acceptor compound.

Preferably, the amino donor compound comprises an amino acid.

Preferably, the amino acceptor compound comprises a saccharide.

The invention also provides application of the fructosamine enzyme-removing enzyme in producing glucosamine and/or ketocarboxylic acid, and the amino acid sequence of the fructosamine enzyme-removing enzyme is shown as SEQ ID NO. 1.

The present invention also provides a method for producing glucosamine and/or ketocarboxylic acid, the method comprising:

Performing amino conversion by using fructosamine dehydrase, or an expression vector or cloning vector for expressing the fructosamine dehydrase, or a transgenic cell line for expressing the fructosamine dehydrase, or genetically engineered bacteria for expressing the fructosamine dehydrase, and using amino acid and saccharide as reaction substrates to obtain glucosamine and/or ketocarboxylic acid; the amino acid of fructosamine denase is shown as SEQ ID NO. 1.

Preferably, the saccharide comprises one or more of starch, glucose, fructose, and fructose-6-phosphate.

Preferably, the amino acid comprises alanine, glutamic acid, aspartic acid or glutamine.

Preferably, the conditions for the amino group conversion include: the pH value is 6-8, and the temperature is 30-50 ℃.

Preferably, the enzyme used in the method further comprises a fructokinase, the amino acid sequence of which is shown in SEQ ID NO. 3.

The invention also provides a fructosamine-degrading enzyme vector, which comprises a skeleton vector and a gene for encoding fructosamine-degrading enzyme; the nucleotide of the gene for encoding fructosamine denase is shown as SEQ ID NO. 2.

The invention also provides a transgenic cell line for expressing fructosamine-removing enzyme, and the nucleotide of the gene for encoding fructosamine-removing enzyme is shown as SEQ ID NO. 2.

The invention also provides a gene engineering bacterium for expressing fructosamine-removing enzyme, and the nucleotide of the gene for encoding fructosamine-removing enzyme is shown as SEQ ID NO. 2.

The invention also provides a production method of fructosamine descanase based on the genetically engineered bacteria, which comprises the following steps: and (3) carrying out liquid culture and induction on the genetically engineered bacteria to obtain fructosamine desaccharase.

The invention also provides a complex enzyme for co-producing glucosamine and ketocarboxylic acid, wherein the complex enzyme comprises fructosamine dehydrase and fructokinase; the amino acid sequence of fructosamine dehydrase is shown as SEQ ID NO. 1; the amino acid sequence of the fructokinase is shown as SEQ ID NO. 3.

The invention also provides a method for co-producing glucosamine and ketocarboxylic acid based on the complex enzyme in the technical scheme, which comprises the following steps: mixing fructose and fructokinase for phosphorylation to obtain fructose-6-phosphate; mixing fructose-6-phosphate, amino acid and fructosamine enzyme, and performing transamination to obtain glucosamine and ketocarboxylic acid.

The present invention provides a method of catalyzing the transfer of an amino group from an amino donor compound to an amino acceptor compound. The method disclosed by the invention has the advantages of sufficient substrate source, no limitation of raw materials, mild preparation conditions, small environmental pollution, high reaction specificity, few impurities in a system, easiness in downstream separation and purification and low production cost. In a specific embodiment of the invention, the fructokinase (GenBank: ESD 97983.1) derived from Escherichia coli (ESCHERICHIA COLI 908658) and fructosamine-removing enzyme (GenBank: AOR 99552.1) derived from Bacillus subtilis (Bacillus subtilis) are subjected to gene sequence synthesis by a gene synthesis method, and are constructed in plasmid pCold II and stored in Escherichia coli JM 109. The invention utilizes the escherichia coli BL21 to successfully and high-solubility express to respectively obtain the fructokinase protein and fructosamine-desaccharase protein, and the expressed biological enzyme protein is purified. The invention verifies the biological activity of the obtained recombinant fructokinase and fructosamine enzyme, and discovers that starch, glucose, fructose and fructose-6-phosphate can be synthesized into glucosamine by sequentially carrying out phosphorylation and transamination through the fructokinase and fructosamine enzyme, and simultaneously, the alpha-ketoglutarate is generated by utilizing glutamic acid, or the pyruvic acid is generated by utilizing the transamination of alanine. The data show that the fructokinase and fructosamine dehydrase have the function of well synthesizing glucosamine and pyruvic acid or alpha-ketoglutaric acid, and have great application potential. The method can greatly reduce the production cost of the glucosamine and/or ketocarboxylic acid, amino acid and/or sugar, and has important significance as raw materials in the pharmaceutical industry and the food industry.

Drawings

FIG. 1 is a diagram showing double digestion of the positive pCold II-FrIB plasmid provided by the invention;

FIG. 2 is a diagram showing double digestion of the positive pCold II-FRK 1 plasmid provided by the invention;

FIG. 3 is an SDS-PAGE electrophoresis of fructokinase and fructosamine-dehydrase protein purification provided by the invention.

Detailed Description

1) Amino acid sequence ：MSQATAKVNREVQAFLQDLKGKTIDHVFFVACGGSSAIMYPSKYVFDRESKSINSDLYSANEFIQRNPVQLGEKSLVILCSHSGNTPETVKAAAFARGKGALTIAMTFKPESPLAQEAQYVAQYDWGDEALAINTNYGVLYQIVFGTLQVLENNTKFQQAIEGLDQLQAVYEKALKQEADNAKQFAKAHEKESIIYTMASGANYGVAYSYSICILMEMQWIHSHAIHAGEYFHGPFEIIDESVPFIILLGLDETRPLEERALTFSKKYGKKLTVLDAASYDFTAIDDSVKGYLAPLVLNRVLRSYADELAEERNHPLSHRRYMWKVEY; or shown as SEQ ID NO.1

In the present invention, the enzyme is fructosamine-removing enzyme (Fructosamine deglycase, frIB), the nucleotide sequence of the gene encoding the enzyme is shown as ：TTATATAACATTATAGTCTAATGCATAATGGTTCTTCATTTTCAGATCAATACTCAACTTTCCACATGTATCTTCTATGAGATAAAGGATGATTTCTCTCCTCTGCCAGCTCGTCTGCATAGCTTCTCAGCACACGATTGAGAACGAGCGGAGCAAGATAGCCTTTAACTGAATCGTCAATTGCAGTGAAGTCGTAAGATGCAGCATCAAGCACAGTGAGCTTTTTGCCATACTTTTTCGAGAAGGTAAGCGCCCGCTCTTCAAGAGGTCTTGTTTCATCTAAACCGAGCAGGATGATAAACGGCACGGATTCATCAATAATTTCAAACGGTCCGTGAAAATATTCTCCGGCATGAATGGCGTGGGAATGAATCCATTGCATTTCCATGAGAATGCAGATGCTGTAGGAGTAAGCGACACCGTAGTTTGCACCGCTTGCCATGGTATAAATAATACTTTCTTTTTCATGGGCTTTTGCAAATTGCTTGGCGTTGTCAGCTTCCTGCTTAAGGGCTTTTTCATATACAGCCTGCAATTGATCTAAGCCTTCAATTGCTTGTTGGAATTTCGTATTGTTTTCTAATACTTGCAGGGTTCCAAAAACGATTTGATACAAAACGCCATAGTTTGTATTGATCGCAAGCGCCTCATCACCCCAATCGTACTGGGCAACATATTGCGCTTCCTGCGCTAAAGGAGACTCCGGTTTAAACGTCATCGCAATCGTAAGTGCACCCTTGCCCCTTGCAAACGCAGCAGCTTTGACTGTCTCCGGGGTATTTCCCGAATGCGAGCACAAAATAACAAGAGACTTTTCACCAAGCTGAACAGGGTTGCGCTGAATAAATTCGTTGGCGCTGTAGAGGTCGGAGTTTATTGATTTTGACTCTCTGTCAAACACATACTTACTCGGATACATAATGGCAGAAGACCCTCCGCATGCGACAAAGAATACATGATCAATGGTTTTCCCTTTCAAATCCTGCAAGAAAGCTTGAACCTCACGATTTACTTTTGCTGTGGCCTGACTCAAATCCTTCACTCCCCGTTTTTATTATATAACGTTATATAACATTATATAT. in SEQ ID NO.2, and the fructosamine-removing enzyme has the function of catalyzing the transfer of amino groups from amino donor compounds to amino acceptor compounds. The fructosamine denase disclosed by the invention is used for amino transfer.

In the present invention, the amino donor compound preferably includes an amino acid. In the present invention, the amino acceptor compound preferably includes a saccharide. In the present invention, the saccharide preferably includes one or more of starch, glucose, fructose, and fructose-6-phosphate.

The method has the advantages of low raw material cost, abundant sources, low production cost, environmental friendliness, safety to human bodies and the like.

The invention also provides a method for producing glucosamine and/or ketocarboxylic acid, which comprises utilizing fructosamine carbohydrase, or an expression vector or a cloning vector for expressing the fructosamine carbohydrase, or a transgenic cell line for expressing the fructosamine carbohydrase, or a genetically engineered bacterium for expressing the fructosamine carbohydrase, and performing amino conversion by taking amino acid and saccharide as reaction substrates to obtain the glucosamine and/or ketocarboxylic acid; the amino acid of fructosamine denase is shown as SEQ ID NO. 1.

In the present invention, the saccharide preferably includes one or more of starch, glucose, fructose, and fructose-6-phosphate. In the production process of the present invention, amino conversion is preferably carried out using fructose-6-phosphate and amino acid as substrates. The amino group of the amino acid is transferred to fructose-6 phosphate to obtain glucosamine, and the amino acid is deaminated to obtain ketocarboxylic acid. The starch, glucose and fructose of the present invention are preferably converted to fructose-6-phosphate prior to the reaction. In the present invention, the amino acid preferably includes alanine, glutamic acid, aspartic acid or glutamine. After conversion of the amino groups, each amino acid will produce the corresponding ketocarboxylic acid. In the present invention, the conditions for the amino group conversion preferably include: the pH value is 6-8, the temperature is 30-50 ℃, more preferably the pH value is 6.7-7.5, and the temperature is 40-50 ℃. In the present invention, the enzyme used in the method preferably further comprises a fructokinase whose amino acid sequence is shown as SEQ ID NO.3 and whose nucleotide sequence is shown as SEQ ID NO.4 at ：MITNCRRPCIANPVVRLYAIDIEKNKESTVRIGIDLGGTKTEVIALGDAGEQLYRHRLPTPRDDYRQTIETIATLVDMAEQATGQRGTVGMGIPGSISPYTGVVKNANSTWLNGQPFDKDLSARLQREVRLANDANCLAVSEAVDGAAAGAQTVFAVIIGTGCGAGVAFNGRAHIGGNGTAGEWGHNPLPWMDEDELRYREEVPCYCGKQGCIETFISGTGFAMDYRRLSGHALKGSEIIRLVEESDPVAELALRRYELRLAKSLAHVVNILDPDVIVLGGGMSNVDRLYQTVGQLIKQFVFGGECETPVRKAKHGDSSGVRGAAWLWPQE; and ：CTCTTGTGGCCATAACCACGCAGCGCCGCGTACGCCGCTGGAATCACCGTGCTTCGCCTTACGCACCGGCGTTTCACATTCGCCGCCGAAGACAAATTGTTTAATCAACTGCCCAACCGTTTGATATAAACGGTCTACATTGCTCATCCCGCCCCCCAGGACAATCACATCCGGATCGAGAATATTCACGACATGTGCCAGCGATTTTGCCAGCCGCAGCTCGTAGCGACGCAATGCCAGTTCCGCTACCGGATCGCTTTCTTCAACCAGGCGGATAATTTCACTGCCTTTCAGCGCATGTCCGCTCAAACGACGATAATCCATCGCGAATCCCGTGCCCGAAATAAAGGTTTCAATACAACCTTGTTTACCGCAATAACAAGGGACTTCCTCGCGATAACGCAGTTCGTCTTCGTCCATCCACGGTAGCGGATTGTGTCCCCACTCACCTGCCGTGCCATTGCCGCCGATATGCGCCCGCCCATTGAATGCCACGCCCGCGCCGCATCCCGTGCCGATAATCACGGCAAATACCGTCTGCGCTCCCGCTGCCGCGCCATCTACTGCTTCTGAAACCGCCAGACAGTTAGCGTCATTTGCCAGCCGCACTTCCCGCTGCAACCTCGCGCTTAAGTCTTTATCGAATGGCTGACCGTTGAGCCAGGTTGAATTGGCATTCTTCACCACACCGGTGTAAGGCGAAATTGAGCCAGGAATGCCCATACCTACCGTTCCGCGCTGCCCCGTCGCCTGCTCCGCCATATCAACCAACGTGGCGATCGTTTCAATAGTCTGCCGGTAATCATCACGCGGCGTGGGCAGACGATGGCGGTACAACTGCTCCCCTGCATCGCCCAGTGCAATCACTTCAGTTTTGGTGCCGCCTAAATCGATACCTATACGCACGGTACTCTCCTTATTTTTTTCAATATCAATAGCGTAGAGACGGACAACCGGATTGGCAATGCAAGGCCGCCGACAATTCGTTATCAT. in the present invention, and when the saccharide is fructose, the catalytic system of the present invention preferably comprises fructosamine-desaccharase and fructokinase, and the use of fructokinase is capable of converting fructose into fructose-6-phosphate, thereby performing the subsequent reaction described above.

The invention also provides a fructosamine-degrading enzyme vector, which comprises a skeleton vector and a gene for encoding fructosamine-degrading enzyme; the nucleotide of the gene for encoding fructosamine denase is shown as SEQ ID NO. 2. In the present invention, the vector preferably comprises an expression vector or a cloning vector, and when the vector is an expression vector, the backbone vector preferably comprises pCold II, pCold I or pUC19, more preferably comprises pCold II.

The invention also provides a transgenic cell line for expressing fructosamine-removing enzyme, and the nucleotide of the gene for encoding fructosamine-removing enzyme is shown as SEQ ID NO. 2. The type and source of the cell line are not particularly limited in the present invention, and conventional commercially available cell lines well known to those skilled in the art may be used.

The invention also provides a gene engineering bacterium for expressing fructosamine-removing enzyme, and the nucleotide of the gene for encoding fructosamine-removing enzyme is shown as SEQ ID NO. 2. In the present invention, the host bacteria include E.coli; the E.coli preferably includes E.coli BL21, DH 5. Alpha. Or Top10, more preferably E.coli BL21.

In the specific embodiment of the invention, the construction of the specific recombinant escherichia coli genetic engineering bacteria is carried out, and the construction method preferably comprises the following steps:

The fructokinase (Fructokinase) and fructosamine-desase (Fructosamine deglycase) genes were found by NCBI (National Center forBiotechnology Information) database. After finding out the gene of biological enzyme which accords with the corresponding substrate, the gene is delivered to a biological company for gene synthesis, so that the enzyme gene is obtained; and the fructokinase gene is named FRK1 and fructosamine-removing enzyme gene is named FrIB.

And (3) selecting pCold II as an escherichia coli expression vector, respectively constructing recombinant expression vectors pCold II-FRK 1 and pCold II-FrIB, respectively converting the recombinant expression plasmids pCold II-FRK 1 and pCold II-FrIB into escherichia coli JM109, and storing the recombinant expression vectors.

In a specific embodiment of the present invention, after obtaining the recombinant expression vector, the method for producing fructosamine-desaccharase preferably comprises: positive recombinant escherichia coli BL21/pCold II-FrIB of the electrotransformation linearization recombinant expression plasmid pCold II-FRK 1 or pCold II-FrIB is cultivated at 37 ℃ and 200rpm until OD ₆₀₀ =0.4-0.6, then is transferred to 15 ℃ for cultivation, and added with isopropyl beta-D-1-thiogalactopyranoside (IPTG) with the final concentration of 0.4mM, and induced to express for 24 hours at the rotating speed of 200rpm.

The invention also provides a method for co-producing glucosamine and ketocarboxylic acid based on the complex enzyme in the technical scheme, which comprises the following steps: mixing fructose and fructokinase for phosphorylation to obtain fructose-6-phosphate; mixing fructose-6-phosphate, amino acid and fructosamine enzyme, and performing transamination to obtain glucosamine and ketocarboxylic acid. In the present invention, the amino acid preferably includes alanine or glutamic acid. In the present invention, when the amino acid is alanine, the ketocarboxylic acid is pyruvic acid; when the amino acid is glutamic acid, the ketocarboxylic acid is α -ketoglutarate. The method for co-producing the glucosamine and the ketocarboxylic acid can be used for preparing the ketocarboxylic acids of different types by changing the types of amino acids.

Specifically, the invention provides a method for co-producing glucosamine and pyruvic acid based on the complex enzyme in the technical scheme, which comprises the following steps: mixing fructose and fructokinase for phosphorylation to obtain fructose-6-phosphate; mixing fructose-6-phosphate, alanine and fructosamine enzyme, and performing transamination to obtain glucosamine and pyruvic acid. The preparation method for co-producing the glucosamine and the pyruvic acid by the enzyme method not only solves the requirement of low-cost glucosamine, but also solves the requirement of the pyruvic acid as a raw material of health-care food in quantity and price. The invention utilizes a biological conversion method, also called a biological catalysis method, and utilizes extracted pure enzyme as a catalyst to complete amino conversion. Meanwhile, by adding alanine as an amino donor, not only is the amino requirement of enzyme catalysis solved, but also the alanine generates a health-care food raw material-pyruvic acid due to the transfer of amino in the preparation process. The invention produces the glucosamine and the pyruvic acid with high yield and low cost by a bioconversion method. The method of the invention improves the production efficiency by controlling the addition amount of the enzyme, reduces the generation of byproducts, obtains two products of glucosamine and pyruvic acid by simple separation, and greatly reduces the prices of the two products.

The invention provides a method for co-producing amino acid and alpha-ketoglutarate based on the complex enzyme in the technical scheme, which comprises the following steps: mixing fructose and fructokinase for phosphorylation to obtain fructose-6-phosphate; mixing fructose-6-phosphate, glutamic acid and fructosamine enzyme, and performing transamination to obtain glucosamine and alpha-ketoglutarate. According to the invention, through adding glutamic acid, the amino group for producing the glucosamine is provided by the glutamic acid, and in the enzymatic preparation process, not only the high-concentration and high-purity glucosamine is prepared, but also the glutamic acid is converted into the alpha-ketoglutaric acid with high added value. Thus, the glucosamine and the alpha-ketoglutarate are prepared by the enzyme method. Has the advantages of low production cost, high product purity, simple preparation steps and the like.

The fructosamine-removing enzyme vector, the transgenic cell line expressing fructosamine-removing enzyme, the genetically engineered bacterium and the application of fructosamine-removing enzyme according to the invention are described in further detail below with reference to specific examples, and the technical scheme of the invention includes but is not limited to the following examples.

The terms used in the present invention generally have meanings commonly understood by those of ordinary skill in the art unless otherwise indicated.

In the following examples, various processes and methods, which are not described in detail, are conventional methods well known in the art.

Example 1

Acquisition of fructokinase and fructosamine desase genes

The fructokinase (Fructokinase) and fructosamine-desase (Fructosamine deglycase) genes were first searched by NCBI (National Center for Biotechnology Information) database. Fructokinase (GenBank: ESD 97983.1) derived from Escherichia coli (ESCHERICHIA COLI 908658) and fructosamine-removing enzyme (GenBank: AOR 99552.1) derived from Bacillus subtilis were obtained by gene mining, and were subjected to gene synthesis by biological company. Wherein, the synthesized gene sequence is introduced with SnaBI and NotI restriction enzyme cutting sites (without signal peptide), the enzyme cutting sites are protective bases, and the effective sequence is the sequence after the enzyme cutting sites. And the sequence is stored in the plasmid pMD-19T to form the recombinant plasmid pMD-19T-FRK1 or pMD-19T-FrIB.

The synthesized gene sequence is transformed, namely, 10 mu l of the connection product is added into competent cells JM109, the mixture is placed on ice for 30min, immediately placed on ice after heat shock for 90s at 42 ℃, after the mixture is placed for 2min, 1ml of LB culture medium without antibiotics is added, shaking culture is carried out for 1h at 37 ℃, the transformed bacterial liquid is coated on LB (Amp) blue white plate, and the culture is carried out at 37 ℃ overnight. Multiple colonies were picked on plates for PCR identification.

At the same time, plasmid extraction was used, and the cloned plasmid identified as positive by PCR was extracted using the omega plasmid extraction kit.

Example 2

Construction, recombinant expression and protein expression of prokaryotic expression vector of fructokinase and fructosamine desase genes

1. Construction of prokaryotic expression vectors

(1) Primer design: the primers were designed starting from the mature peptide sequence following the signal peptide.

Fructosamine dehydrase primer:

Forward primer:

5’-TACGTAAATATATTGTAATATCAGATTACGT-3’(SEQ ID NO.5)

reverse primer:

5’-GCGGCCGCGTTATATAACATTATAGTCTAATGCA-3’(SEQ ID NO.6)

underlined are cleavage sites, and the enzymes used are SnaBI and NotI

Fructokinase primer:

Forward primer:

5’-TACGTAGAGAACACCGGTATTGGTGCGTCGC-3’(SEQ ID NO.7)

reverse primer:

5’-GCGGCCGCGCTCTTGTGGCCATAACCACGCAGCG-3’(SEQ ID NO.8)

underlined are cleavage sites, and the enzymes used are SnaBI and NotI

(2) PCR reactions were performed using the cloning vector pMD-19T-FRK1 or pMD-19T-FrIB as a template, annealing at 62℃and 35cycles.

(3) PCR products of corresponding biological enzyme genes of SnaBI and NotI double enzyme digestion and plasmid pCold II. The cleavage system is shown in Table 1.

Table 1 double enzyme digestion System

Composition of the components	Usage amount
		Purification of PCR products/plasmids	30μl
10*quitcutbuffer	5μl
		QuitCutSnaBI	1μl
QuitCutNotI	1μl
		ddH₂O	13μl
Total volume of	50μl

Enzyme cutting at 37deg.C for 2hr.

(4) Ligation, transformation and double restriction identification are carried out as in the conventional method.

The plasmid was extracted from this clone and full length sequencing was performed from both AOX3 and AOX5 ends to further verify the correctness of the inserted gene of interest.

2. Expression of recombinant plasmid in E.coli BL21

1) Screening of transformed E.coli and Positive transformants

According to the instruction manual of the E.coli expression system, 10. Mu.l of the ligation product was added to competent cells DE3, placed on ice for 30min, immediately placed on ice after heat shock at 42℃for 90s, placed for 2min, 1ml of LB medium without antibiotics was added, shake cultured at 37℃for 1h, and the transformed bacterial solution was spread on LB (Amp) blue white plates and cultured at 37℃overnight. Multiple colonies were picked on plates for PCR identification. The control of the empty vector transformed bacteria further verifies that the target gene has been transferred into escherichia coli BL21.

2) Inducible expression of recombinant plasmids in E.coli

The recombinant escherichia coli is cultured in a culture medium (250 mL triangular flask) of 25-50 mLLB until the OD is 0.4-0.6, then the culture is carried out at 15 ℃ and the isopropyl beta-D-1-thiogalactopyranoside (IPTG) with the final concentration of 0.4mM is added, and the induction expression is carried out for 24 hours at the rotating speed of 200rpm. Then, the cells were sonicated to extract the recombinant enzyme, and SDS-PAGE and enzyme activity measurement of the culture medium were performed simultaneously.

FIG. 1 is a double restriction map of a positive pCold II-FrIB plasmid, M is a 10000bp Marker; FIG. 2 is a double restriction map of positive pCold II-FRK 1 plasmid, M is 10000bp Marker; the two figures have obvious bands at about 1082bp and 993bp respectively, which are consistent with the theoretical value, and prove that the expression vectors of the two genes have been successfully constructed.

Example 3

Purification of fructokinase and fructosamine desase proteins

Purification of the recombinant enzyme and SDS-PAGE gel electrophoresis analysis thereof

Protein purification reference GE HEALTHCARE guide, SDS-PAGE analysis was performed according to molecular cloning Experimental guidelines (third edition) using a gel concentration of 12.5% and loading of 5-25. Mu.L. Proteins were stained with Coomassie brilliant blue R-250.

Wherein the native-SDS-PAGE experimental procedure:

A. Adding 5-10 mu L of sample buffer solution [0.1mol/L Tris (Tris-HC 1) ] into 5-10 mu L of enzyme solution, and pH 6.8;2% SDS (weight: volume), 10% glycerol (volume: volume) and 0.01% bromophenol blue (weight: volume) are placed in a water bath at 37 ℃ for 5-10 min, and then loaded for electrophoretic separation. And (3) injection: the lack of mercaptoethanol in the sample extract is used to moderately denature proteases during electrophoresis to restore the protease activity after electrophoresis. Beta-mercaptoethanol: for opening disulfide bonds, the quaternary or tertiary structure of the protein is destroyed. Is colorless transparent liquid with special odor, is inflammable, and is easy to dissolve in water, alcohol, ether and other organic solvents.

B. gel preparation and electrophoresis: adding 0.2% Gelatin in the process of preparing the separation gel, uniformly mixing, filling the gel, and solidifying to obtain the Gelatin-SDS-PAGE (substrate gel). The density of polyacrylamide in the concentrated gel is 5%, the density of polyacrylamide in the separation gel is 12%, and the thickness is 1mm ³. And (5) sample adding and running electrophoresis. And (3) injection: gelatin is added during the preparation of the gel, and therefore, is already crosslinked in the gel and does not migrate under the action of an electric field during electrophoresis.

C. SDS was removed: after electrophoresis, the separation gel is soaked in renaturation buffer solution [2% Triton X-100, 50mmol/LTris-HC1, pH 7.5] for 2-3 times, each time for 5-10 min.

D. renaturation: the separation gel was placed in a buffer [50mmol/LTris-HC1, pH7.5] and allowed to stand at 37℃for 3 hours for enzymatic reaction.

E. Dyeing and decoloring: the background was clear by staining with coomassie brilliant blue for 30min and then changing the decolorization solution (5% acetic acid +10% methanol) once for several hours. And (3) injection: the color of the gel background after dyeing and decoloring treatment is blue-black, and the color of the protease reaction part becomes light. The size of the region in the gel where protease reaction occurs and the light transmittance at that region are proportional to protease activity.

FIG. 3 is an SDS-PAGE electrophoresis of fructokinase and fructosamine-dehydrase protein purification. As shown in fig. 3, lane 1 is a protein Marker; lane 2 is an SDS-PAGE electrophoretogram of fructosamine desase FrIB; lane 3 is an SDS-PAGE electrophoretogram of fructokinase FRK 1. The two proteins had distinct bands at around 39.7kDa (fructosamine dehydrase) and 36.4kDa (fructokinase), respectively, which were consistent with theory demonstrating that both proteins have been successfully expressed and purified.

Example 4

Activity detection of purified fructokinase and fructosamine descanase

1. Activity detection of fructokinase

The enzyme activity was measured by the ELISA method, the composition of 400. Mu.L of the reaction mixture was ：30mmol/L Hepes-NaOH(pH 7.5),1mmol/LMgCl₂,0.6mmol/LNa₂EDTA,9mmol/LKCl,1mmol/LNAD,1mmol/LATP,2mmol/L fructose, 1U Glc-6-PDH (mesenteroides at fromLeuconost ℃), 1Uphosphoglucoisomerase (PGI, sigma), and the reaction was started after 80. Mu.L of the enzyme extract was added at 25℃to determine the absorbance at 340 nm. Fructokinase activity is expressed as the nad+ yield per minute, i.e., the change in nad+ absorbance.

2. Detection of fructosamine descanase Activity

The reaction system:

The 1mL reaction system contains 15mM glutamic acid, 20mM fructose-6-phosphoric acid, 0.2mL fructosamine carbohydrase, 2.5mM EDTA and 100mM buffer solutions with different pH values (Na ₂HPO₄-NaH₂PO₄, pH 2.0-10.0), the reaction system is placed in a 1.5mL centrifuge tube to be uniformly mixed, then the mixture is placed in a PCR (polymerase chain reaction) to react for 20min at 37 ℃, the reaction is stopped by heating for 5min at 95 ℃ after the reaction is finished, and the supernatant is centrifugally taken to detect the yield of alpha-ketoglutarate by using High Performance Liquid Chromatography (HPLC), so that the fructosamine carbohydrase enzyme activity is calculated.

Detection conditions:

the alpha-ketoglutaric acid was quantitatively analyzed by High Performance Liquid Chromatography (HPLC). The chromatographic column is a Hypersil BDS chromatographic column, the mobile phase is 0.1 mol.L ^-1(NH₄)H₂PO₄, the pH is 2.65, the flow rate is 1.0mL/min, the column temperature is 40 ℃, and the detection wavelength is 215nm. The content of alpha-ketoglutaric acid in the reaction liquid is calculated according to the peak area by an external standard method by taking the standard substance alpha-ketoglutaric acid as a reference.

Definition of enzyme activity:

The amount of enzyme required to catalyze the conversion of 1. Mu. Mol of glutamic acid to alpha-ketoglutarate per minute at an optimum reaction temperature of 37℃is defined as one unit of enzyme activity, i.e., 1U.

The enzyme activity of the fructokinase is 1.6221U through measurement; the enzyme activity of fructosamine dehydrase is 2.7859U.

Example 5

Synthetic reaction system for co-production of glucosamine and alpha-ketoglutaric acid

The reaction system:

1mM fructose; 100mM HEPES buffer (pH 7.0); 10mM magnesium chloride; 15mM glutamic acid; 2.5mM EDTA;10U fructosamine dehydrase; 10U fructokinase. The reaction was carried out for 1 hour in total using this reaction system. By quantitatively analyzing the glucosamine, the reaction process is controlled, and glutamic acid is converted into alpha-ketoglutarate due to amino transfer while the glucosamine is generated by the reaction.

Detection conditions:

The glucosamine was quantitatively analyzed by High Performance Liquid Chromatography (HPLC). The chromatographic column is an amino column, the mobile phase is an 80% acetonitrile water solution, the flow rate is 0.6mL/min, the column temperature is 40 ℃, and the detector is a differential refraction detector. The glucosamine retention time was about 12.7 minutes. The glucosamine concentration is proportional to the intensity of the response of the HPLC characteristic peak of glucosamine.

After the reaction time of 1 hour, it was found that the glucosamine concentration was 0.65mM by the above-mentioned experimental procedure.

Example 6

Synthesis reaction system of glucosamine and pyruvic acid

The reaction system:

1mM fructose; 100mM HEPES buffer (pH 7.0); 10mM magnesium chloride; 15mM alanine; 2.5mM EDTA;10U fructosamine dehydrase; 10U fructokinase. The reaction system is utilized to react for 1 hour in total, then detection is carried out, the end point of the synthesis reaction is determined by quantitatively analyzing the glucosamine, and finally, the health-care food raw material-pyruvic acid with natural characteristics is prepared while the glucosamine is prepared through separation.

Detection conditions:

After the reaction time of 1 hour, it was found that the glucosamine concentration was 0.78mM by the above-mentioned experimental procedure.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Sequence listing

<110> Wuhan Bai Ammonia sink Biotechnology Co., ltd

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Phe Gly Thr Leu Gln Val Leu Glu Asn Asn Thr Lys Phe Gln Gln Ala

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Ile Glu Gly Leu Asp Gln Leu Gln Ala Val Tyr Glu Lys Ala Leu Lys

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Claims

1. A method for producing glucosamine and/or ketocarboxylic acid, the method comprising:

Performing amino conversion by using fructosamine dehydrase or a transgenic cell line expressing the fructosamine dehydrase or genetically engineered bacteria expressing the fructosamine dehydrase and amino acid and saccharides as reaction substrates to obtain glucosamine and/or ketocarboxylic acid; the amino acid of fructosamine decase is shown as SEQ ID NO. 1;

The saccharide is fructose-6-phosphoric acid; the amino acid is alanine or glutamic acid; the ketocarboxylic acid is pyruvic acid or alpha-ketoglutaric acid;

The fructose-6-phosphoric acid is obtained by converting fructose; the transformation uses fructokinase, and the amino acid sequence of the fructokinase is shown as SEQ ID NO. 3.

2. The method of claim 1, wherein the conditions for amino conversion comprise: the pH value is 6-8, and the temperature is 30-50 ℃.

3. A method for co-producing glucosamine and ketocarboxylic acid based on a complex enzyme, wherein the complex enzyme is fructosamine dehydrase and fructokinase; the method comprises the following steps: mixing fructose and fructokinase for phosphorylation to obtain fructose-6-phosphate; mixing fructose-6-phosphate, amino acid and fructosamine enzyme, and performing transamination to obtain glucosamine and ketocarboxylic acid; the amino acid is alanine or glutamic acid; the ketocarboxylic acid is pyruvic acid or alpha-ketoglutaric acid;

the amino acid sequence of fructosamine dehydrase is shown as SEQ ID NO. 1; the amino acid sequence of the fructokinase is shown as SEQ ID NO. 3.