CN113584007A - Fructosamine deglycosidase vector, transgenic cell line and genetic engineering bacteria expressing fructosamine deglycosidase and application of fructosamine deglycosidase - Google Patents

Fructosamine deglycosidase vector, transgenic cell line and genetic engineering bacteria expressing fructosamine deglycosidase and application of fructosamine deglycosidase Download PDF

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CN113584007A
CN113584007A CN202110872810.6A CN202110872810A CN113584007A CN 113584007 A CN113584007 A CN 113584007A CN 202110872810 A CN202110872810 A CN 202110872810A CN 113584007 A CN113584007 A CN 113584007A
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CN113584007B (en
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王怀英
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Wuhan Baiammonia Huiji Biotechnology Co ltd
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Abstract

The invention relates to a fructosamine deglycosidase vector, a transgenic cell line expressing the fructosamine deglycosidase, a genetic engineering bacterium and application of the fructosamine deglycosidase, belonging to the technical field of production by a biological enzyme method. 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 amino donor compounds to amino acceptor compounds is catalyzed by enzymes (fructosamine desaccharidases), or expression vectors or cloning vectors expressing said enzymes, or transgenic cell lines expressing said enzymes, or genetically engineered bacteria expressing said enzymes. The method has the advantages of sufficient related substrate sources, no limitation of raw materials, mild preparation conditions, little environmental pollution, high reaction specificity, less impurities in the system, easy downstream separation and purification and low production cost.

Description

Fructosamine deglycosidase vector, transgenic cell line and genetic engineering bacteria expressing fructosamine deglycosidase and application of fructosamine deglycosidase
Technical Field
The invention relates to the technical field of production by a biological enzyme method, in particular to a fructosamine deglycosidase vector, a transgenic cell line expressing the fructosamine deglycosidase, a genetic engineering bacterium and application of the fructosamine deglycosidase.
Background
Glucosamine (GlcN), i.e. 2-amino-2-deoxy-D-glucose. Glucosamine has important physiological functions for human bodies, is widely applied to the fields of foods, health care products, medicines and the like, can be used as a substance for treating osteoarticular diseases in medical clinic, can also be used as a medicine for treating rheumatoid arthritis, and can be used as an additive in foods to be applied to infant foods. With the further improvement of quality of life and the aggravation of aging problems of the population, the global demand for medical and food nutrition health products of glucosamine is continuously increasing.
The traditional glucosamine preparation method has two types, one is the preparation method by hydrolyzing chitin, but the process has the defects that the raw materials are influenced by seasons, the hydrolysis process is complex, the acid and alkali consumption is large, the environment is polluted and the like. Secondly, the N-acetylglucosamine can be produced by a microbial fermentation method, and then the glucosamine is prepared by hydrolysis. The process of microbial fermentation has been widely used, and in recent years, the production of glucosamine has been developed by using escherichia coli, bacillus subtilis and fungi which are modified by metabolic engineering. But the yield is low, the cost is high and the refining process is complex.
Disclosure of Invention
The invention aims to provide a fructosamine deglycosidase vector, a transgenic cell line expressing the fructosamine deglycosidase, a genetically engineered bacterium and application of the fructosamine deglycosidase. The method has the advantages of sufficient related substrate sources, no limitation of raw materials, mild preparation conditions, little environmental pollution, high reaction specificity, less impurities in the system, easy 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 amino to be transferred from an amino donor compound to an amino acceptor compound by using an enzyme, or an expression vector or a cloning vector for expressing the enzyme, or a transgenic cell line for expressing the enzyme, or a genetically engineered bacterium for expressing the enzyme; the enzyme contains:
1) an amino acid sequence shown as SEQ ID NO. 1; or,
2) an amino acid sequence of an enzyme which is formed by deletion, substitution, insertion or mutation of amino acids on the basis of the amino acid sequence shown in SEQ ID No.1 and has the activity of catalyzing the transfer of amino groups 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 the application of the fructosamine deglycosidase in producing glucosamine and/or ketocarboxylic acid, wherein the amino acid sequence of the fructosamine deglycosidase is shown as SEQ ID NO. 1.
The present invention also provides a method of producing glucosamine and/or ketocarboxylic acid comprising:
amino conversion is carried out by utilizing fructosamine deglycosidase, or an expression vector or a cloning vector for expressing the fructosamine deglycosidase, or a transgenic cell line for expressing the fructosamine deglycosidase, or a genetic engineering bacterium for expressing the fructosamine deglycosidase, and amino acid and saccharide are used as reaction substrates to obtain glucosamine and/or ketocarboxylic acid; the amino acid of the fructosamine deglycosidase is shown as SEQ ID NO. 1.
Preferably, the saccharide includes 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 conversion include: the pH value is 6-8, and the temperature is 30-50 ℃.
Preferably, the enzyme used in the method further comprises fructokinase, and the amino acid sequence of the fructokinase is shown as SEQ ID NO. 3.
The invention also provides a fructosamine deglycosidase carrier, which comprises a skeleton carrier and a gene for coding the fructosamine deglycosidase; the nucleotide of the gene for coding fructosamine desugarise is shown as SEQ ID NO. 2.
The invention also provides a transgenic cell line expressing fructosamine deglycosidase, and the nucleotide of the gene encoding fructosamine deglycosidase is shown in SEQ ID NO. 2.
The invention also provides a gene engineering bacterium for expressing fructosamine deglycosidase, and the nucleotide of the gene for coding the fructosamine deglycosidase is shown as SEQ ID NO. 2.
The invention also provides a production method of fructosamine deglycosidase based on the above technical scheme, which comprises the following steps: and carrying out liquid culture and induction on the genetic engineering bacteria to obtain the fructosamine deglycosidase.
The invention also provides a complex enzyme for coproducing glucosamine and ketocarboxylic acid, which comprises fructosamine desugarise and fructokinase; the amino acid sequence of the fructosamine deglycosidase is shown as SEQ ID NO. 1; the amino acid sequence of the fructokinase is shown in SEQ ID NO. 3.
The invention also provides a method for co-producing glucosamine and ketocarboxylic acid based on the complex enzyme, which comprises the following steps: mixing fructose and fructokinase for phosphorylation to obtain fructose-6-phosphate; mixing fructose-6-phosphate, amino acid and fructosamine desugar, 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 has the advantages of sufficient related substrate sources, no limitation of raw materials, mild preparation conditions, little environmental pollution, high reaction specificity, less impurities in the system, easy downstream separation and purification and low production cost. In the specific embodiment of the present invention, the present invention has been carried out by gene sequence synthesis of fructokinase (GenBank: ESD97983.1) derived from Escherichia coli (Escherichia coli 908658) and fructosamine deglycosidase (GenBank: AOR99552.1) derived from Bacillus subtilis (Bacillus subtilis) by means of gene synthesis method, and constructed in plasmid pCold II and stored in Escherichia coli JM 109. The invention utilizes escherichia coli BL21 to successfully express in high solubility to respectively obtain the fructokinase protein and the fructosamine desugarase protein, and purifies the expressed biological enzyme protein. The invention carries out biological activity verification on the obtained recombinant fructokinase and fructosamine deglycosidase, and finds that starch, glucose, fructose and fructose-6-phosphate can be synthesized into glucosamine by sequentially carrying out phosphorylation and transamination through the fructokinase and the fructosamine deglycosidase, and simultaneously alpha-ketoglutaric acid is generated by utilizing glutamic acid, or pyruvic acid is generated by utilizing transamination of alanine. The data show that the fructokinase and the fructosamine deglycosidase have the function of well synthesizing glucosamine and pyruvate or alpha-ketoglutarate and have larger application potential. The method can greatly reduce the production cost of glucosamine and/or ketocarboxylic acid, amino acid and/or saccharide, and has important significance when being used as raw materials in the pharmaceutical industry and the food production industry.
Drawings
FIG. 1 is a diagram of a double-enzymatic cleavage of a positive pCold II-FrIB plasmid provided by the present invention;
FIG. 2 is a diagram of a double-enzymatic cleavage of the positive pCold II-FRK 1 plasmid provided by the present invention;
FIG. 3 is an SDS-PAGE electrophoresis of fructokinase and fructosamine dehydrogenase protein purification provided by the present invention.
Detailed Description
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 amino to be transferred from an amino donor compound to an amino acceptor compound by using an enzyme, or an expression vector or a cloning vector for expressing the enzyme, or a transgenic cell line for expressing the enzyme, or a genetically engineered bacterium for expressing the enzyme; the enzyme contains:
1) the amino acid sequence shown as SEQ ID NO. 1: MSQATAKVNREVQAFLQDLKGKTIDHVFFVACGGSSAIMYPSKYVFDRESKSINSDLYSANEFIQRNPVQLGEKSLVILCSHSGNTPETVKAAAFARGKGALTIAMTFKPESPLAQEAQYVAQYDWGDEALAINTNYGVLYQIVFGTLQVLENNTKFQQAIEGLDQLQAVYEKALKQEADNAKQFAKAHEKESIIYTMASGANYGVAYSYSICILMEMQWIHSHAIHAGEYFHGPFEIIDESVPFIILLGLDETRPLEERALTFSKKYGKKLTVLDAASYDFTAIDDSVKGYLAPLVLNRVLRSYADELAEERNHPLSHRRYMWKVEY, respectively; or,
2) an amino acid sequence of an enzyme which is formed by deletion, substitution, insertion or mutation of amino acids on the basis of the amino acid sequence shown in SEQ ID No.1 and has the activity of catalyzing the transfer of amino groups from an amino donor compound to an amino acceptor compound.
In the invention, the nucleotide sequence of the gene of the enzyme coded by Fructosamine deglycosidase (FrIB) is shown as SEQ ID NO. 2: TTATATAACATTATAGTCTAATGCATAATGGTTCTTCATTTTCAGATCAATACTCAACTTTCCACATGTATCTTCTATGAGATAAAGGATGATTTCTCTCCTCTGCCAGCTCGTCTGCATAGCTTCTCAGCACACGATTGAGAACGAGCGGAGCAAGATAGCCTTTAACTGAATCGTCAATTGCAGTGAAGTCGTAAGATGCAGCATCAAGCACAGTGAGCTTTTTGCCATACTTTTTCGAGAAGGTAAGCGCCCGCTCTTCAAGAGGTCTTGTTTCATCTAAACCGAGCAGGATGATAAACGGCACGGATTCATCAATAATTTCAAACGGTCCGTGAAAATATTCTCCGGCATGAATGGCGTGGGAATGAATCCATTGCATTTCCATGAGAATGCAGATGCTGTAGGAGTAAGCGACACCGTAGTTTGCACCGCTTGCCATGGTATAAATAATACTTTCTTTTTCATGGGCTTTTGCAAATTGCTTGGCGTTGTCAGCTTCCTGCTTAAGGGCTTTTTCATATACAGCCTGCAATTGATCTAAGCCTTCAATTGCTTGTTGGAATTTCGTATTGTTTTCTAATACTTGCAGGGTTCCAAAAACGATTTGATACAAAACGCCATAGTTTGTATTGATCGCAAGCGCCTCATCACCCCAATCGTACTGGGCAACATATTGCGCTTCCTGCGCTAAAGGAGACTCCGGTTTAAACGTCATCGCAATCGTAAGTGCACCCTTGCCCCTTGCAAACGCAGCAGCTTTGACTGTCTCCGGGGTATTTCCCGAATGCGAGCACAAAATAACAAGAGACTTTTCACCAAGCTGAACAGGGTTGCGCTGAATAAATTCGTTGGCGCTGTAGAGGTCGGAGTTTATTGATTTTGACTCTCTGTCAAACACATACTTACTCGGATACATAATGGCAGAAGACCCTCCGCATGCGACAAAGAATACATGATCAATGGTTTTCCCTTTCAAATCCTGCAAGAAAGCTTGAACCTCACGATTTACTTTTGCTGTGGCCTGACTCAAATCCTTCACTCCCCGTTTTTATTATATAACGTTATATAACATTATATAT are provided. The fructosamine deglycosidase of the present invention has the effect of catalyzing the transfer of an amino group from an amino donor compound to an amino acceptor compound. The fructosamine deglycosidase of the present invention performs transamination.
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 cheap raw materials, abundant sources, low production cost, environmental friendliness, safety to human bodies and the like.
The invention also provides the application of the fructosamine deglycosidase in producing glucosamine and/or ketocarboxylic acid, wherein the amino acid sequence of the fructosamine deglycosidase is shown as SEQ ID NO. 1.
The invention also provides a method for producing glucosamine and/or ketocarboxylic acid, which comprises the steps of performing amino conversion by utilizing fructosamine dehydrogenase, or an expression vector or a cloning vector for expressing the fructosamine dehydrogenase, or a transgenic cell line for expressing the fructosamine dehydrogenase, or a genetically engineered bacterium for expressing the fructosamine dehydrogenase, and taking amino acid and sugar as reaction substrates to obtain glucosamine and/or ketocarboxylic acid; the amino acid of the fructosamine deglycosidase 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, it is preferable to carry out amino conversion using fructose-6-phosphate and an amino acid as substrates. The amino group of the amino acid is transferred to fructose-6-phosphate to obtain glucosamine, and the amino group of the amino acid is removed to obtain ketocarboxylic acid. The starch, glucose and fructose of the present invention are preferably converted to fructose-6-phosphate before the above reaction. In the present invention, the amino acid preferably includes alanine, glutamic acid, aspartic acid or glutamine. After the amino group conversion, each amino acid will form the corresponding ketocarboxylic acid. In the present invention, the conditions for the amino 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 fructokinase, and the amino acid sequence of the fructokinase is shown in SEQ ID No. 3: MITNCRRPCIANPVVRLYAIDIEKNKESTVRIGIDLGGTKTEVIALGDAGEQLYRHRLPTPRDDYRQTIETIATLVDMAEQATGQRGTVGMGIPGSISPYTGVVKNANSTWLNGQPFDKDLSARLQREVRLANDANCLAVSEAVDGAAAGAQTVFAVIIGTGCGAGVAFNGRAHIGGNGTAGEWGHNPLPWMDEDELRYREEVPCYCGKQGCIETFISGTGFAMDYRRLSGHALKGSEIIRLVEESDPVAELALRRYELRLAKSLAHVVNILDPDVIVLGGGMSNVDRLYQTVGQLIKQFVFGGECETPVRKAKHGDSSGVRGAAWLWPQE, respectively; the nucleotide sequence is shown as SEQ ID NO. 4: CTCTTGTGGCCATAACCACGCAGCGCCGCGTACGCCGCTGGAATCACCGTGCTTCGCCTTACGCACCGGCGTTTCACATTCGCCGCCGAAGACAAATTGTTTAATCAACTGCCCAACCGTTTGATATAAACGGTCTACATTGCTCATCCCGCCCCCCAGGACAATCACATCCGGATCGAGAATATTCACGACATGTGCCAGCGATTTTGCCAGCCGCAGCTCGTAGCGACGCAATGCCAGTTCCGCTACCGGATCGCTTTCTTCAACCAGGCGGATAATTTCACTGCCTTTCAGCGCATGTCCGCTCAAACGACGATAATCCATCGCGAATCCCGTGCCCGAAATAAAGGTTTCAATACAACCTTGTTTACCGCAATAACAAGGGACTTCCTCGCGATAACGCAGTTCGTCTTCGTCCATCCACGGTAGCGGATTGTGTCCCCACTCACCTGCCGTGCCATTGCCGCCGATATGCGCCCGCCCATTGAATGCCACGCCCGCGCCGCATCCCGTGCCGATAATCACGGCAAATACCGTCTGCGCTCCCGCTGCCGCGCCATCTACTGCTTCTGAAACCGCCAGACAGTTAGCGTCATTTGCCAGCCGCACTTCCCGCTGCAACCTCGCGCTTAAGTCTTTATCGAATGGCTGACCGTTGAGCCAGGTTGAATTGGCATTCTTCACCACACCGGTGTAAGGCGAAATTGAGCCAGGAATGCCCATACCTACCGTTCCGCGCTGCCCCGTCGCCTGCTCCGCCATATCAACCAACGTGGCGATCGTTTCAATAGTCTGCCGGTAATCATCACGCGGCGTGGGCAGACGATGGCGGTACAACTGCTCCCCTGCATCGCCCAGTGCAATCACTTCAGTTTTGGTGCCGCCTAAATCGATACCTATACGCACGGTACTCTCCTTATTTTTTTCAATATCAATAGCGTAGAGACGGACAACCGGATTGGCAATGCAAGGCCGCCGACAATTCGTTATCAT are provided. In the present invention, when the saccharide is fructose, the catalytic system of the present invention preferably contains fructosamine deglycosidase and fructokinase, the use of which can convert fructose into fructose-6-phosphate, and further perform the above-described subsequent reaction.
The invention also provides a fructosamine deglycosidase carrier, which comprises a skeleton carrier and a gene for coding the fructosamine deglycosidase; the nucleotide of the gene for coding fructosamine desugarise is shown as SEQ ID NO. 2. In the present invention, the vector preferably includes an expression vector or a cloning vector, and when the vector is an expression vector, the backbone vector preferably includes pCold ii, pCold i or pUC19, and more preferably includes pCold ii.
The invention also provides a transgenic cell line expressing fructosamine deglycosidase, and the nucleotide of the gene encoding fructosamine deglycosidase is shown in SEQ ID NO. 2. The present invention is not particularly limited in kind and source of the cell line, and a conventional commercially available cell line well known to those skilled in the art may be used.
The invention also provides a gene engineering bacterium for expressing fructosamine deglycosidase, and the nucleotide of the gene for coding the fructosamine deglycosidase is shown as SEQ ID NO. 2. In the present invention, the host bacterium includes Escherichia coli; the Escherichia coli preferably includes Escherichia coli BL21, DH5 α or Top10, more preferably Escherichia coli BL 21.
In the 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:
fructokinase (Fructokinase) and Fructosamine deglycosidase (Fructosamine deglycosidase) genes were searched for by NCBI (national Center for Biotechnology information) database. After finding out the gene of the biological enzyme which accords with the corresponding substrate, handing the gene to a biological company for gene synthesis so as to obtain the enzyme gene; the fructokinase gene is named as FRK1, and the fructosamine desugarine gene is named as FrIB.
Selecting pCold II as an Escherichia coli expression vector, respectively constructing recombinant expression vectors pCold II-FRK 1 and pCold II-FrIB, and respectively transforming the recombinant expression plasmids pCold II-FRK 1 and pCold II-FrIB into Escherichia coli JM109 for storing the recombinant expression vectors.
The invention also provides a production method of fructosamine deglycosidase based on the above technical scheme, which comprises the following steps: and carrying out liquid culture and induction on the genetic engineering bacteria to obtain the fructosamine deglycosidase.
In a specific embodiment of the present invention, after obtaining the recombinant expression vector, the method for producing fructosamine deglycosidase preferably comprises: electrically transformed positive recombinant Escherichia coli BL21/pCold II-FrIB with linearized recombinant expression plasmid pCold II-FRK 1 or pCold II-FrIB was cultured at 37 deg.C and 200rpm to OD600Then transferred to 15 ℃ for culture, added with isoproyl beta-D-1-thiogalactopyranoside (IPTG) with the final concentration of 0.4mM, and induced for expression for 24h at the rotating speed of 200 rpm.
The invention also provides a complex enzyme for coproducing glucosamine and ketocarboxylic acid, which comprises fructosamine desugarise and fructokinase; the amino acid sequence of the fructosamine deglycosidase is shown as SEQ ID NO. 1; the amino acid sequence of the fructokinase is shown in SEQ ID NO. 3.
The invention also provides a method for co-producing glucosamine and ketocarboxylic acid based on the complex enzyme, which comprises the following steps: mixing fructose and fructokinase for phosphorylation to obtain fructose-6-phosphate; mixing fructose-6-phosphate, amino acid and fructosamine desugar, 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 alpha-ketoglutaric acid. The method for co-producing glucosamine and ketocarboxylic acid can realize the preparation of ketocarboxylic acid with different types by changing the types of amino acid.
Specifically, the invention provides a method for co-producing glucosamine and pyruvic acid based on the complex enzyme, which comprises the following steps: mixing fructose and fructokinase for phosphorylation to obtain fructose-6-phosphate; mixing fructose-6-phosphate, alanine and fructosamine desugarise, and performing transamination to obtain glucosamine and pyruvic acid. The preparation method for the enzyme method to coproduce the glucosamine and the pyruvic acid not only solves the requirement of the glucosamine with low cost, but also solves the quantity and price requirements of the pyruvic acid as the raw material of the health food. The invention utilizes a biotransformation method, also called a biocatalysis method, and utilizes the extracted pure enzyme as a catalyst to complete the amino conversion. Simultaneously, the alanine is added as an amino donor, so that the requirement of enzyme catalysis on the amino is met, and the alanine generates a health food raw material, namely pyruvic acid, due to the transfer of the amino in the preparation process. The invention produces the glucosamine and the pyruvic acid with high yield and low cost by a biotransformation method. The method 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 price of the two products.
The invention provides a method for co-producing amino acid and alpha-ketoglutaric acid based on the complex enzyme, which comprises the following steps: mixing fructose and fructokinase for phosphorylation to obtain fructose-6-phosphate; mixing fructose-6-phosphate, glutamic acid and fructosamine deglycosidase, and performing transamination to obtain glucosamine and alpha-ketoglutaric acid. According to the invention, glutamic acid is added to provide amino for producing glucosamine, and during the preparation process of the enzyme method, the glucosamine with high concentration and high purity is prepared, and the glutamic acid is converted to generate the alpha-ketoglutaric acid with high added value. Thus, the enzyme method coproduces glucosamine and alpha-ketoglutaric acid. Has the advantages of low production cost, high product purity, simple preparation steps and the like.
The fructosamine deglycosidase vector, the transgenic cell line and the genetically engineered bacteria expressing the fructosamine deglycosidase, and the application of the fructosamine deglycosidase of the present invention will be described in further detail with reference to the following specific examples, and the technical solutions of the present invention include, but are not limited to, the following examples.
Terms used in the present invention have generally meanings as commonly understood by one of ordinary skill in the art, unless otherwise specified.
In the following examples, various procedures and methods not described in detail are conventional methods well known in the art.
Example 1
Obtaining of fructokinase and fructosamine desugar enzyme genes
The Fructokinase (Fructokinase) and Fructosamine deglycosidase (Fructosamine deglycosidase) genes were first searched for by the NCBI (national Center for Biotechnology information) database. Fructokinase (GenBank: ESD97983.1) derived from Escherichia coli (Escherichia coli 908658) and fructosamine deglycosidase (GenBank: AOR99552.1) derived from Bacillus subtilis (Bacillus subtilis) were obtained by gene excavation and submitted to a bio-company for gene synthesis. Wherein SnaBI and NotI restriction enzyme sites (without signal peptide) are introduced into the synthesized gene sequence, the restriction enzyme sites are protection bases, and the effective sequence is a sequence behind the restriction enzyme sites. And the sequence was stored in plasmid pMD-19T to form recombinant plasmid pMD-19T-FRK1 or pMD-19T-FrIB.
And (3) transforming the synthesized gene sequence, namely adding 10 mu l of the ligation product into a competent cell JM109, placing on ice for 30min, immediately placing on ice after heat shock for 90s at 42 ℃, placing for 2min, adding 1ml of LB culture medium without antibiotics, performing shake culture at 37 ℃ for 1h, coating the transformed bacterial liquid on an LB (Amp) blue-white plate, and performing culture at 37 ℃ overnight. A plurality of colonies were picked on the plate for PCR identification.
Meanwhile, plasmid extraction is used, and a plasmid extraction kit of an omega company is used for extracting clone plasmids which are identified as positive by PCR.
Example 2
Construction of prokaryotic expression vector of fructokinase and fructosamine desugar enzyme gene, recombinant expression and protein expression thereof
1. Construction of prokaryotic expression vector
(1) Designing a primer: primers were designed starting from the mature peptide sequence following the signal peptide.
Fructosamine desugarise primers:
a forward primer:
5’-TACGTAAATATATTGTAATATCAGATTACGT-3’(SEQ ID NO.5)
reverse primer:
5’-GCGGCCGCGTTATATAACATTATAGTCTAATGCA-3’(SEQ ID NO.6)
the restriction sites are underlined and the enzymes used are SnaB I and Not I
Fructokinase primer:
a forward primer:
5’-TACGTAGAGAACACCGGTATTGGTGCGTCGC-3’(SEQ ID NO.7)
reverse primer:
5’-GCGGCCGCGCTCTTGTGGCCATAACCACGCAGCG-3’(SEQ ID NO.8)
the restriction sites are underlined and the enzymes used are SnaB I and Not I
(2) PCR reaction, using cloning vector pMD-19T-FRK1 or pMD-19T-FrIB as template, annealing at 62 deg.C, 35 cycles.
(3) PCR products of the corresponding bioenzyme genes digested by SnaBI and NotI and plasmid pCold II. The cleavage system is shown in Table 1.
TABLE 1 double enzyme digestion System
Composition (I) Amount of the composition used
Purification of PCR products/plasmids 30μl
10*quitcutbuffer 5μl
QuitCutSnaBI 1μl
QuitCutNotI 1μl
ddH2O 13μl
Total volume 50μl
The enzyme was cleaved at 37 ℃ for 2 hr.
(4) Ligation, transformation and double-enzyme cleavage identification were performed as in the conventional manner.
The plasmid is extracted from the clone, and full-length sequencing is carried out on both ends of AOX3 and AOX5, so that the correctness of the inserted target gene is further verified.
2. Expression of recombinant plasmid in Escherichia coli BL21
1) Transformation of Escherichia coli and screening of Positive transformants
According to the operation manual of an escherichia coli expression system, 10 mul of the ligation product is added into a competent cell DE3, the cell is placed on ice for 30min, immediately placed on ice after being thermally shocked for 90s at 42 ℃, placed for 2min, added with 1ml of LB culture medium without antibiotics, subjected to shake cultivation for 1h at 37 ℃, and the transformed bacterial liquid is coated on an LB (Amp) blue white plate and cultured overnight at 37 ℃. A plurality of colonies were picked on the plate for PCR identification. The empty vector transformation strain is used as a control, and the target gene is further verified to be transferred into escherichia coli BL 21.
2) Inducible expression of recombinant plasmids in E.coli
The recombinant Escherichia coli is cultured in a 25-50 mLLB culture medium (250mL triangular flask) until the OD is 0.4-0.6, then transferred to 15 ℃ for culture, added with isoproyl beta-D-1-thiogalactopyranoside (IPTG) with the final concentration of 0.4mM, and induced for expression for 24h at the rotating speed of 200 rpm. Then, the cells were disrupted by ultrasonic waves to extract a recombinant enzyme, and SDS-PAGE of the culture solution and enzyme activity measurement were carried out simultaneously.
FIG. 1 is a diagram of a positive pCold II-FrIB plasmid double-enzyme cleavage, M being a Marker of 10000 bp; FIG. 2 is a diagram of a positive pCold II-FRK 1 plasmid double-enzyme cutting, M is a Marker of 10000 bp; the two images have obvious bands at about 1082bp and 993bp respectively, which are consistent with theoretical values, and prove that the expression vectors of the two genes are successfully constructed.
Example 3
Purification of fructokinase and fructosamine desugarase proteins
Purification of recombinant enzymes and SDS-PAGE gel electrophoresis analysis thereof
Protein purification reference is made to the GE Healthcare guide, SDS-PAGE analysis according to the molecular cloning Experimental guide (third edition), using a gel concentration of 12.5% and a loading of 5-25. mu.L. The protein was stained with Coomassie Brilliant blue R-250.
Wherein the native-SDS-PAGE experimental steps are as follows:
A. adding 5-10 μ L sample buffer [0.1mol/L Tris-HC1 ] with pH of 6.8 into 5-10 μ L enzyme solution; placing 2% SDS (weight: volume), 10% glycerol (volume: volume) and 0.01% bromophenol blue (weight: volume) in water bath at 37 ℃ for 5-10 min, and carrying out loading electrophoretic separation. Note: the reason why mercaptoethanol is not added to the sample extract during sample extraction is to moderately denature proteases during electrophoresis so that the activities of the proteases can be recovered after electrophoresis is finished. Beta-mercaptoethanol: for opening disulfide bonds, the quaternary or tertiary structure of the protein is disrupted. Is a colorless transparent liquid with special odor, is flammable and easily soluble in water, alcohol, ether and other organic solvents.
B. Preparing glue and performing electrophoresis: adding 0.2% Gelatin in the process of preparing the separation gel, uniformly mixing, then pouring the gel, and solidifying to obtain Gelatin-SDS-PAGE (substrate gel). The polyacrylamide density of the concentrated gel is 5%, the polyacrylamide density of the separation gel is 12%, and the thickness of the separation gel is 1mm3. And (4) sample loading and running electrophoresis. Note: gelatin is added during the preparation of the gel, so that the gelatin is crosslinked in the gel and cannot migrate under the action of an electric field during electrophoresis.
C. And (3) SDS removal: after electrophoresis, the separation gel is soaked and washed for 2-3 times, 5-10 min each time, in a renaturation buffer solution [ 2% TritonX-100, 50mmol/LTris-HC1, pH7.5 ].
D. Renaturation: the gel was incubated in a buffer [50mmol/LTris-HC1, pH7.5] at 37 ℃ for 3 hours.
E. Dyeing and decoloring: staining with Coomassie Brilliant blue for 30min, followed by a change of destaining solution (5% acetic acid + 10% methanol) for several hours until the background is clear. Note: the background color of the dyed and decolored gel is blue black, and the color of the protease reaction part is lightened. The size of the region in the gel in which the protease reaction is exhibited and the light transmittance at that site are proportional to the protease activity.
FIG. 3 is an SDS-PAGE electrophoresis of fructokinase and fructosamine deglycosidase protein purification. As shown in fig. 3, lane 1 is a protein Marker; lane 2 is an SDS-PAGE electrophoresis of fructosamine dehydrogenase FrIB; lane 3 is an SDS-PAGE of fructokinase FRK 1. The two proteins showed distinct bands around 39.7kDa (fructosamine deglycosidase) and 36.4kDa (fructokinase), respectively, which are consistent with theoretical values, demonstrating that both proteins have been successfully expressed and purified.
Example 4
Activity detection of purified fructokinase and fructosamine deglycosidase
1. Detection of fructokinase Activity
The enzyme activity was measured by enzyme-linked assay, and 400. mu.L of the reaction solution consisted of: 30mmol/L Hepes-NaOH (pH 7.5), 1mmol/LMgCl2，0.6mmol/LNa2EDTA, 9mmol/LKCl, 1mmol/LNAD, 1mmol/LATP, 2mmol/L fructose, 1U of Glc-6-P DH (from Leuconost ℃ C. sensteroides), 1Uphos gluco isomerase (PGI, sigma), the reaction was started after adding 80. mu.L of the enzyme extract at 25 ℃ and the absorbance at 340nm was measured. Fructokinase activity is expressed as the amount of NAD + produced per minute, i.e., the change in NAD + absorbance.
2. Activity detection of fructosamine deglycosidase
Reaction system:
1mL of the reaction system contained 15mM of glutamic acid, 20mM of fructose-6-phosphate, 0.2mL of fructosamine deglycosidase, 2.5mM of EDTA and 100mM of buffer solutions (Na) of various pH values2HPO4-NaH2PO4And the pH value is 2.0-10.0), the reaction system is placed in a 1.5mL centrifuge tube to be mixed uniformly, then the mixture is placed in a PCR to react for 20min at 37 ℃, the reaction is stopped after the reaction is finished and heated for 5min at 95 ℃, the reaction is stopped, the supernatant is centrifuged, and the yield of alpha-ketoglutaric acid is detected by using a High Performance Liquid Chromatography (HPLC), so that the fructosamine desugarise enzyme activity is calculated.
Detection conditions are as follows:
high Performance Liquid Chromatography (HPLC) was used for quantitative analysis of alpha-ketoglutaric acid. The chromatographic column is Hypersil BDS chromatographic column, and the mobile phase is 0.1 mol.L-1(NH4)H2PO4The pH was 2.65, the flow rate was 1.0mL/min, the column temperature was 40 ℃ and the detection wavelength was 215 nm. And (3) calculating the content of the alpha-ketoglutaric acid in the reaction liquid by peak area according to an external standard method by taking the standard 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-ketoglutaric acid per minute at an optimum reaction temperature of 37 ℃ is defined as one unit of enzyme activity, i.e., 1U.
Through determination, the enzyme activity of the fructokinase is 1.6221U; the enzymatic activity of fructosamine deglycosidase is 2.7859U.
Example 5
Synthetic reaction system for co-production of glucosamine and alpha-ketoglutaric acid
Reaction system:
1mM fructose; 100mM HEPES buffer (pH 7.0); 10mM magnesium chloride; 15mM glutamic acid; 2.5mM EDTA; 10U fructosamine deglycosidase; 10U of fructokinase. The reaction was carried out for 1 hour in total using this reaction system. Through the quantitative analysis of glucosamine, the reaction process is controlled, and glutamic acid is converted into alpha-ketoglutaric acid due to transamination while the glucosamine is generated through the reaction.
Detection conditions are as follows:
glucosamine was quantitatively analyzed by High Performance Liquid Chromatography (HPLC). The chromatographic column is an amino column, the mobile phase is 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. Glucosamine concentration is proportional to the response intensity of the HPLC characteristic peak of glucosamine.
Through the above experimental process, it was detected that glucosamine was produced at 0.65mM after 1 hour of the reaction.
Example 6
Synthesis reaction system of glucosamine and pyruvic acid
Reaction system:
1mM fructose; 100mM HEPES buffer (pH 7.0); 10mM magnesium chloride; 15mM alanine; 2.5mM EDTA; 10U fructosamine deglycosidase; 10U of fructokinase. The reaction system is used for detecting after reacting for 1 hour totally, the end point of the synthetic reaction is determined by quantitatively analyzing the glucosamine, and finally, the pyruvic acid which is a health food raw material with natural characteristics is prepared while the glucosamine is prepared by separation.
Detection conditions are as follows:
glucosamine was quantitatively analyzed by High Performance Liquid Chromatography (HPLC). The chromatographic column is an amino column, the mobile phase is 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. Glucosamine concentration is proportional to the response intensity of the HPLC characteristic peak of glucosamine.
Through the above experimental process, it was detected that glucosamine was produced at 0.78mM after 1 hour of the reaction.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> Wuhan Bai-Amiki Biotechnology Co., Ltd
<120> fructosamine deglycosidase vector, transgenic cell line and genetic engineering bacteria expressing fructosamine deglycosidase, and application of fructosamine deglycosidase
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<211> 328
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<213> Artificial Sequence (Artificial Sequence)
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Met Ser Gln Ala Thr Ala Lys Val Asn Arg Glu Val Gln Ala Phe Leu
1 5 10 15
Gln Asp Leu Lys Gly Lys Thr Ile Asp His Val Phe Phe Val Ala Cys
20 25 30
Gly Gly Ser Ser Ala Ile Met Tyr Pro Ser Lys Tyr Val Phe Asp Arg
35 40 45
Glu Ser Lys Ser Ile Asn Ser Asp Leu Tyr Ser Ala Asn Glu Phe Ile
50 55 60
Gln Arg Asn Pro Val Gln Leu Gly Glu Lys Ser Leu Val Ile Leu Cys
65 70 75 80
Ser His Ser Gly Asn Thr Pro Glu Thr Val Lys Ala Ala Ala Phe Ala
85 90 95
Arg Gly Lys Gly Ala Leu Thr Ile Ala Met Thr Phe Lys Pro Glu Ser
100 105 110
Pro Leu Ala Gln Glu Ala Gln Tyr Val Ala Gln Tyr Asp Trp Gly Asp
115 120 125
Glu Ala Leu Ala Ile Asn Thr Asn Tyr Gly Val Leu Tyr Gln Ile Val
130 135 140
Phe Gly Thr Leu Gln Val Leu Glu Asn Asn Thr Lys Phe Gln Gln Ala
145 150 155 160
Ile Glu Gly Leu Asp Gln Leu Gln Ala Val Tyr Glu Lys Ala Leu Lys
165 170 175
Gln Glu Ala Asp Asn Ala Lys Gln Phe Ala Lys Ala His Glu Lys Glu
180 185 190
Ser Ile Ile Tyr Thr Met Ala Ser Gly Ala Asn Tyr Gly Val Ala Tyr
195 200 205
Ser Tyr Ser Ile Cys Ile Leu Met Glu Met Gln Trp Ile His Ser His
210 215 220
Ala Ile His Ala Gly Glu Tyr Phe His Gly Pro Phe Glu Ile Ile Asp
225 230 235 240
Glu Ser Val Pro Phe Ile Ile Leu Leu Gly Leu Asp Glu Thr Arg Pro
245 250 255
Leu Glu Glu Arg Ala Leu Thr Phe Ser Lys Lys Tyr Gly Lys Lys Leu
260 265 270
Thr Val Leu Asp Ala Ala Ser Tyr Asp Phe Thr Ala Ile Asp Asp Ser
275 280 285
Val Lys Gly Tyr Leu Ala Pro Leu Val Leu Asn Arg Val Leu Arg Ser
290 295 300
Tyr Ala Asp Glu Leu Ala Glu Glu Arg Asn His Pro Leu Ser His Arg
305 310 315 320
Arg Tyr Met Trp Lys Val Glu Tyr
325
<210> 2
<211> 1082
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
ttatataaca ttatagtcta atgcataatg gttcttcatt ttcagatcaa tactcaactt 60
tccacatgta tcttctatga gataaaggat gatttctctc ctctgccagc tcgtctgcat 120
agcttctcag cacacgattg agaacgagcg gagcaagata gcctttaact gaatcgtcaa 180
ttgcagtgaa gtcgtaagat gcagcatcaa gcacagtgag ctttttgcca tactttttcg 240
agaaggtaag cgcccgctct tcaagaggtc ttgtttcatc taaaccgagc aggatgataa 300
acggcacgga ttcatcaata atttcaaacg gtccgtgaaa atattctccg gcatgaatgg 360
cgtgggaatg aatccattgc atttccatga gaatgcagat gctgtaggag taagcgacac 420
cgtagtttgc accgcttgcc atggtataaa taatactttc tttttcatgg gcttttgcaa 480
attgcttggc gttgtcagct tcctgcttaa gggctttttc atatacagcc tgcaattgat 540
ctaagccttc aattgcttgt tggaatttcg tattgttttc taatacttgc agggttccaa 600
aaacgatttg atacaaaacg ccatagtttg tattgatcgc aagcgcctca tcaccccaat 660
cgtactgggc aacatattgc gcttcctgcg ctaaaggaga ctccggttta aacgtcatcg 720
caatcgtaag tgcacccttg ccccttgcaa acgcagcagc tttgactgtc tccggggtat 780
ttcccgaatg cgagcacaaa ataacaagag acttttcacc aagctgaaca gggttgcgct 840
gaataaattc gttggcgctg tagaggtcgg agtttattga ttttgactct ctgtcaaaca 900
catacttact cggatacata atggcagaag accctccgca tgcgacaaag aatacatgat 960
caatggtttt ccctttcaaa tcctgcaaga aagcttgaac ctcacgattt acttttgctg 1020
tggcctgact caaatccttc actccccgtt tttattatat aacgttatat aacattatat 1080
at 1082
<210> 3
<211> 331
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 3
Met Ile Thr Asn Cys Arg Arg Pro Cys Ile Ala Asn Pro Val Val Arg
1 5 10 15
Leu Tyr Ala Ile Asp Ile Glu Lys Asn Lys Glu Ser Thr Val Arg Ile
20 25 30
Gly Ile Asp Leu Gly Gly Thr Lys Thr Glu Val Ile Ala Leu Gly Asp
35 40 45
Ala Gly Glu Gln Leu Tyr Arg His Arg Leu Pro Thr Pro Arg Asp Asp
50 55 60
Tyr Arg Gln Thr Ile Glu Thr Ile Ala Thr Leu Val Asp Met Ala Glu
65 70 75 80
Gln Ala Thr Gly Gln Arg Gly Thr Val Gly Met Gly Ile Pro Gly Ser
85 90 95
Ile Ser Pro Tyr Thr Gly Val Val Lys Asn Ala Asn Ser Thr Trp Leu
100 105 110
Asn Gly Gln Pro Phe Asp Lys Asp Leu Ser Ala Arg Leu Gln Arg Glu
115 120 125
Val Arg Leu Ala Asn Asp Ala Asn Cys Leu Ala Val Ser Glu Ala Val
130 135 140
Asp Gly Ala Ala Ala Gly Ala Gln Thr Val Phe Ala Val Ile Ile Gly
145 150 155 160
Thr Gly Cys Gly Ala Gly Val Ala Phe Asn Gly Arg Ala His Ile Gly
165 170 175
Gly Asn Gly Thr Ala Gly Glu Trp Gly His Asn Pro Leu Pro Trp Met
180 185 190
Asp Glu Asp Glu Leu Arg Tyr Arg Glu Glu Val Pro Cys Tyr Cys Gly
195 200 205
Lys Gln Gly Cys Ile Glu Thr Phe Ile Ser Gly Thr Gly Phe Ala Met
210 215 220
Asp Tyr Arg Arg Leu Ser Gly His Ala Leu Lys Gly Ser Glu Ile Ile
225 230 235 240
Arg Leu Val Glu Glu Ser Asp Pro Val Ala Glu Leu Ala Leu Arg Arg
245 250 255
Tyr Glu Leu Arg Leu Ala Lys Ser Leu Ala His Val Val Asn Ile Leu
260 265 270
Asp Pro Asp Val Ile Val Leu Gly Gly Gly Met Ser Asn Val Asp Arg
275 280 285
Leu Tyr Gln Thr Val Gly Gln Leu Ile Lys Gln Phe Val Phe Gly Gly
290 295 300
Glu Cys Glu Thr Pro Val Arg Lys Ala Lys His Gly Asp Ser Ser Gly
305 310 315 320
Val Arg Gly Ala Ala Trp Leu Trp Pro Gln Glu
325 330
<210> 4
<211> 993
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
ctcttgtggc cataaccacg cagcgccgcg tacgccgctg gaatcaccgt gcttcgcctt 60
acgcaccggc gtttcacatt cgccgccgaa gacaaattgt ttaatcaact gcccaaccgt 120
ttgatataaa cggtctacat tgctcatccc gccccccagg acaatcacat ccggatcgag 180
aatattcacg acatgtgcca gcgattttgc cagccgcagc tcgtagcgac gcaatgccag 240
ttccgctacc ggatcgcttt cttcaaccag gcggataatt tcactgcctt tcagcgcatg 300
tccgctcaaa cgacgataat ccatcgcgaa tcccgtgccc gaaataaagg tttcaataca 360
accttgttta ccgcaataac aagggacttc ctcgcgataa cgcagttcgt cttcgtccat 420
ccacggtagc ggattgtgtc cccactcacc tgccgtgcca ttgccgccga tatgcgcccg 480
cccattgaat gccacgcccg cgccgcatcc cgtgccgata atcacggcaa ataccgtctg 540
cgctcccgct gccgcgccat ctactgcttc tgaaaccgcc agacagttag cgtcatttgc 600
cagccgcact tcccgctgca acctcgcgct taagtcttta tcgaatggct gaccgttgag 660
ccaggttgaa ttggcattct tcaccacacc ggtgtaaggc gaaattgagc caggaatgcc 720
catacctacc gttccgcgct gccccgtcgc ctgctccgcc atatcaacca acgtggcgat 780
cgtttcaata gtctgccggt aatcatcacg cggcgtgggc agacgatggc ggtacaactg 840
ctcccctgca tcgcccagtg caatcacttc agttttggtg ccgcctaaat cgatacctat 900
acgcacggta ctctccttat ttttttcaat atcaatagcg tagagacgga caaccggatt 960
ggcaatgcaa ggccgccgac aattcgttat cat 993
<210> 5
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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tacgtaaata tattgtaata tcagattacg t 31
<210> 6
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<213> Artificial Sequence (Artificial Sequence)
<400> 6
gcggccgcgt tatataacat tatagtctaa tgca 34
<210> 7
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
tacgtagaga acaccggtat tggtgcgtcg c 31
<210> 8
<211> 34
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
gcggccgcgc tcttgtggcc ataaccacgc agcg 34

Claims (15)

1. A method of catalyzing the transfer of an amino group from an amino donor compound to an amino acceptor compound, the method comprising: catalyzing amino to be transferred from an amino donor compound to an amino acceptor compound by using an enzyme, or an expression vector or a cloning vector for expressing the enzyme, or a transgenic cell line for expressing the enzyme, or a genetically engineered bacterium for expressing the enzyme; the enzyme contains:
1) an amino acid sequence shown as SEQ ID NO. 1; or,
2) an amino acid sequence of an enzyme which is formed by deletion, substitution, insertion or mutation of amino acids on the basis of the amino acid sequence shown in SEQ ID No.1 and has the activity of catalyzing the transfer of amino groups from an amino donor compound to an amino acceptor compound.
2. The method of claim 1, wherein the amino donor compound comprises an amino acid.
3. The method of claim 1 or 2, wherein the amino acceptor compound comprises a saccharide.
4. Use of a fructosamine deglycosidase having an amino acid sequence as set forth in SEQ ID No.1 for the production of glucosamine and/or ketocarboxylic acids.
5. A method of producing glucosamine and/or ketocarboxylic acid comprising:
amino conversion is carried out by utilizing fructosamine deglycosidase, or an expression vector or a cloning vector for expressing the fructosamine deglycosidase, or a transgenic cell line for expressing the fructosamine deglycosidase, or a genetic engineering bacterium for expressing the fructosamine deglycosidase, and amino acid and saccharide are used as reaction substrates to obtain glucosamine and/or ketocarboxylic acid; the amino acid of the fructosamine deglycosidase is shown as SEQ ID NO. 1.
6. The method according to claim 5, wherein the saccharide comprises one or more of starch, glucose, fructose, and fructose-6-phosphate.
7. The use of claim 5 or 6, wherein the amino acid comprises alanine, glutamic acid, aspartic acid or glutamine.
8. The method of claim 5, wherein the amino conversion conditions comprise: the pH value is 6-8, and the temperature is 30-50 ℃.
9. The method according to claim 5, wherein the enzyme used in the method further comprises fructokinase, the amino acid sequence of which is shown in SEQ ID No. 3.
10. A fructosamine deglycosidase vector, characterized in that, the vector comprises skeleton vector and gene encoding fructosamine deglycosidase; the nucleotide of the gene for coding fructosamine desugarise is shown as SEQ ID NO. 2.
11. A transgenic cell line expressing fructosamine deglycosidase, the nucleotide of the gene encoding fructosamine deglycosidase is shown in SEQ ID NO. 2.
12. A gene engineering bacterium for expressing fructosamine deglycosidase, the nucleotide of the gene coding for fructosamine deglycosidase is shown in SEQ ID NO. 2.
13. The method for producing fructosamine deglycosidase based on the genetically engineered bacterium of claim 12, comprising the steps of: and carrying out liquid culture and induction on the genetic engineering bacteria to obtain the fructosamine deglycosidase.
14. A complex enzyme for co-producing glucosamine and ketocarboxylic acid, which is characterized by comprising fructosamine deglycosidase and fructokinase; the amino acid sequence of the fructosamine deglycosidase is shown as SEQ ID NO. 1; the amino acid sequence of the fructokinase is shown in SEQ ID NO. 3.
15. A process for co-producing glucosamine and ketocarboxylic acid based on the complex enzyme of claim 14, comprising the steps of: mixing fructose and fructokinase for phosphorylation to obtain fructose-6-phosphate; mixing fructose-6-phosphate, amino acid and fructosamine desugar, and performing transamination to obtain glucosamine and ketocarboxylic acid.
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Citations (3)

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Publication number Priority date Publication date Assignee Title
US5387109A (en) * 1992-06-05 1995-02-07 Nakano Vinegar Co., Ltd. Fructosylamine deglycase and a method of producing it
US20110143394A1 (en) * 2006-02-14 2011-06-16 Universiteit Leiden Methods and Means for Metabolic Engineering and Improved Product Formation by Micro-Organisms
CN112852774A (en) * 2019-11-28 2021-05-28 中国科学院大连化学物理研究所 Phosphofructosyl aminotransferase encoding gene and preparation and application of enzyme

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Publication number Priority date Publication date Assignee Title
US5387109A (en) * 1992-06-05 1995-02-07 Nakano Vinegar Co., Ltd. Fructosylamine deglycase and a method of producing it
US20110143394A1 (en) * 2006-02-14 2011-06-16 Universiteit Leiden Methods and Means for Metabolic Engineering and Improved Product Formation by Micro-Organisms
CN112852774A (en) * 2019-11-28 2021-05-28 中国科学院大连化学物理研究所 Phosphofructosyl aminotransferase encoding gene and preparation and application of enzyme

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Title
"fructokinase [Escherichia coli 908658]"，GenBank: ESD97983.1", 《GENBANK》 *
"Fructosamine deglycase FrlB [Bacillus subtilis]"，GenBank: AOR99552.1", 《GENBANK》 *
ELSA WIAME等: "Identification of enzymes acting on alpha-glycated amino acids in Bacillus subtilis", 《FEBS LETT.》, vol. 577, no. 3, pages 470 *

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