Enzymatic preparation method of glucosamine

A glucosamine and catalytic preparation technology, applied in the field of glucosamine catalyzed by biological enzymes in vitro, enzymatic preparation of glucosamine, can solve the problems of difficulty in controlling the genetic stability of engineering bacteria and high difficulty in transforming microbial metabolic pathways, and achieve production costs Low cost, cheap raw materials, environment-friendly effect

Pending Publication Date: 2021-12-21
JIANGSU AOXIN BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although this method has the advantages of not being limited by raw material sources, high production efficiency, and less environmental pollution, it also has disadvantages such as high difficulty in modifying microbial metabolic pathways, difficulty in controlling the genetic stability of engineered bacteria, and easy production of metabolic by-products.

Method used

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  • Enzymatic preparation method of glucosamine

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Embodiment 1, construction of engineered expression strain containing glucose isomerase gene and transaminase gene

[0039] According to Bacillus coagulans ( B. coagulans) of glucose isomerase, also known as xylose isomerase gene nucleotide sequence (GenBank number KYC85466) and Bacillus subtilis ( B. subtilis ) transaminase gene nucleotide sequence (GenBank number QHF59041) was synthesized separately, connected with pColdII plasmid through enzyme digestion and ligation reaction, transformed into competent cells of Escherichia coli DH5α strain, and coated with ampicillin-containing antibiotic (30μg / ml) After culturing at 37°C for 12 hours, positive transformants were picked, identified and sequenced. Inoculate the verified positive single clone into 5 mL of LB liquid medium containing 30 μg / mL ampicillin antibiotic, culture overnight at 37°C, extract two recombinant plasmids, and transform them into expression hosts E. coli In BL21(DE3), a recombinant strain was ob...

Embodiment 2

[0040] Embodiment 2, the recombinant expression preparation of glucose isomerase and transaminase

[0041] 1) Seed culture: the recombinant strain preserved on the slant E. coli BL21(DE3) / pColdII-GI and recombinant strains E. coli BL21(DE3) / pColdII-AT were respectively inoculated into LB liquid medium containing 30 μg / ml ampicillin, and cultured at 37°C for 8-10 hours to obtain seed liquid;

[0042] 2) Fermentation culture: Inoculate the seed solution with an inoculum size of 1% volume concentration into LB liquid medium containing 30 μg / ml ampicillin, and culture it at 37°C until OD 600 The value is 0.5, then transferred to 15°C for culture and added isopropyl-β-D-galactoside (IPTG) with a final concentration of 0.1mM, the rotation speed was 160 rpm, and the expression was induced for 24h. Amide gel electrophoresis (SDS-PAGE) analysis of the total protein of the whole bacteria showed that the genetically engineered bacteria had obvious recombinantly expressed protein ba...

Embodiment 3

[0043] Embodiment 3, an enzyme-catalyzed preparation method of glucosamine,

[0044] The method comprises: using the glucose isomerase prepared in Example 2 to catalyze D-glucose into D-fructose; For glucosamine.

[0045] The amino donor compound is selected from D-alanine, isopropylamine, tert-butylamine and phenethylamine; the weak oxidant compound is selected from one of phosphite, organic peroxyacid and copper oxide. The temperature of the catalytic reaction is 35°C; the pH of the catalytic reaction is 6.7. When the two catalytic reaction steps are carried out simultaneously, the catalytic reaction time is 20h. The concentration of substrate glucose in the reaction system is 8g / L; the concentration of glucose isomerase in the reaction system is 4U / mL; the concentration of transaminase is 4U / mL.

[0046] The concentration of the amino donor compound in the reaction system was 1 mM; the concentration of the weak oxidant compound was 8 mM. The reaction system contains cof...

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Abstract

The invention relates to an enzymatic preparation method of glucosamine, and belongs to the technical field of bioengineering. The method comprises the following steps: converting D-glucose into D-fructose by adopting glucose isomerase catalysis; and converting D-fructose into glucosamine using transaminase, an amino donor compound and a weak oxidant compound, wherein the glucose isomerase is derived from bacillus coagulans, flavobacterium dendrimer, streptomyces olivaceus, bacillus coagulans, flavobacterium dendrimer and streptomyces olivaceus; and the transaminase is derived from bacillus subtilis, bacillus velezensis, bacillus pumilus, bacillus licheniformis, streptococcus salivarius and lactobacillus masi. According to the method for preparing the glucosamine by catalyzing the D-glucose in vitro by utilizing the biological enzyme, the initial raw materials are glucose isomerase and transaminase required by the D-glucose or D-fructose, and the final product glucosamine can be directly obtained by constructing escherichia coli genetic engineering expression bacteria and catalyzing by adopting a 'one-pot method'. The method has the advantages of cheap raw materials, low production cost, environmental friendliness and the like.

Description

technical field [0001] The invention relates to an enzyme-catalyzed preparation method of glucosamine, in particular to a method for preparing glucosamine by biological enzyme catalysis in vitro, and belongs to the technical field of bioengineering. Background technique [0002] Glucosamine is a compound in which the 2-hydroxyl group in the D-glucose molecule is replaced by an amino group. The chemical name is 2-amino-2-deoxy-D-glucose. It is easily soluble in water and hydrophilic solvents. It is an important functional Monosaccharide. Glucosamine exists in almost all organisms including bacteria, fungi, plants and animals, and is the main component of glycoproteins and proteoglycans, as well as chitosan and chitin. [0003] Glucosamine and its derivatives are widely used and have important uses in medicine, food, cosmetics and other fields. In the pharmaceutical industry, glucosamine sulfate can be used as a raw material for the treatment of rheumatoid arthritis by stimu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/26C12P19/24C12P19/02
CPCC12P19/26C12P19/24C12P19/02
Inventor 陈延静詹金明王松叶
Owner JIANGSU AOXIN BIOTECHNOLOGY CO LTD
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