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Glucosamine-6 phosphate synthase mutant

A technology of phosphate synthase and glucosamine, which is applied in the field of glucosamine-6 ​​phosphate synthase mutants, can solve the problem of low catalytic activity and achieve the effect of improving catalytic activity

Pending Publication Date: 2019-08-23
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The glucosamine-6 ​​phosphate synthase is a double substrate binding enzyme belonging to the aminotransferase class with low catalytic activity

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] Example 1 Determination of Mutation Sites

[0016] The catalytic mechanism of GlmS follows an ordered reaction mechanism. Fructose 6-phosphate is first combined with glutamine. With the hydrolysis of glutamine, amino transfer and isomerization of amino sugar, GlmS releases glutamic acid and GlcN-6- p. The GlmS of Escherichia coli is a homodimer, the isomerase domains on two different monomers are inside the dimer, and the outside of the dimer are two glutamine-binding domains. G471, R249, and A38 were identified as possible enzyme activity-enhancing sites by analyzing the PDB crystal structure of GlmS derived from E. coli (PDB code 1JXA).

Embodiment 2

[0017] The preparation of embodiment 2 mutant G471S, R249W, A38E

[0018] (1) Site-directed mutation

[0019] Using rapid PCR technology, the plasmid pET28a-glmS-gna1 expressing the wild-type GlmS gene (see CN103589696A for the construction method of pET28a-glmS-gna1) was used as a template to perform site-directed mutation.

[0020] Primers for introducing the G471S mutation:

[0021] Forward primer: 5'-GCTGTTCCTGGCCGTGGCGATC-3'

[0022] Reverse primer: 5'-GATCGCCACGGCCAGGAACAGC-3'

[0023] Primers for introducing the R249W mutation

[0024] Forward primer: 5'-CGGGCGATAAAGGCATTTACTGGCACTACATGCAGA-3'

[0025] Reverse primer: 5'-TCTGCATGTAGTGCCAGTAAATGCCTTTATCGCCCG-3'

[0026] Primers for introducing the A38E mutation

[0027] Forward primer: 5'-CCGTTGTTGATGAAGAAGGTCATATGACCCGCC-3'

[0028] Reverse primer: 5'-GGCGGGTCATATGACCTTCTTCATCAACAACGG-3'

[0029] The PCR reaction system is: 5*PrimerStar Buffer 10μL, dNTPs (2.5mM each) 4μL, forward primer (10μM) 1μL, reverse prim...

Embodiment 3

[0037] Example 3 Fermentative production of acetylglucosamine

[0038] Cultivate recombinant Escherichia coli with LB slant medium. After culturing for 12 hours, use an inoculation loop to take 1 loop and intervene in a 250mL Erlenmeyer flask containing 20mL seed medium for cultivation. Add kanamycin to the seed medium at 50 μg / mL. Cultivate at 37°C and 200rpm for 12h. The seeds were transferred to the fermentation medium with a 5% inoculum size, cultivated at 37°C and 200rpm, and the OD 600 When it was equal to 0.6, 0.4mM IPTG was added to induce, and cultured for 16h after induction. The output of acetylglucosamine in the final fermentation supernatant was measured, and the results showed that the acetylglucosamine output in the fermentation supernatant of the recombinant bacteria expressing G471S was 36.975 mg / L, which was 22.62% higher than that of the starting bacteria; the fermentation of the recombinant bacteria expressing R249W The yield in the supernatant reached 14...

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PUM

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Abstract

The invention discloses a glucosamine-6 phosphate synthase mutant, and belongs to the fields of genetic engineering and enzyme engineering. A method for site-saturation mutagenesis of glucosamine-6-phosphate synthase is adopted and provides the glucosamine-6 phosphate synthase mutant derived from E.coli. Compared with wild-type glucosamine-6-phosphate transferase, the catalytic activity of the mutant to fructose-6-phosphate is significantly improved, the yield of acetylglucosamine is higher, and the mutant is more suitable for industrial production.

Description

technical field [0001] The invention relates to a mutant of glucosamine-6 ​​phosphate synthase, belonging to the fields of genetic engineering and enzyme engineering. Background technique [0002] Acetyl glucosamine is a kind of monosaccharide in organisms, which widely exists in bacteria, yeast, mold, plants and animals. In the human body, acetylglucosamine is the synthetic precursor of the disaccharide unit of glycosaminoglycan, which plays an important role in the repair and maintenance of cartilage and joint tissue functions. Therefore, acetyl glucosamine is widely used as a drug and nutritional dietary supplement to treat and repair joint damage. In addition, acetylglucosamine also has many applications in the field of cosmetics and pharmaceuticals. At present, acetyl glucosamine is mainly produced by acid-decomposing chitin in shrimp shells or crab shells. The waste liquid produced by this method is relatively serious for environmental pollution, and the obtained pro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/70C12N1/21C12P19/26C12R1/19
CPCC12N9/1096C12N15/70C12P19/26C12Y206/01016
Inventor 刘龙陈坚堵国成宋阳顾洋李江华赵雅雯
Owner JIANGNAN UNIV
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