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Cyclodextrin glucosyltransferase mutant with improved disproportionation specific activity and AA-2G yield

A glucose-based and cyclodextrin technology, applied in the fields of genetic engineering and enzyme engineering, can solve problems such as low conversion rate

Active Publication Date: 2020-08-14
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the reaction system of preparing AA-2G with maltodextrin as the glycosyl donor, there are more glucose and maltose, and compared with VC, the small molecule sugar has the advantage of being an acceptor, thus competing with VC, leading to the transformation low rate

Method used

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  • Cyclodextrin glucosyltransferase mutant with improved disproportionation specific activity and AA-2G yield
  • Cyclodextrin glucosyltransferase mutant with improved disproportionation specific activity and AA-2G yield
  • Cyclodextrin glucosyltransferase mutant with improved disproportionation specific activity and AA-2G yield

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: Preparation of Cyclodextrin Glucosyltransferase Single Mutation Recombinant Bacteria

[0034] (1) Cyclodextrin glucosyltransferase single mutation

[0035] According to the nucleotide sequence of the cyclodextrin glucosyltransferase shown in SEQ ID NO.5 of the amino acid sequence, design and synthesize primers that introduce Y195F and Y260F mutations, and perform site-directed mutation on the cyclodextrin glucosyltransferase gene, Single mutation mutants with mutations at the 195th and 260th positions were obtained respectively.

[0036] Utilize rapid PCR technology, use the expression vector pET20b(+) / cgt as a template (the vector already contains the promoter and signal peptide, and the signal peptide is followed by the target gene sequence), the construction method of the expression vector pET20b(+) / cgt is described in Li Zhaofeng's "Expression of Paenibacillus softening α-cyclodextrin glucosyltransferase in Escherichia coli and analysis of its product sp...

Embodiment 2

[0056] Example 2: Preparation of Cyclodextrin Glucosyltransferase Double Mutant Recombinant Bacteria

[0057] On the basis of single mutant Y195F, double mutants were constructed by using site-directed mutagenesis primers for Y260F mutation. Refer to Example 1 for the specific implementation method. Using the rapid PCR technology and using the recombinant plasmid cgt / pET20b(+) as a template, the construction method of the recombinant plasmid cgt / pET20b(+) is described in Li Zhaofeng's "Paenibacillus softening α-cyclodextrin glucose The expression of base transferase in Escherichia coli and its product specificity analysis" (publication date 2009) in the literature.

[0058] The site-directed mutagenesis primers for introducing the Y260F mutation are:

[0059] Forward primer: CGGGGAATGG TTC CTTGGCGCGGATCAAAC, SEQ ID NO. 8,

[0060] Reverse primer: GTTTGATCCGCGCCAAG GAA CCATTCCCCCG, SEQ ID NO.9.

[0061] For the preparation method of the recombinant bacteria, refer to Exam...

Embodiment 3

[0062] Embodiment 3: mutant recombinant bacteria ferment and produce enzyme

[0063] Pick the recombinant bacterial strain that embodiment 1 and 2 obtain and grow in LB liquid culture medium (containing 100 μ g / mL ampicillin) 8-10h, connect seed fermented liquid to TB medium (containing 100 μ g / mL ampicillin) by 5% inoculum size ), after culturing in a shaker at 25°C for 60 hours, the fermentation broth was centrifuged at 4°C and 8000 rpm for 10 minutes to remove bacteria, and the centrifuged supernatant was collected to obtain a crude enzyme liquid.

[0064] Escherichia coli BL21(DE3) containing the wild-type enzyme was fermented in the same way to produce the enzyme.

[0065] The enzyme activities of the wild type, Y195F, Y260F, and Y195F / Y260F were measured respectively, and the results are shown in Table 1. The enzyme activity of the mutant Y195F was slightly lower than that of the wild type, and the enzyme activity of the mutant Y260F and Y195F / Y260F were respectively inc...

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Abstract

The invention discloses a cyclodextrin glucosyltransferase mutant with improved disproportionation specific activity and AA-2G yield, and belongs to the field of gene engineering and enzyme engineering. Cyclodextrin glucosyltransferase from paenibacillus macerans JFB05-01 is mutated, the disproportionation specific activity of the obtained mutant is remarkably improved, wherein the yield of AA-2Gproduced by the mutant Y195F / Y260F is 22g / L, and compared with that of wild enzymes, the yield is improved by 46.7%. The mutant improves the conversion rate, reduces the production cost, and is suitable for industrial production.

Description

technical field [0001] The invention relates to a cyclodextrin glucosyltransferase mutant with improved disproportionation specific activity and AA-2G yield, and belongs to the fields of genetic engineering and enzyme engineering. Background technique [0002] Vitamin C (Vitamin C, VC), also known as L-Ascorbic Acid (L-AA), is a water-soluble vitamin that plays an important role in maintaining and promoting human health and animal growth. However, due to its extremely unstable chemical structure, the scope of application is limited. The study found that the VC glycosyl derivative 2-O-α-D-glucopyranosyl-L-ascorbic acid (AA-2G) has the advantages of safety, strong stability and easy degradation in the body to produce L-AA, which can be better It can be absorbed and utilized by humans and animals in a timely manner, so it has more advantages. [0003] At present, AA-2G is mainly biocatalyzed by glycosyltransferases, among which cyclodextrin glucosyltransferase (CGTase) is the...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C12N15/70C12N1/21C12P19/60
CPCC12N9/1074C12N15/70C12P19/60C12Y204/01019
Inventor 吴敬王蕾陶秀梅宿玲恰孔德民
Owner JIANGNAN UNIV
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