Cyclodextrin glucosyltransferase mutants with improved disproportionation specific activity and aa-2g production

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: 2022-03-25
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 mutants with improved disproportionation specific activity and aa-2g production
  • Cyclodextrin glucosyltransferase mutants with improved disproportionation specific activity and aa-2g production
  • Cyclodextrin glucosyltransferase mutants with improved disproportionation specific activity and aa-2g production

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: Preparation of cyclodextrin glucosyltransferase single mutant recombinant bacteria

[0034] (1) Single mutation of cyclodextrin glucosyltransferase

[0035] According to the nucleotide sequence of the cyclodextrin glucosyltransferase shown in the amino acid sequence position SEQ ID NO. 2, the primers with Y195F and Y260F mutations were designed and synthesized, and the cyclodextrin glucosyltransferase gene was site-directed mutagenesis, Single mutants mutated at positions 195 and 260 were obtained, respectively.

[0036] Using rapid PCR technology, using the expression vector pET20b(+) / cgt as a template (the vector already contains a promoter and a signal peptide, and the sequence of the target gene after the signal peptide), the construction method of the expression vector pET20b(+) / cgt is described in Li Zhaofeng, "The expression of Paenibacillus softening α-cyclodextrin glucosyltransferase in Escherichia coli and its product specificity analysis" (publish...

Embodiment 2

[0056] Example 2: Preparation of cyclodextrin glucosyltransferase double mutant recombinant bacteria

[0057] On the basis of the single mutant Y195F, the double mutant was constructed by using the site-directed mutagenesis primer of Y260F mutation. For specific embodiments, see Example 1, using the rapid PCR technology, 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" Expression of basal transferase in Escherichia coli and analysis of its product specificity" (published in 2009).

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

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

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

[0061] For the preparation method of the recombinant bacteria, see Example 1, and the recombinant bacteria ob...

Embodiment 3

[0062] Example 3: Fermentation of mutant recombinant bacteria to produce enzymes

[0063] The recombinant strains obtained in Examples 1 and 2 were picked and grown for 8-10 h in LB liquid medium (containing 100 μg / mL ampicillin), and the seed fermentation broth was connected to TB medium (containing 100 μg / mL ampicillin by 5% inoculum size). ), after culturing in a shaker at 25 °C for 60 h, the fermentation broth was centrifuged at 4 °C and 8000 rpm for 10 min to remove bacteria, and the centrifuged supernatant was collected to obtain crude enzyme liquid.

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

[0065] The enzymatic activities of wild-type, Y195F, Y260F, Y195F / Y260F were measured respectively. The results are shown in Table 1. The enzymatic activity of mutant Y195F was slightly lower than that of wild-type, and the enzymatic activities of mutant Y260F and Y195F / Y260F were increased compared with wil...

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Abstract

The invention discloses a cyclodextrin glucosyltransferase mutant with improved disproportionation specific activity and AA-2G yield, belonging to the fields of genetic engineering and enzyme engineering. The present invention mutates the cyclodextrin glucosyltransferase derived from Paenibacillusmacerans JFB05-01, and the obtained mutant has significantly improved disproportionation specific activity, wherein, the yield of AA-2G produced by the mutant Y195F / Y260F is 22g / L, which is higher than that of the wild enzyme 46.7%, the conversion rate is improved, the production cost is reduced, and it is suitable for industrialized production.

Description

technical field [0001] The invention relates to a cyclodextrin glucosyltransferase mutant with improved disproportionation specific activity and AA-2G yield, belonging 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 very 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 vivo to produce L-AA. It can be absorbed and utilized by humans and animals, so it is more advantageous. [0003] At present, AA-2G is mainly biocatalyzed by glycosyltransferases, of which cyclodextrin glycosyltransferase (CGTase) is the most commonly used catalytic enzyme. There are man...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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