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A group of cyclodextrine glucosyltransferases and encoding gene and application thereof

A cyclodextrin and gene technology, applied in the directions of transferase, application, genetic engineering, etc., can solve the problems of high price, small production scale, limitation of γ-cyclodextrin production and application, etc.

Active Publication Date: 2013-08-07
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The application prospect of γ-cyclodextrin is wider than that of β-cyclodextrin, but at present, due to the small scale of production, the price is high, which limits the production and application of γ-cyclodextrin

Method used

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  • A group of cyclodextrine glucosyltransferases and encoding gene and application thereof
  • A group of cyclodextrine glucosyltransferases and encoding gene and application thereof
  • A group of cyclodextrine glucosyltransferases and encoding gene and application thereof

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Experimental program
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Effect test

Embodiment 1

[0070] Embodiment 1, the discovery of mutated protein and its coding gene

[0071] A strain of Bacillus macerans screened from nature can produce cyclodextrin glycosyltransferase, and the cyclodextrin glycosyltransferase gene in the strain is shown in sequence 2 of the sequence listing, encoding sequence 1 of the sequence listing proteins indicated.

[0072] The protein shown in Sequence 1 is named α-CGTase-1 protein, and the gene encoding it is named α-CGTase-1 gene (as shown in Sequence 2). The protein shown in Sequence 3 was named α-CGTase-WT protein (sequence published in NCBI), and the gene encoding it was named α-CGTase-WT gene (shown in Sequence 4).

[0073] The difference between Sequence 1 and Sequence 3 lies only in the 590th amino acid residue (M / V) and the 677th amino acid residue (S / G). The difference between sequence 2 and sequence 4 lies in the 1768th nucleotide (A / G) and the 2029th nucleotide (A / G).

[0074] Using the double-stranded DNA shown in Sequence 1 ...

Embodiment 2

[0075] Embodiment 2, enzyme activity identification

[0076] 1. Artificially synthesize the following double-stranded DNA molecules:

[0077] (1) Double-stranded DNA molecule 1: The difference from the DNA molecule shown in Sequence 2 of the sequence table is that the 583-585 nucleotides (TAC) from the 5' end will be deleted, and the 499-501 nucleotides from the 5' end will be deleted. The nucleotide at position 1 is mutated from TAC to CAC; correspondingly, the 195th tyrosine in sequence 1 is deleted, and the 167th amino acid residue is mutated from tyrosine to histidine. The protein encoded by double-stranded DNA molecule 1 is named protein 1.

[0078] (2) Double-stranded DNA molecule 2: The difference from the DNA molecule shown in Sequence 2 of the sequence table is that the 583-585 nucleotides from the 5' end are mutated from TAC to TTC, and the 499- The 501st nucleotide is mutated from TAC to CAC; correspondingly, the 195th amino acid residue in sequence 1 is mutated f...

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Abstract

The invention discloses a group of cyclodextrine glucosyltransferases and an encoding gene and application of the group of cyclodextrine glucosyltransferases.. The cyclodextrine glucosyltransferases provided by the invention are characterized in that a protein as shown in a sequence 1 in a sequence table is the protein obtained by mutating as shown in (a) and (b): (a) mutating from tyrosine to histidine since the 167th amino acid residue at the N tail end as shown in the sequence 1 in the sequence table; and (b) mutating from the 195th amino acid residue at N tail end as shown in the sequence 1 in the sequence table. By adoption of the protein, the influence of the 195th amino acid in a catalytic center on the catalyzing specificity of Alpha-CGTase is researched, which brings a significant value for the study on the catalytic mechanism of the CGTase.

Description

technical field [0001] The invention relates to a group of cyclodextrin glucosyltransferases and their coding genes and applications. Background technique [0002] The English name of cyclodextrin is cyclodextrin, referred to as CD. Cyclodextrin is the product of cyclodextrin glucosyltransferase (CGTase) acting on starch. It is a cyclic oligosaccharide with more than six glucose linked by α-1,4-glycosidic bonds. The most common and most studied is α-cyclodextrin (α-CD), β-cyclodextrin (β-CD), γ-cyclodextrin (γ-CD), respectively composed of six, seven and eight glucose molecules, are relatively large and relatively flexible molecules. [0003] The formation of cyclodextrin is mainly based on starch as raw material, through the glycosyl conversion of cyclodextrin glucano-transferase (CGTases for short). The specificity of CGTases is low, and three products of α-CD, β-CD and γ-CD are produced simultaneously in most cases. The proportions of the three products produced by di...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/63C12N5/10C12N1/21C12P19/18C12R1/19
Inventor 钞亚鹏钱世钧谢婷
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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