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Acid phosphatase mutant, encoding gene, vector and application

A technology of acid phosphatase and mutant, which is applied to vectors and application fields containing the gene, can solve the problems of high cost of I+G and low enzyme activity, and achieve the effect of high practical value and broad market application prospects.

Active Publication Date: 2014-02-05
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] However, the main problem of the current acid phosphatase EB-AP / PTase is that the enzyme activity is relatively low, resulting in high cost of enzymatic production of I+G

Method used

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  • Acid phosphatase mutant, encoding gene, vector and application
  • Acid phosphatase mutant, encoding gene, vector and application
  • Acid phosphatase mutant, encoding gene, vector and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1: Determination of protein crystal structure and saturation mutation point of acid phosphatase (EB-AP / PTase)

[0019] Analysis of the protein crystal structure of acid phosphatase (PDB protein database ID: 1EOI), the active sites of acid phosphatase are: Lys115, Arg122, Ser148, Gly149, His150, Arg183, His189, Asp193. Inosine was used as the substrate for molecular docking. The following three mutation points were determined: aspartic acid at position 108, glutamic acid at position 104, and asparagine at position 143.

Embodiment 2

[0020] Embodiment 2: Construction of acid phosphatase mutant

[0021] (1) Construction of acid phosphatase (EB-AP / PTase) recombinant Escherichia coli

[0022] According to the sequence shown in SEQ ID No: 4, it was synthesized by a total chemical synthesis method, cloned into the expression vector pET28b, and the recombinant expression plasmid pET28b-AP / PT was transferred into E. coli E.coli BL21 (DE3). Recombinant Escherichia coli E.coli BL21(DE3) / pET28b-AP / PT was successfully constructed by enzyme digestion and colony PCR identification.

[0023] (2) Molecular modification of E.coli BL21(DE3) / pET28b-AP / PTase

[0024] A. For E.coli BL21(DE3) / pET28b-AP / PTase, amplify the target gene with primers (108):

[0025] Upstream primer: GAGGACGCCGGANNNCTTGCAACTCGT

[0026] Downstream primer: ACGAGTTGCAAGNNNTCCGGCGTCCTC

[0027] Using the plasmid of E.coli BL21(DE3) / pET28b-AP / PTase as a template and Prime STAR as a polymerase, PCR amplification was carried out with the participation...

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Abstract

The invention provides an acid phosphatase mutant, an encoding gene thereof, a vector containing the gene and application. The mutant is obtained through point mutation of an amino acid sequence shown as SEQ ID No. 1, and the point mutation comprises mutation of 108th site into serine and / or mutation of 143rd site into leucine. The beneficial effects mainly comprise that by employing a half-rationality design method, the acid phosphatase (EB-AP / PTase) gene is subjected to multi-round site saturation mutagenesis for obtaining acid phosphatase mutants AP / PT-D108S and AP / PT-D108S / N143L with improved enzyme activity, and the acid phosphatase mutants AP / PT-D108S and AP / PT-D108S / N143L have relatively high practical value and wide market application prospect.

Description

(1) Technical field [0001] The present invention relates to an acid phosphatase mutant and its coding gene, as well as the carrier containing the gene and its application. (2) Background technology [0002] Taste nucleotide I+G, as a new generation of food freshness enhancer, is made by mixing 5'-inosinic acid (5'-IMP) and 5'-guanylic acid (5'-GMP) at a ratio of 1:1. I+G has a more delicious taste effect than sodium glutamate (monosodium glutamate), and it can also be mixed with monosodium glutamate, which has a multiplied synergistic effect, and can significantly reduce product costs, and is widely used in the field of food processing. [0003] The taste effect of I+G depends on its chemical structure. Studies have found that only the hydroxyl group on the carbon atom at the 5'-position of the nucleoside and the phosphoric acid group are esterified to show umami taste activity, while the 2'- and 3'-position Hydroxyl phosphorylation of carbon atoms is inactive. Therefore, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/16C12N15/55C12N15/70C12P19/32
CPCC12N9/16C12Y301/03002
Inventor 郑裕国孙丽慧沈爱萍沈寅初
Owner ZHEJIANG UNIV OF TECH
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