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Sucrose phosphorylase mutant and application thereof

A phosphorylase mutant, sucrose phosphorylase technology, applied in the field of enzyme engineering and microbial engineering, can solve problems such as poor thermal stability

Active Publication Date: 2020-09-04
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But most SPases are not thermophilic enzymes
Existing SPases have poor thermal stability. For example, the wild-type SPase derived from streptococcus mutants has only 11.3% of the initial activity after being treated at 55°C for 20 minutes (see references for details: Fujii K, Iiboshi M, Yanase M , et al.Enhancing the Thermal Stability of Sucrose Phosphorylase from Streptococcus mutans by Random Mutagenesis[J].Journal of Applied Glycoence,2006,53(2):91-97.)

Method used

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  • Sucrose phosphorylase mutant and application thereof
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  • Sucrose phosphorylase mutant and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1: Expression of sucrose phosphorylase wild enzyme

[0044](1) Obtain the gene sequence of the SPase gene (GenBank accession number: D90314) of Leuconostoc enteritidis ATCC 12291 from NCBI, optimize it according to the codon preference of E. NcoI and XhoI were added to both ends of the optimized SPase gene, and sent to Jinweizhi Biotechnology Co., Ltd. to synthesize the sucrose phosphorylase whose coding nucleotide sequence is shown in SEQ ID NO.1.

[0045] (2) Construction and transformation of gene expression vector

[0046] The sucrose phosphorylase SPase and the pET-28a vector whose coding nucleotide sequence is shown in SEQ ID NO.1 are double-digested with restriction endonucleases NcoI and XhoI, and the digested product is connected with Solution I to obtain a recombinant vector pET-28a-SPase, and then transfer the recombinant vector pET-28a-SPase into Escherichia coli BL21 (DE3) for expression, pick 4 transformants to extract the plasmid and verify it wi...

Embodiment 2

[0047] Example 2: Preparation and expression of sucrose phosphorylase mutants

[0048] Specific steps are as follows:

[0049] Utilizing the whole plasmid PCR technology, the recombinant plasmid pET-28a-spase obtained in Example 1 was used as a template for site-directed mutation, and mutants I31F, Q453G, G252L, A232M, T152G, N158C, T219L, S360A, N249A, T263L and T31F\T219L\S360A\T263L gene recombinant plasmids pET-28a-SPase1~pET-28a-SPase11;

[0050] Wherein, the mutation I31F is obtained by mutating the 31st amino acid of the sucrose phosphorylase shown in SEQ ID NO.22 from isoleucine to phenylalanine, and the primers used are as follows:

[0051] I31F-1: 5'-GTTCTGAAAGAAGAC TTC GGTGACGCTA-3' (SEQ ID NO. 2);

[0052] I31F-2: 5'-ACCGATAGCG TCACC GAA GTCTTCTTTC-3' (SEQ ID NO. 3);

[0053] The mutation Q453G is obtained by mutating the 453rd amino acid of sucrose phosphorylase with the amino acid sequence shown in SEQ ID NO.22 from glutamine to glycine, and the primers us...

Embodiment 3

[0093] Embodiment 3: Separation and purification of different sucrose phosphorylase mutants

[0094] Specific steps are as follows:

[0095] The crude enzyme solutions containing sucrose phosphorylase mutants I31F, T219L, S360A, T263L and T31F\T219L\S360A\T263L obtained in Example 2 were purified respectively, and a His tag was attached to the SPase C-terminus, and the nickel column affinity Proteins were purified by chromatography.

[0096] (1) Prepare fresh crude enzyme solutions containing sucrose phosphorylase mutants I31F, T219L, S360A, T263L and T31F\T219L\S360A\T263L in advance and place them on ice (the purification process should be carried out at low temperature as much as possible);

[0097] (2) Use binding buffer at 1 mL·min -1 The column was equilibrated at a flow rate, and then the crude enzyme solution containing sucrose phosphorylase mutants I31F, T219L, S360A, T263L and T31F\T219L\S360A\T263L was mixed at 0.5mL·min -1 Load the sample at a flow rate, and was...

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Abstract

The present invention discloses a sucrose phosphorylase mutant and an application thereof, and belongs to the technical fields of enzyme engineering and microbial engineering. A thermal stability of the sucrose phosphorylase mutant is significantly improved compared to that of a wild type. The sucrose phosphorylase mutants of T219L and I31F / T219L / S360A / T263L have better thermal stability at 50 DEGC and have a half-life of about 50 min, and the half-life of the sucrose phosphorylase mutant is about 35 min higher than that of the wild type.

Description

technical field [0001] The invention relates to a mutant of sucrose phosphorylase and application thereof, and belongs to the technical fields of enzyme engineering and microbial engineering. Background technique [0002] Sucrose phosphorylase (EC 2.4.1.7, Sucrose phosphorylase, SPase) catalyzes the phosphorylation of sucrose to generate α-D-glucose-1-phosphate and D-fructose and its reversible reactions. The enzyme has the ability to transfer glucosyl groups, and can transfer a glucosyl group of a sucrose molecule to a different acceptor. In recent years, the use of this enzyme to produce fine chemicals such as α-arbutin and α-glucosyl glycerol (αGG) has received more and more attention. [0003] For industrial applications, the conversion of carbohydrates is best performed at 60 °C or higher to avoid microbial contamination. But most SPases are not thermophilic enzymes. Existing SPases have poor thermal stability. For example, the wild-type SPase derived from streptococ...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C12N15/70C12N1/21A23L29/00A23L33/10A23L33/13A23L33/195A61K38/45A61K8/66A61Q19/00C12R1/19
CPCA23V2002/00A61K8/66A61K38/45A61K2800/10A61Q19/00A23L29/06A23L33/10A23L33/13A23L33/195C12N9/1051C12N15/70C12Y204/01007A23V2250/546
Inventor 陈献忠夏媛媛李晓玉沈微曹钰杨海泉
Owner JIANGNAN UNIV
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