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A kind of sucrose isomerase mutant with improved stability and its construction method

A technology for sucrose isomerase and mutants, which is applied in the fields of genetic engineering and enzyme engineering, can solve the problem of low thermal stability of sucrose isomerase mutants, and achieve flexibility, unaffected enzyme activity, and thermal stability Improved effect

Active Publication Date: 2022-05-06
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] In order to solve the problem of low thermal stability of the current sucrose isomerase mutant, the present invention provides a sucrose isomerase mutant, which is the 280th sequence of the sucrose isomerase whose amino acid sequence is shown in SEQ ID NO.1 and / or amino acid at position 499 are mutated

Method used

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  • A kind of sucrose isomerase mutant with improved stability and its construction method
  • A kind of sucrose isomerase mutant with improved stability and its construction method
  • A kind of sucrose isomerase mutant with improved stability and its construction method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Embodiment 1: Construction of the recombinant plasmid containing sucrose isomerase mutant

[0046] Specific steps are as follows:

[0047] (1) Construction of recombinant plasmid containing wild-type sucrose isomerase pdsi

[0048] The chemically synthesized wild-type sucrose isomerase pdsi whose nucleotide sequence is shown in SEQ ID NO.2 was ligated with the pXMJ19 vector after digestion with HindⅢ enzyme and EcoRI enzyme to prepare the recombinant vector pXMJ19-pdsi.

[0049] (2) Obtaining recombinant vectors containing mutants:

[0050] Using the whole plasmid PCR technology, the recombinant vector pXMJ19-pdsi prepared in step (1) was used as a template for site-directed mutagenesis to obtain recombinant plasmids pXMJ19-A100E, pXMJ19-I205M, pXMJ19-S563L, pXMJ19-N578M, and pXMJ19-G152P, pXMJ19-S328F, pXMJ19-E76R, pXMJ19-S563R, pXMJ19-V280L, pXMJ19-S499F, pXMJ19-V280L / S499F.

[0051] The primer sequences designed respectively are as follows:

[0052] A100E-F: GAGTAT...

Embodiment 2

[0074] Example 2: Construction of recombinant Escherichia coli engineering bacteria producing sucrose isomerase mutants and expression, isolation and purification of sucrose isomerase

[0075] Specific steps are as follows:

[0076] (1) The recombinant plasmids pXMJ19-pdsi, pXMJ19-A100E, pXMJ19-I205M, pXMJ19-S563L, pXMJ19-N578M, pXMJ19-G152P, pXMJ19-S328F, pXMJ19-E76R, pXMJ19-S563R, V280L, pXMJ19-S499F, pXMJ19-V280L / S499F were transformed into C.glutamium ATCC13032 competent cells by electroporation, and genetic engineering bacteria were prepared respectively: C.glutamium13032 / pXMJ19-pdsi, C.glutamium13032 / pXMJ19-A100E, C.glutamium13032 / pXMJ19-A100E, C. .glutamium13032 / pXMJ19-I205M,C.glutamium13032 / pXMJ19-S563L,C.glutamium13032 / pXMJ19-N578M,C.glutamium13032 / pXMJ19-G152P,C.glutamium13032 / pXMJ19-S328F,C.glutamium13032 / pXMJ19-E76R,C.glutamium13032 / pXMJ19-S563R, C. glutamium13032 / pXMJ19-V280L, C. glutamium13032 / pXMJ19-S499F, C. glutamium13032 / pXMJ19-V280L / S499F.

[0077] (2) In...

Embodiment 3

[0089]Embodiment 3: Enzymatic properties of sucrose isomerase mutant

[0090] 1. Thermal stability

[0091] Take the pure enzyme solution containing wild-type pdsi, the pure enzyme solution containing V280L, the pure enzyme solution containing S499F, and the pure enzyme solution containing V280L / S499F prepared in step (2) of Example 2 and place them in a constant temperature water bath at 45°C , take samples at intervals, measure its residual enzyme activity according to the sucrose isomerase enzyme activity assay method, compare its thermal stability, and obtain the half-life results of wild-type pdsi and its mutants as shown in Table 2 and image 3 shown.

[0092] Table 2 Half-life of different sucrose isomerases

[0093]

[0094] 2. Optimal pH

[0095] The pure enzyme solution containing wild-type pdsi, the pure enzyme solution containing V280L, the pure enzyme solution containing S499F, and the pure enzyme solution containing V280L / S499F prepared in step (2) of Examp...

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Abstract

The invention discloses a sucrose isomerase mutant with improved stability and a construction method thereof, belonging to the fields of genetic engineering and enzyme engineering. The invention screens and obtains two mutant strains V280L and S499F with improved single-point thermostability and a combined mutant V280L / S499L on the basis of high-enzyme activity disperse Pantoea-derived sucrose isomerase. While the heat stability of the three mutants was significantly improved, the enzyme activity was not affected. These mutants in the present invention are more suitable for industrial production than natural sucrose isomerase, and have very huge application prospects and industrial value.

Description

technical field [0001] The invention relates to a sucrose isomerase mutant with improved stability and a construction method thereof, belonging to the technical fields of genetic engineering and enzyme engineering. Background technique [0002] Sucrose isomerase (EC5.4.99.11), also known as isomaltulose synthase, sucrose mutase, belongs to the α-amylase 13 family, is an isomerase with high industrial application value, can Isomaltulose and trehalulose are produced from isomaltulose and trehalulose with high efficiency. Isomaltulose is a new functional sweetener in recent years. It is the most ideal sucrose substitute sugar for diabetic patients. The application prospect of sucrose isomerase is currently the most effective enzyme for the industrial production of isomaltulose by biological enzymatic method. [0003] Currently reported sucrose isomerases show limited thermostability in biocatalytic processes, for example, the half-life of sucrose isomerase from Klebsiella sp.L...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/90C12N15/61C12N15/70C12N15/77C12N1/21C12P19/24C12P19/12C12R1/19C12R1/125C12R1/15C12R1/865
CPCC12N9/90C12N15/70C12N15/77C12P19/24C12P19/12C12Y504/99011
Inventor 张显胡孟凯唐梓桐饶志明王一迈蔡超凡卢杨徐美娟杨套伟
Owner JIANGNAN UNIV
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