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Sucrose isomerase mutant and method for producing isomaltulose

A technology of sucrose isomerase and isomaltulose, which is applied in the field of separation of immobilized enzymes, achieves the effects of saving economic costs, easy implementation of enzyme production efficiency, and reduced operation difficulty

Active Publication Date: 2016-04-13
森大(天津)生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The DNA nucleic acid sequence adopts the recognized IUPAC nomenclature

Method used

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  • Sucrose isomerase mutant and method for producing isomaltulose
  • Sucrose isomerase mutant and method for producing isomaltulose
  • Sucrose isomerase mutant and method for producing isomaltulose

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1: Cloning and expression of sucrose isomerase gene in Bacillus licheniformis

[0047] 1. Construction of recombinant strains

[0048] Using the genomic DNA of Pantoeadispersa UQ68J as a template, primers were designed according to the sequence of the sucrose isomerase gene sim (GenBank accession number: AY223549.1) of P. SEQ ID NO: 14) and R1 (SEQ ID NO: 15) amplify the sim gene (nucleotide sequence SEQ ID NO: 1) that does not contain a signal peptide, see SEQ ID NO: 2 for the amino acid sequence. PCR amplification system and reaction conditions reference HSDNA Polymerase instruction sheet (TaKaRa). The PCR product was cloned into the plasmid pMD18-Tsimple to obtain the recombinant plasmid pMD18-T-sim with the sim gene. After double digestion with XbaI and SmaI, the recombinant plasmid was cloned into the corresponding site of the expression vector pHY-WZX (DandanNiuandZhengxiangWang. JIndMicrobiolBiotechnol, 2007, 34:357-362) to obtain the recombinant expr...

Embodiment 2

[0062] Example 2: Construction and screening of sucrose isomerase mutants

[0063] Taking Tyr307Asp as an example, using the plasmid pMD18-T-sim as a template, primers F1 (SEQ ID NO: 15) and R2 (SEQ ID NO: 18) were used to amplify by conventional PCR method to obtain PCR product 1. Then use primers F2 (SEQ ID NO: 17) and R1 (SEQ ID NO: 16) to amplify by conventional PCR method to obtain PCR product 2.

[0064] The above two reactions are carried out synchronously. The two amplification products, PCR product 1 and PCR product 2, are purified and recovered, mixed equimolarly and added to the second-step reaction system. No primers are added to the reaction system, and the others are the same as conventional PCR. The reaction system is circulated for 5 to 10 times.

[0065] Using the amplified product of the second-step reaction system as a template, add primers F1 and R1, and perform 25 to 30 cycles in the same way as the conventional PCR reaction system. The amplified product...

Embodiment 3

[0073] Example 3: Enzyme activity and catalytic product analysis of sucrose isomerase mutants

[0074] The enzyme activity assay was carried out according to the method of Example 1, and the specific results are shown in Table 3. It can be seen from the table that the mutation did not cause a significant decrease in enzyme activity, and all 11 mutants maintained good biological activity.

[0075] With 10 mL of sucrose at a concentration of 50% as the substrate, the fermentation liquid of the sucrose isomerase mutant was added at 30°C, and the reaction time was extended to 100 min to complete the conversion of sucrose and facilitate the analysis of the product ratio. The obtained reaction liquid was diluted 10 times, Detection by HPLC method. Taking the recombinant bacterium CBBD302 (pHY-sim) obtained in Example 1 as a control, the results are shown in Table 3. Mutation of Gln to Glu at position 310, a negatively charged acidic amino acid mutated into an uncharged polar amino...

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Abstract

The invention belongs to the technical field of enzyme engineering and particularly relates to a method for obtaining sucrose isomerase through site-directed mutation so as to improve the product specificity of the sucrose isomerase, and a method for preparing immobilized sucrose isomerase and achieving separation of the immobilized enzyme through a non-aqueous phase catalytic system. The content of isomaltulose in a catalytic product of the sucrose isomerase is substantially increased and increased to 99.16% from 90.28%, and the enzyme preparation process is easy to implement and is high in enzyme producing efficiency; due to the sucrose isomerase immobilization technology, the sucrose isomerase can easily keep high catalytic activity and low enzyme amount loss in the repeated catalysis process, multi-batch continuous use can be performed, operating difficulty can be greatly reduced, and economic cost is saved.

Description

Technical field: [0001] The invention belongs to the technical field of enzyme engineering, and in particular relates to obtaining a sucrose isomerase by site-directed mutation so as to improve its product specificity, and preparing immobilized sucrose isomerase and utilizing a non-aqueous phase catalytic system to realize the separation of the immobilized enzyme. Background technique: [0002] Isomaltulose, also known as Palatinose, is a reducing disaccharide, which is an isomer of sucrose. It was first discovered in sugar beet production by Weidenhagen et al. in 1957. Isomaltulose has sweetness characteristics and mouthfeel similar to sucrose, but its sweetness is low, only 52% of sucrose. Compared with sucrose, its outstanding advantages are mainly reflected in: (1) low cariogenic properties; 2) It is suitable for diabetics; (3) It selectively stimulates the growth of bifidobacteria in the human intestinal tract; (4) It has extremely low hygroscopicity, strong stability, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/90C12N15/61C12N15/63C12P19/24C12P19/12
CPCC12N9/90C12P19/12C12P19/24C12Y504/99022
Inventor 路福平王正祥
Owner 森大(天津)生物科技有限公司
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