High-temperature-resistant glucose isomerase mutant and application thereof

A technology of glucose isomerase and mutant is applied in the application field of isomerizing glucose to produce ultra-high D-fructose concentration and high fructose syrup, and achieves the effect of significant technical progress

Active Publication Date: 2018-05-18
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there have been some heat-resistant GI reports, such as Thermotoga maritima and Thermusthermophiles, etc., whose optimum te

Method used

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  • High-temperature-resistant glucose isomerase mutant and application thereof
  • High-temperature-resistant glucose isomerase mutant and application thereof
  • High-temperature-resistant glucose isomerase mutant and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1: Construction and screening of ToGI single point mutants

[0021] 1. Mutant construction

[0022] According to the parental sequence of ToGI (the amino acid sequence is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.2), the mutation primers for site-directed mutation were designed, and the recombinant vector pET28b / ToGI was used as a template by using rapid PCR technology. To introduce a single mutation at position 89, the primers are:

[0023] Forward primer CCGATGGTT NNN GCTAACCTGTTC (the underline is the mutated base)

[0024] reverse primer CAGGTTAGC NNN AACCATCGGAACTTTC (the underline is the mutated base)

[0025] PCR reaction system: 2×Phanta Max Buffer (containing Mg 2+ ) 25μL, dNTPs 10mM, forward primer 2μL, reverse primer 2μL, template DNA 1μL, Phanta Max Super-Fidelity DNA Polymerase 50U, add ddH 2 0 to 50 μL.

[0026] The PCR amplification conditions were 95°C for 3min; (95°C for 15s, 50°C for 15s, 61°C for 6.5min) ...

Embodiment 2

[0037] Example 2: Construction and screening of two-site mutants of glucose isomerase

[0038] According to the single mutant ToGI-1 sequence constructed in Example 1, the mutation primers for site-directed mutation were designed, and the rapid PCR technology was used to use the recombinant vector pET28b / ToGI-1 as a template to introduce a single mutation at position 136. The primers were:

[0039] Forward primer CGTTGTT NNN CCGGGTCGTG (the underline is the mutated base)

[0040] reverse primer GACCCGG NNN AACAACGTAGATTTC (the underline is the mutated base)

[0041] PCR reaction system: 2×Phanta Max Buffer (containing Mg 2+ ) 25μL, dNTPs 10mM, forward primer 2μL, reverse primer 2μL, template DNA 1μL, Phanta Max Super-Fidelity DNA Polymerase 50U, add ddH 2 0 to 50 μL.

[0042] The PCR amplification conditions were 95°C for 3min; (95°C for 15s, 50°C for 15s, 62°C for 6.5min) for 30 cycles; 72°C for 5min.

[0043] The PCR product was transformed into E.coliBL21 (DE3) comp...

Embodiment 3

[0049] Example 3: Construction and screening of three-site mutants of glucose isomerase

[0050] According to the mutant ToGI-2 sequence constructed in Example 2, the mutation primers for site-directed mutagenesis were designed, using the rapid PCR technology, using the recombinant vector pET28b / ToGI-2 as a template, and introducing a single mutation at position 352, the primers were:

[0051] Forward primer CGTGCT NNN GCTCTGAAAG (the underline is the mutated base)

[0052] reverse primer CAGAGC NNN AGCACGTTCAC (the underline is the mutated base)

[0053] PCR reaction system: 2×Phanta Max Buffer (containing Mg 2+ ) 25μL, dNTPs 10mM, forward primer 2μL, reverse primer 2μL, template DNA 1μL, Phanta Max Super-Fidelity DNA Polymerase 50U, add ddH 2 0 to 50 μL.

[0054] The PCR amplification conditions were 95°C for 3min; (95°C for 15s, 50°C for 15s, 60°C for 6.5min) 30 cycles; 72°C for 5min.

[0055] The PCR product was transformed into E.coliBL21 (DE3) competent cells, an...

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Abstract

The invention discloses a high-temperature-resistant glucose isomerase mutant and application of the mutant in catalyzing the isomerization of D-glucose to prepare D-fructose. The mutant is obtained through single or multiple mutations of one or more of a 89th position, a 136th position and a 352th position of an amino acid sequence shown in SEQ ID NO.1. By means of the mutant, the efficient expression of high-conversion-rate glucose isomerase genes is achieved, and the enzyme activity is 7.23 U/mg. The optimal reaction temperature of the ToGI mutant is 110 DEG C, and in high fructose syrup prepared by using catalysts to catalyze 400 g/L D-glucose at the high temperature of 100 DEG C, the concentration of D-fructose is as high as 61.3%.

Description

(1) Technical field [0001] The present invention relates to a glucose isomerase mutant, in particular to a method for preparing a glucose isomerase mutant with ultra-high reaction temperature using gene mutation technology, and the stable isomerization of the mutant at ultra-high temperature The application of glucose to produce high fructose syrup with ultra-high D-fructose concentration. (2) Background technology [0002] Glucose isomerase (GI for short, EC 5.3.1.5) is mainly used to catalyze the isomerization of D-glucose to generate D-fructose in vitro, and is the key enzyme for the industrial production of high fructose syrup by biotransformation. According to the primary structure of GI, it can be divided into two classes, namely class I and class II enzymes. Compared with class I GI, the N-terminal of the peptide chain of class II GI contains 40-50 extra amino acid residues (Deng H. et al., Bioprocess and Biosystems Engineering, 37:1211-1219, 2014). [0003] High fr...

Claims

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Application Information

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IPC IPC(8): C12N9/92C12P19/02
CPCC12N9/92C12P19/02C12Y503/01005
Inventor 柳志强贾东旭郑裕国刘子健王腾金利群王远山
Owner ZHEJIANG UNIV OF TECH
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