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Glucose isomerase mutant and application thereof

A technology for glucose isomerase and mutants, which is applied in the field of high conversion rate glucose isomerase mutants, can solve the problems of low cell yield in culture conditions, unsuitable enzyme production for industrial production, etc., and achieves the effect of outstanding technological progress.

Active Publication Date: 2012-05-09
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the harsh culture conditions, low cell yield and isomerization temperature above 90°C of thermophilic bacteria, a large number of by-products and pigments are produced, and its enzyme production is not suitable for industrial production.

Method used

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  • Glucose isomerase mutant and application thereof
  • Glucose isomerase mutant and application thereof
  • Glucose isomerase mutant and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Embodiment 1, the construction of glucose isomerase single point mutant

[0023] According to Thermobifida fusca glucose isomerase gene NCBI number: CP000088, the parental sequence was synthesized by chemical total synthesis, cloned into pMD18-T, and the vector xylA / pMD18-T was constructed. Design the mutation primers for site-directed mutation according to the parental sequence, use the rapid PCR technology, use the vector xylA / pMD18-T as the template, and the single mutation P284S primer is:

[0024] Primer B-ST-1: 5'-AGTGGCTACGACGGA TCG CGCCACTTCGACTTCAAGACC-3' (the underline is the mutated base)

[0025] Primer B-ST-2: 5'-GGTCTTGAAGTCGAAGTGGCG CGA TCCGTCGTAGCCACT-3' (the underline is the mutated base)

[0026] PCR reaction system: 2×PrimeSTARTM GC Buffer (containing Mg2+) 25μL, dNTPs (2.5mmol / L each) 4μL, forward primer (10μM) 1μL, reverse primer (10μM) 1μL, template DNA 1μL, PrimeSTARTM HS DNA Polymerase (2.5U / μl) 0.5μL, add double distilled water to 50μL.

...

Embodiment 2

[0029] Embodiment 2, the construction of double-site mutant of glucose isomerase

[0030] According to Thermobifida fusca glucose isomerase gene NCBI number: CP000088, the parental sequence was synthesized by chemical total synthesis, cloned into pMD18-T, and the vector xylA / pMD18-T was constructed. Design primers for site-directed mutagenesis based on the parental sequence, use rapid PCR technology, use the vector xylA / pMD18-T as a template, and double-mutate F243I and H244T, the site-directed mutation primers are:

[0031] Primer A-IT-1: 5'-TGGCACGGCAAACTG ATCACC ATCGACCTCAACGGC-3' (the underline is the mutated base)

[0032] Primer A-IT-2: 5'-GCCGTTGAGG TCGATGGTGA TCAGTTTGCC GTGCCA-3' (underline is the mutated base)

[0033] PCR reaction system: 2×PrimeSTARTM GC Buffer (containing Mg2+) 25μL, dNTPs (2.5mmol / L each) 4μL, forward primer (10μM) 1μL, reverse primer (10μM) 1μL, template DNA 1μL, PrimeSTARTM HS DNA Polymerase (2.5U / μl) 0.5μL, add double distilled water to 50μ...

Embodiment 3

[0036] Embodiment 3, the construction of four-site mutant of glucose isomerase

[0037] According to Thermobifida fusca glucose isomerase gene NCBI number: CP000088, the parental sequence was synthesized by chemical total synthesis, cloned into pMD18-T, and the vector xylA / pMD18-T was constructed. Design primers for site-directed mutagenesis based on the parental sequence, use rapid PCR technology, use the vector xylA / pMD18-T as a template, and double-mutate F243I / H244T, the site-directed mutation primers are:

[0038] Primer A-IT-1: 5'-TGGCACGGCAAACTG ATCACC ATCGACCTCAACGGC-3' (the underline is the mutated base)

[0039] Primer A-IT-2: 5'-GCCGTTGAGG TCGAT GGTGA T CAGTTTGCC GTGCCA-3' (the underline is the mutated base)

[0040] Then use the vector xylA(F243I / H244T) / pT7-7 as a template, double mutation P284S / H286T, and the site-directed mutagenesis primers are:

[0041] Primer B-ST-1: 5'-AGTGGCTACGACGGA TCG CGC ACC TTCGACTTCAAGACC-3' (the underline is the mutated base...

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Abstract

The invention discloses a glucose isomerase mutant and application thereof, belonging to the technical field of enzyme genetic engineering and enzyme engineering. According to the invention, a high temperature glucose isomerase gene (NCBI coding: CP000088) is obtained from total DNA of thermobifida fusca, and after site-directed mutagenesis, high efficiency expression of the glucose isomerase gene with a high conversion rate is realized, with the plasmid pT7-7 or a vector that can express glucose isomerase as an expression vector and E. coli BL21 (DE3) or a bacterial strain that can express glucose isomerase as an expression host; the glucose isomerase gene has altogether 1158 nucleotide and encodes 385 amino acids; expression plasmid is constructed in the invention, glucose isomerase is expressed through conversion of bacteria or yeast, and an obtained recombinase mutant has activity of glucose isomerase and has a conversion rate of 60% at a temperature of 70 DEG C, 7% higher than the conversion rate of a parent; optimum temperature of the recombinant glucose isomerase is 80 DEG C, an optimum pH is 10, and the half life of the recombinant glucose isomerase at a temperature of 70 DEG C is no less than 30 h. The recombinant glucose isomerase is particularly applicable to production of F55 high-fructose syrup in the industry of foodstuffs.

Description

technical field [0001] The invention relates to a glucose isomerase, in particular to a glucose isomerase mutant with high conversion rate and its production and application. technical background [0002] Glucose isomerase (GI), also known as xylose isomerase (XI) (EC5.3.1.5), can catalyze the isomerization of aldoses such as D-glucose to corresponding ketoses such as D-fructose. structuring reaction. Therefore, it is one of the key enzymes for large-scale production of high fructose syrup from starch in industrial production. [0003] High fructose syrup is also called high fructose syrup or isomerized syrup. It is composed of glucose and fructose by isomerizing the saccharification liquid obtained by enzymatically saccharifying starch through the isomerization of glucose isomerase. A mixed sugar syrup. High fructose syrup has the same sweetness as sucrose, pure flavor, rich nutrition, and has synergistic effect, cool and sweet taste, low calorie, no caries: good antisep...

Claims

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Application Information

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IPC IPC(8): C12N9/92C12P19/24C12N15/56C12N15/10C12N15/70C12R1/645
Inventor 吴敬陈晟邓辉陈坚
Owner JIANGNAN UNIV
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