Glucose isomerase and gene, mutant, engineering bacteria and application thereof

A technology of glucose isomerase and genetically engineered bacteria, applied in the field of genetic engineering, can solve the problem of low reaction temperature, achieve high yield, simple process, and easy industrial application

Inactive Publication Date: 2015-07-01
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to provide a glucose isomerase, coding gene, recombinant vector, genetically engineered bacteria and its mutants, and the application of glucose isomerase genetically engineered bacteria in catalyzing the isomerization of D-glucose to prepare D-fructose, The high-temperature-resistant glucose isomerase highly expressed by recombinant Escherichia coli provided by the present invention solves the problem of low optimum reaction temperature of general glucose isomerase. The conversion rate reached 55.4%, providing good technical support for the large-scale production of high fructose syrup (HFCS-55)

Method used

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  • Glucose isomerase and gene, mutant, engineering bacteria and application thereof
  • Glucose isomerase and gene, mutant, engineering bacteria and application thereof
  • Glucose isomerase and gene, mutant, engineering bacteria and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0043] Example 1: Gene synthesis of glucose isomerase

[0044] The gene of glucose isomerase is derived from the ctg00047 sequence fragment of the whole genome shotgun sequencing of Thermoanaerobacter ethanolicus CCSD1, wherein the bases from 7706bp to 9022bp are the sequence encoding the glucose isomerase gene (GenBank No.EEU61835.1, GI 256748793). In order to express the protein with His-tag after the gene is connected to the vector pET-28b, its stop codon was excised, and its sequence and common restriction endonuclease were compared with the codon preference of B. subtilis 168 as a reference The sequences of the recognition sites BamH I, Xho I, Pst I, Hind III and Nco I were optimized. The sequence of the newly designed glucose isomerase gene (tegi) is shown in SEQ ID NO.1. The gene synthesis work was entrusted to Shanghai Xuguan Completed by Biotechnology Development Co., Ltd., the gene was synthesized and connected to the cloning vector pES.

Embodiment 2

[0045] Example 2: Construction of glucose isomerase recombinant Bacillus subtilis

[0046] On the basis of Example 1, primers were designed respectively for different Bacillus subtilis expression vectors, using PCR technology, with the synthetic nucleotide sequence (shown in SEQ ID NO.1) as template, glucose isomerase gene ( tegi) for amplification. For expression vector pP43NMK, design primer tegi-NMK-F: 5'-AAAA CTGCAG ATGGAATACTTCAAAAACG-3' and tegi-NMK-R:5'-CCC AAGCTT TTATTCAGAGAAAAGGTATTGG-3', and introduce Pst I and Hind III restriction enzyme sites respectively; for expression vector pMA0911, design primer tegi-MA-F: 5'-CCG GAATTC ATGGAATACTTCAAAAACG-3' and tegi-MA-R:5'-CGC GGATCC TTATTCAGAGAAAAGGTATTGG-3', and introduced EcoR I and BamH I restriction enzyme sites respectively.

[0047]The PCR reaction system (50 μL) was: 5 μL of 10×Pfu PCR buffer, 8 μL of dNTP Mixture; 1 μL of template DNA; 2 μL of upstream and downstream primers; 0.5 μL of Pfu DNA polymerase; 3...

Embodiment 3

[0050] Embodiment 3: the enzyme activity assay of recombinant Bacillus subtilis

[0051] Recombinant bacteria B. subtilis WB800 / pP43NMK-tegi and B. subtilis WB800 / pMA0911-tegi after enzyme digestion and sequencing verification were inoculated into 50 mL LB liquid medium (containing kanamycin 40 μg / mL), 37 °C , 150r / min shaking culture to OD 600 =0.8~1.0; the culture solution was inoculated into fresh 100mL LB liquid medium containing 40μg / mL kanamycin with 2% (v / v) inoculation amount, and cultured with shaking at 37°C and 150r / min for 10~24h.

[0052] Take the cell culture solution, centrifuge at 1000r / min for 5min, take the cell pellet and supernatant respectively, add 20μL SDS buffer solution to mix, heat in a boiling water bath for 5min, and perform SDS-PAGE analysis, and use the empty host without carrier as a control, As a result, no obvious glucose isomerase protein band was found; correspondingly, no enzyme activity of glucose isomerase was detected. It indicated that...

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Abstract

The invention discloses a glucose isomerase, an encoding gene, a recombinant vector, a genetically engineered bacteria and a mutant thereof and an application of the glucose isomerase and the mutant thereof in preparation of D-fructose for catalyzing D-glucose isomerization. A recombinant escherichia coli capable of efficiently expressing high-temperature resistant glucose isomerase, provided by the invention, solves the problem of low optimal reaction temperature of a common glucose isomerase, is applied to production of the glucose isomerase and has the advantages of high yield, simple process and convenient industrial application; the strain can be directly used for the high fructose syrup production without cell disruption; after finishing fermentation, the total enzyme activity for D-fructose at the temperature of 90 DEG C is 414.3U / g, the conversion rate of D-fructose is 55.4%, the remnant enzyme activity at the temperature of 90 DEG C after storage for 24h is 68% and the equilibrium of reaction is shortened to 1.5h; a good technical support for the large-scale production of the high fructose syrup and for the use of high fructose syrup as an important food additive can be provided.

Description

(1) Technical field [0001] The invention relates to a glucose isomerase gene, a mutant, a glucose isomerase-producing genetically engineered bacterium, a construction method and application thereof, and belongs to the field of genetic engineering. (2) Technical background [0002] Glucose isomerase (Glucose isomerase, referred to as GI, EC 5.3.1.5), also known as xylose isomerase (Xylose isomerase, XI), can catalyze the isomerization reaction of D-glucose and D-xylose, which are converted into D-fructose and D-xylulose are one of the important biocatalysts in the food industry, especially in the process of biotransformation to prepare high fructose syrup. [0003] High fructose syrup (HFCS) is a mixture of glucose and fructose, also known as fructose syrup, and is an important sweetener. According to the content of fructose, high fructose syrup can be divided into three types, HFCS-42 (fructose 42%, glucose 53%, polysaccharide 5%), HFCS-55 (fructose 55%, glucose 42%, polysa...

Claims

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Application Information

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IPC IPC(8): C12N9/92C12N15/61C12N1/21C12P19/24C12P19/02C12R1/19
CPCC12N9/92C12P19/02C12P19/24
Inventor 柳志强郑裕国郑微周海岩刘成龙廖承军陈德水程新平毛宝兴
Owner ZHEJIANG UNIV OF TECH
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