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

A technology of glucose isomerase and genetically engineered bacteria, applied in the direction of genetic engineering, isomerase, application, etc., to achieve the effect of high enzyme activity and excellent thermal stability

Active Publication Date: 2017-04-19
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] At present, there have been some heat-resistant GI reports, such as Thermotoga maritima and Thermusthermophiles, etc., whose optimum temperatures reach 105°C and 95°C respectively, but these enzymes have not been made into enzyme preparations and successfully put on the market

Method used

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

Examples

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Effect test

Embodiment 1

[0026] Example 1: Gene synthesis of glucose isomerase

[0027] The glucose isomerase gene sequence was screened using the protein PDB database and NCBI database, and the glucose isomerase gene (GenBank accession no.WP_016329521.1) derived from Thermus oshimai was obtained. According to the amino acid sequence of the glucose isomerase, and codon optimization according to the codon preference of Escherichia coli, the nucleotide sequence of the glucose isomerase was synthesized by the conventional operation of genetic engineering, such as SEQ ID Shown in NO.2; the amino acid sequence encoding the enzyme is shown in SEQ ID NO.1. Add 6×his-tag tags at the end of the nucleic acid sequence, add restriction sites Xba I and Xho I at both ends, clone the gene into the Xba I and Xho I sites corresponding to pET28b(+), and obtain the recombinant expression plasmid pET28b / ToGI.

Embodiment 2

[0028] Embodiment 2: Transformation of recombinant plasmid and screening of recombinant bacteria

[0029] The recombinant expression plasmid pET28b / ToGI obtained in Example 1 was transformed into Escherichia coli BL21 (DE3) recipient bacteria, spread on an agar plate containing a final concentration of 50 μg / mL kanamycin, and cultured overnight at 37°C. On the second day, clones were randomly selected from the colonies grown on the plate, and the plasmids were extracted for identification by agarose gel electrophoresis to obtain genetically engineered bacteria containing the glucose isomerase gene.

Embodiment 3

[0030] Embodiment 3: Induced expression of recombinant engineering bacteria

[0031] LB liquid medium composition: tryptone 10g / L, yeast powder 5g / L, NaCl 10g / L, solvent is water, pH value is natural; LB solid medium is added 20g / L agar in LB liquid medium; 121℃ high pressure Sterilize for 20 minutes; add kanamycin at a final concentration of 50 μg / mL before use.

[0032] Inoculate the genetically engineered bacteria containing the glucose isomerase gene obtained in Example 2 into LB liquid medium containing a final concentration of 50 μg / mL kanamycin, and cultivate the OD at 37°C and 150r / min. 600 About 0.6-0.8 to obtain the seed solution; inoculate the seed solution with a volume concentration of 2% inoculum into fresh LB medium containing a final concentration of 50 μg / mL kanamycin, and cultivate OD at 37°C and 150r / min 600 To 0.6-0.8, add IPTG with a final concentration of 0.1mM to the culture medium, induce expression at 28°C for 10h, centrifuge at 8000r / min at 4°C for 1...

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Abstract

The invention discloses a glucose isomerase, a gene, a vector, an engineering bacteria and the application thereof in preparing D-fructose through catalyzing the D-glucose isomerization. The amino acid sequence of the glucose isomerase is shown in SEQ ID NO. 1. The invention provides a novel high-temperature-resistant glucose isomerase. The glucose isomerase is higher in enzyme activity (2.42 U / mg) and excellent in thermal stability. At the temperature of 85 DEG C, 20 mM of a manganese salt additive is added and 80% of the initial enzyme activity is still retained even after the temperature is maintained for 48 hours. Therefore, the glucose isomerase has an industrial application potential in the biocatalytic production of high fructose syrup at a high temperature.

Description

[0001] (1) Technical field [0002] The invention relates to a glucose isomerase, a gene, a recombinant vector containing the gene, a recombinant genetically engineered bacterium obtained by transforming the recombinant vector, and the application of isomerizing glucose at high temperature to produce high fructose syrup. [0003] (2) Background technology [0004] 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). [0005] High fructose corn syrup (HFCS for short) is a mixture of glucose and fr...

Claims

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Application Information

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IPC IPC(8): C12N9/92C12N15/61C12N15/70C12N1/21C12P19/24C12P19/02C12R1/19
CPCC12N9/92C12P19/02C12P19/24C12Y503/01005
Inventor 郑裕国贾东旭周霖
Owner ZHEJIANG UNIV OF TECH
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