Glucose isomerase genes, encoding enzymes, vectors and engineering bacteria and application

A technology of glucose isomerase and genetically engineered bacteria, applied in the field of genetic engineering, can solve the problems of weak acid resistance, difficult to meet the needs of HFCS-55 production, etc., and achieves the effects of high yield, convenient industrial application and simple process

Inactive Publication Date: 2015-01-21
ZHEJIANG UNIV OF TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, some high-temperature-resistant glucose isomerases have weak acid resistance, and will catalyze the formation of colored substance

Method used

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  • Glucose isomerase genes, encoding enzymes, vectors and engineering bacteria and application
  • Glucose isomerase genes, encoding enzymes, vectors and engineering bacteria and application
  • Glucose isomerase genes, encoding enzymes, vectors and engineering bacteria and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Gene synthesis of glucose isomerase

[0036] The gene of glucose isomerase is derived from the whole genome sequence of Thermotoga petrophila RKU-1 (DSM NO.13995; JCM NO.10881), in which bases from 1149014bp to 1150348bp are the sequence encoding the glucose isomerase gene (GenBank No. YP_001244714, GI 148270254). In order to express the protein with the His-tag after the gene is connected to the vector pET-28b, its stop codon was excised, and the common restriction endonuclease recognition site BamH I was used in its sequence according to the codon degeneracy , Xho I, Pst I, Hind III and Nco I were sequence optimized, and the sequence of the newly designed glucose isomerase gene (tpgi) is shown in SEQ ID NO.1. The gene synthesis was entrusted to Shanghai Xuguan Biotechnology Development Co., Ltd. After completion, the gene was synthesized and connected to the cloning vector pES.

Embodiment 2

[0037] Example 2: Construction of glucose isomerase recombinant Bacillus subtilis

[0038] 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 ( tpgi) for amplification. For expression vector pP43NMK, design primer tpgi-NMK-F (5'-AAAA CTGCAG ATGATGGAATTTTTCCCTG-3') and tpgi-NMK-R (5'-CCC AAGCTT TTAACGAAGTTCAGCGATTG-3'), and introduce Pst I and Hind III restriction enzyme sites respectively; for expression vector pMA0911, design primer tpgi-MA-F (5'-CCG GAATTC ATGATGGAATTTTTCCCTG-3') and tpgi-MA-R (5'-CGC GGATCC TTAACGAAGTTCAGCGATTG-3'), and introduced EcoR I and BamH I restriction enzyme sites respectively.

[0039] 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...

Embodiment 3

[0042] Example 3: Protein electrophoresis and enzyme activity assay of recombinant Bacillus subtilis

[0043] Recombinant bacteria B. subtilis WB800 / pP43NMK-tpgi and B. subtilis WB800 / pMA0911-tpgi after enzyme digestion verification 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.

[0044] 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 isomeras...

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Abstract

The invention discloses glucose isomerase genes, encoding enzymes, recombinant vectors and genetic engineering bacteria and application of glucose isomerase gene engineering bacteria in aspects of preparation of D-fructose by virtue of catalysis of D-glucose isomerization. According to the recombinant Escherichia coli for efficiently expressing high-temperature-resistant glucose isomerase, the problem that general glucose isomerase is low in optimal reaction temperature is solved; the glucose isomerase produced by using the strain has the advantages of high yield, simple process, convenient industrial application and the like, the strain can be directly used for producing high fructose syrup, and cell disruption is not needed; and moreover, after fermentation is ended, the total enzyme activity of D-glucose reaches 715U/L under the condition of 85 DEG C, and the conversion rate of the D-fructose is 55 percent, so that a good technical support is provided for performing large-scale production of the high fructose syrup and taking the high fructose syrup as an important food additive.

Description

(1) Technical field [0001] The invention relates to a genetically engineered bacterium producing glucose isomerase (GI for short, EC5.3.1.5), a construction method and application thereof, and belongs to the field of genetic engineering. (2) Background technology [0002] Glucose isomerase (Glucose isomerase, referred to as GI, EC5.3.1.5), also known as xylose isomerase (Xylose isomerase, XI), can catalyze the isomerization reaction of D-glucose and D-xylose, respectively converting It is D-fructose and D-xylulose, and it is one of the important biocatalysts in the field of food industry, especially it plays a key role 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 (fr...

Claims

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Application Information

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IPC IPC(8): C12N9/90C12N15/61C12N15/70C12N1/21C12P19/24C12R1/19
CPCC12N9/92C12P19/24C12Y503/01005
Inventor 郑裕国周海岩柳志强刘成龙廖承军陈德水程新平毛宝兴张为宏
Owner ZHEJIANG UNIV OF TECH
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