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Glucose oxidase mutant for improving specific activity as well as coding gene and application of glucose oxidase mutant

A glucose oxidase and mutant technology is applied to the glucose oxidase mutant and its encoding gene and application fields, which can solve the problems of low specific activity of GOD, restricting industrial application and high production cost, and achieves reduction of production cost and application effect. Good, improve the effect of specific activity

Active Publication Date: 2018-08-07
GUANGDONG VTR BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Wild-type Aspergillus niger has low GOD specific activity and high production cost, which limits its industrial application

Method used

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  • Glucose oxidase mutant for improving specific activity as well as coding gene and application of glucose oxidase mutant
  • Glucose oxidase mutant for improving specific activity as well as coding gene and application of glucose oxidase mutant
  • Glucose oxidase mutant for improving specific activity as well as coding gene and application of glucose oxidase mutant

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] The cloning of the glucose oxidase (GOD) gene of embodiment 1, aspergillus niger (Aspergillus niger)

[0026] Aspergillus niger was inserted into LB medium, and after 24 hours of culture, its genomic DNA was extracted. Two primers (GODF: 5'-cggaattcagcaatggcatcgaagccagcctc-3' and GODR: ​​5'-atagtttagcggccgctcactgcatggaagcataatc-3') were designed based on the reported sequence of Aspergillus niger glucose oxidase (Genebank: FJ979866.1) for the amplification of glucose oxidase in Aspergillus niger enzyme gene. The amplified PCR products were purified and recovered, and respectively connected to expression vectors pPICzαA and pPGAPzαA to obtain expression vectors pPICzαA-GOD and pGAPzαA-GOD.

Embodiment 2

[0027] Embodiment 2, rational site-directed mutation

[0028] Use the above pPICzαA-GOD as a template and perform PCR amplification with the primers in the table. The specific amplification reaction system is as follows:

[0029] Q5 High Fidelity Taq Enzyme MIX

23uL

Corresponding mutant primer 1 (50uM)

1uL

Corresponding mutant primer 2 (50uM)

1uL

pPICzαA-GOD (20ng)

2uL

Add water to

50uL

[0030] When the mutation site is V20Y, the corresponding mutant primer 1: 5'-gttgccggccgcacttacgactacatcatcgct-3'; the corresponding mutant primer 2: 5'-agcgatgatgtagtcgtaagtgcggccggcaac-3'. When the mutation site is T34V, the corresponding mutant primer 1: 5'-tctgactggactcaccgtcgctgcccgtctgacgg-3'; the corresponding mutant primer 2: 5'-ccgtcagacgggcagcgacggtgagtccagtcaga-3'. When the mutation site is D70Q, the corresponding mutant primer 1: 5'-gacctgaacgcttacggtcagatttttggcagcagtgtg-3'; the corresponding mutant primer 2: 5'-cacac...

Embodiment 3

[0033] Embodiment 3, high-throughput screening high specific activity mutant strain

[0034] Pick the recombinant yeast transformants obtained in Example 2 to a 24-well plate one by one with a toothpick, add 1 mL of medium containing BMGY to each well, culture at 30° C., 220 rpm for about 24 hours, and centrifuge to remove the supernatant. Then add 1.6mL BMMY medium respectively for induction culture. After culturing for 24 hours, the supernatant was collected by centrifugation, and 200 μL of the above supernatant was taken out to a 96-well plate, and the enzyme activity of glucose oxidase was measured. After high-throughput screening, six effective mutation sites were obtained: V20Y, T34V, D70Q, V106I, Q257K, and G517A. The relative specific activities of these six mutants are shown in Table 1.

[0035] Table 1 Relative specific activity of original glucose oxidase and mutant glucose oxidase

[0036] serial number

[0037] It can be seen from Table 1 that the abo...

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Abstract

The invention discloses a glucose oxidase mutant for improving specific activity as well as a coding gene and application of the glucose oxidase mutant, relating to the genetic engineering field. Genes of glucose oxidase (GOD) of Aspergillus niger GIM 3.452(CICC 2377) is subjected to site-specific mutagenesis by virtue of a site-specific mutagenesis technique and an error-prone PCR method, and mutation sites include loci 10, 20, 34, 70, 106, 110, 167, 257, 305, 362 and 517. The specific activity of the obtained mutant is higher than that of an original strain, and the mutant can well meet theapplication requirements in the fields of foods, medicines, feeds, textile industry and the like and has very wide application prospects.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a glucose oxidase mutant with improved specific activity, its coding gene and application. Background technique [0002] Glucose oxidase (glucose oxidase, GOD) can specifically catalyze β-D-glucose to generate gluconic acid and hydrogen peroxide under aerobic conditions. Due to the special enzymatic properties of GOD, it is widely used in food, feed, medicine and other fields. GOD is widely distributed in animals, plants and microorganisms. Compared with animals and plants, microorganisms have the advantages of fast growth and reproduction and wide sources. Therefore, GOD mainly comes from microorganisms. [0003] GOD from microbial sources is mainly Aspergillus niger and Penicillium. Compared with Penicillium, GOD produced by Aspergillus niger has the advantages of good thermal stability and strong substrate specificity. The wild-type Aspergillus niger has low GOD specific a...

Claims

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Application Information

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IPC IPC(8): C12N9/04C12N15/53A23K20/189A21D8/04
CPCA21D8/042A23K20/189C12N9/0006C12Y101/03004
Inventor 聂金梅李阳源刘金山唐业王建荣
Owner GUANGDONG VTR BIO TECH
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