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High-specific activity L-glutamate oxidase gene multisite mutant, and preparation method and application thereof

A technology of glutamate oxidase and multi-site mutation, which is applied in the field of microorganisms and can solve problems such as application limitations and expensive L-glutamate oxidase

Active Publication Date: 2017-01-04
SHANGHAI ACAD OF AGRI SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, mainly because the source of L-glutamate oxidase products has not been fundamentally resolved, the price of L-glutamate oxidase is still very expensive, resulting in its application being limited

Method used

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  • High-specific activity L-glutamate oxidase gene multisite mutant, and preparation method and application thereof
  • High-specific activity L-glutamate oxidase gene multisite mutant, and preparation method and application thereof
  • High-specific activity L-glutamate oxidase gene multisite mutant, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1 DNA molecular rearrangement (DNA Shuffling) of L-glutamate oxidase (SDLDOX) gene derived from amylase Streptomyces chromogenes

[0049] 1.1 Gene synthesis of L-glutamic acid oxidase (SDLDOX) derived from amylase Streptomyces chromogenes

[0050] By gene synthesis method (Nucleic Acids Research, 2004, 32, e98) synthetic amylase L-glutamic acid oxidase gene (SDLDOX) of Streptomyces chromogenes, used primer is as follows:

[0051] SDLG1: GGATCCATGACTGAAACTCCACGTGATAATTCTGCAACTCGTGCACGTTGGCAAACTTGT (shown in SEQ ID NO.1)

[0052] SDLG2: CATCAGGACCAACAAGAAGCAGTTCACGTGCCAGCTT GAGACAAGTTTGCCAACGTGCAC (shown in SEQ ID NO.2)

[0053] SDLG3: GCTTCTTGTTGGTCCTGATGACAAGGATCTGAAACTGT CCTATCTGCATACTCTGATTGA (shown in SEQ ID NO.3)

[0054] SDLG4: CTTCTTACGTGGATGATGAGTTGGACCCAG ACGACCAG TATCAATCAGAGTATGCAGATA (shown in SEQ ID NO.4)

[0055] SDLG5: CTCATCATCCACGTAAGAAGATCCTGGTCATTGGTGCT GGTATCACTGGTCTGGTTGCTG (shown in SEQ ID NO.5)

[0056] SDLG6: ATGATAGTGACATCGTAACCAGCATC...

Embodiment 2

[0110] Example 2 High specific activity L-glutamic acid oxidase (SDLDOX) gene screening

[0111] The rearranged L-glutamic acid oxidase gene fragment recovered in Example 1 was constructed between the prokaryotic expression vector pG251 (CN1338515) promoter and the t1t2 terminator after BamH I and SacI double digestion. Ampicillin resistance gene. Transform Escherichia coli strain DH5α by electroporation to obtain mutant expression library with a capacity of 10 8 , and then use a large number of plasmid extraction kits (Omega, USA) for plasmid extraction.

[0112] Take 1 μl of a large amount of extracted plasmid and transfer it into Escherichia coli DH5α, spread it on the antibiotic medium containing 100mg / L ampicillin, and cultivate it at 37°C for 16 hours, and select resistant transformants for L-glutamic acid oxidase activity screening. The specific library screening method is as follows: transformants are picked into a 40-well bacterial culture plate, 50 μl of pH 8.0 pho...

Embodiment 3

[0115] Example 3 Acquisition of High Specific Activity L-Glutamic Acid Oxidase Gene Mutant

[0116]Using the step-by-step sequencing method to carry out DNA sequencing on the full sequence of the five high specific activity L-glutamic acid oxidase genes screened in Example 2, it was shown that there were changes in 21 nucleotide sites T27C, T33A, A95C , A158C, A431T, G464A, C497A, T633A, T714C, C744A, C956A, C957G, C1085T, T1293C, T1410G, A1440C, T1479A, C1588A, C1701T, A1728T, A15F42A, H( G155D, T166K, T319K, A362V, S567F, K600R), the specific results are shown in Table 1.

[0117] Table 1 Nucleotide and amino acid changes of L-glutamate oxidase gene mutants

[0118]

[0119] Using the L-glutamic acid oxidase (SDLDOX) gene SDLGOXM2 with mutated 3 sites as a template, all the other 6 screened mutation sites were mutated to complete 9 site mutations. Primers used for mutation are as follows.

[0120] Primers for the H54P mutation:

[0121] 54Z: GCCGCCTCGGCCCCACCCCCCA (sh...

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Abstract

The invention relates to a high-specific activity L-glutamate oxidase gene multisite mutant, and a preparation method and application thereof. The nucleotide sequence of the mutant is shown as SEQ ID No 64; the amino acid sequence of encoded protein is shown as SEQ ID No 65. A molecular in vitro recombination technique is used for screening high-specific activity L-glutamate oxidase gene; through vector building, enzyme activity screening, multisite mutation techniques and the like, the high-specific activity L-glutamate oxidase gene multisite mutant is obtained through preparation. The multisite mutant contains 9 different mutation sites on the same gene, so that the oxidation specificity and the specific activity of the multisite mutant on L-glutamic acid are greatly improved; the multisite mutant can be used for detecting the content of the L-glutamic acid in food.

Description

technical field [0001] The invention belongs to the field of microorganisms, and relates to a high specific activity L-glutamic acid oxidase gene multi-site mutant derived from Streptomyces amylase chromogenes and its preparation method and application. Background technique [0002] L-glutamate oxidase (L-Glutamate oxidase, LGOX) can specifically catalyze the oxidation of glutamic acid into α-ketoglutaric acid and hydrogen peroxide. amino acid content. However, mainly because the source of the L-glutamic acid oxidase product has not been fundamentally resolved, the price of the L-glutamic acid oxidase is still very expensive, resulting in its application being limited. [0003] In 1983, Japanese Kamei et al first discovered an enzyme that could catalyze the oxidation of glutamic acid to produce α-ketoglutarate and hydrogen peroxide from the extract of wheat bran culture medium of Streptomyces violascens. It is a typical oxidase rather than a dehydrogenase, and NAD+ and NAD...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N9/06C12N15/70C12Q1/26
Inventor 彭日荷姚泉洪王荣谈田永生王丽娟丁卫星严培兰王波孙斌
Owner SHANGHAI ACAD OF AGRI SCI
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