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Catalase mutant and application thereof

A catalase and mutant technology, applied in the field of catalase mutants and their preparation, can solve problems such as insufficient research on enzymatic characteristics and catalytic activity, and achieve improved molar conversion rate of products and high industrialization and application prospects, the effect of improving enzyme activity

Active Publication Date: 2022-07-15
SHANDONG YANGCHENG BIOLOGY TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, few manganese catalases have been found so far, so their enzymatic characteristics and catalytic activities have not been studied much.

Method used

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  • Catalase mutant and application thereof
  • Catalase mutant and application thereof
  • Catalase mutant and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Construction of initial catalase gene recombinant Escherichia coli

[0041] 1. For the catalase gene derived from Bacillus mojavensis LDFZ001, our laboratory has constructed a recombinant vector pET28a-BmCAT derived from the catalase gene of Bacillus mojavensis LDFZ001 in the early stage, and its amino acid sequence is as shown in SEQ ID NO.1 shown.

[0042] 2. For catalase Cat derived from Bacillus subtilis R5 Bsu , according to the amino acid sequence published in the literature (AbeeraShaeer, et al., Structural and functional analyses of a novel manganese-catalase from Bacillus subtilis R5.Int J Biol Macromol, 180(2021): 222-233), namely SEQ ID NO.3, Based on this, the whole gene synthesis was carried out by Huada Company, the synthetic gene sequence was SEQ ID NO.4, and restriction endonuclease sites BamH I and Xho I were introduced at both ends of the gene, catalase Cat Bsu The digested fragment was ligated with the digested fragment of the vector pET28...

Embodiment 2

[0044] Example 2 Construction of random mutant library of initial catalase BmCAT by error-prone PCR

[0045] 1. Using the original catalase recombinant plasmid pET28a-BmCAT as a template, a random mutant library was constructed by error-prone PCR technology. The error-prone PCR kit was purchased from Clontech (Clontech, PT3393-1); the single-point mutation kit was purchased from Nanjing Novizan Biotechnology Co., Ltd. (Novizan, C214).

[0046] 2. The error-prone PCR kit (Clontech, PT3393-1) was used to carry out error-prone PCR amplification with plasmid pET28a-BmMnCAT as template and BmCAT-F and BmCAT-R as primers. The primer sequences are as follows:

[0047] BmCAT-F: acagcaaatgggtcgcggatccATGTTTAAACATACGAAAATGC

[0048] BmCAT-R: gtggtggtggtggtggtgctcgagTTACTCACGCCCAGGAAGCG

[0049] where lowercase letters are homology arm sequences.

[0050] The PCR reaction system is 50 μL, template DNA (final concentration is about 1 ng / μL) 1 μL, forward primer (10 nM) and reverse pri...

Embodiment 3

[0054] Example 3 High Throughput Screening of Mutant Libraries

[0055] 1. Preparation of Mutant Crude Enzyme Liquid Wet Bacteria

[0056] The mutant recombinant Escherichia coli monoclone obtained in Example 2 was picked into a sterile 96 deep-well plate, and each well contained 1 mL of LB liquid medium containing 50 μg / mL kanamycin, and cultured overnight at 37°C and 180 rpm. 8h, then transfer 500 μL of bacterial liquid at a ratio of 1:1 to another LB liquid medium containing 500 μL of kanamycin and a final concentration of 0.2 mM IPTG in each well, and continue to culture at 20 °C and 180 rpm. After 16 hours, the cells were collected by centrifugation at 8000 rpm at room temperature for 5 min to obtain 543 recombinant Escherichia coli wet cells containing mutant genes, namely mutant wet bacteria.

[0057] 2. Primary screening

[0058] The catalase activity detection was carried out using a catalase test kit (Sangon Biotechnology, D7995598-0100).

[0059] Standard enzyme ...

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Abstract

The invention discloses a catalase mutant and application thereof. The mutant is mutated at least at one of the following positions of an initial catalase amino acid sequence: 67th isoleucine is mutated into phenylalanine, 102nd methionine is mutated into leucine, 200th valine is mutated into phenylalanine, and 251st alanine is mutated into serine, and the initial catalase amino acid sequence is shown as SEQ ID NO.1. The invention further discloses a preparation method of the mutant. Compared with initial catalase BmCAT, the amino acid sequence of the catalase mutant is replaced, and the formed mutant remarkably improves the capability of catalyzing hydrogen peroxide. In the process of biosynthesizing pyruvic acid by using lactate oxidase, hydrogen peroxide generated in the reaction process is eliminated by using whole-cell catalysis for expressing the catalase mutant, the use amount of enzyme is small, the elimination rate of hydrogen peroxide is remarkably improved, the maximum molar conversion rate of the product pyruvic acid is 99.7%, and the method has a very high industrial application prospect.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a catalase mutant and a preparation method and application thereof. Background technique [0002] In living organisms, cells are accompanied by the production of hydrogen peroxide due to the oxidative decomposition of substances and the transfer of electrons in the process of metabolism. Hydrogen peroxide is toxic to cells, and catalase in the body is the key enzyme responsible for decomposing hydrogen peroxide and eliminating its toxicity to cells. Hydrogen peroxide in living organisms is often produced in reactions catalyzed by some oxidase enzymes. Therefore, in the process of using some oxidases as catalysts to produce compounds, the participation of catalase is usually required to eliminate the production of hydrogen peroxide in the reaction process. , reduce the inhibition of enzyme activity by hydrogen peroxide. Also in the printing and dyeing industry, hydrogen ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/08C12N15/53C12N15/70C12N1/21C12P7/40C12R1/19
CPCC12N9/0065C12Y111/01006C12N15/70C12P7/40Y02E50/10
Inventor 张娟冯志彬
Owner SHANDONG YANGCHENG BIOLOGY TECH CO LTD
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