Recombined microorganism glutamine transaminase gene and preparation method thereof

A transaminase gene and recombinant microorganism technology, applied in the fields of protein expression and genetic engineering, can solve the problems of difficult screening of high-yield transglutaminase-producing strains, large-scale industrial production of restriction enzymes, and failure to reach the level of industrialization. less foaming, reduced foaming, and low-cost effects

Inactive Publication Date: 2014-02-05
SHAOXING UNIV YUANPEI COLLEGE
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Problems solved by technology

At present, the complete gene encoding TGase and the amino acid sequence of TGase protein have been cloned and sequenced, and the TGase activity level obtained by them is very low, which is far from reaching the level of industrialization; the transglutaminase from microbial sources is not affected by season and season because of its production. Raw material limitation is one of the most likely large-scale production applicat

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  • Recombined microorganism glutamine transaminase gene and preparation method thereof
  • Recombined microorganism glutamine transaminase gene and preparation method thereof
  • Recombined microorganism glutamine transaminase gene and preparation method thereof

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Embodiment Construction

[0023] 1. Isolation and cloning of microbial transglutaminase gene: Genomic DNA was extracted from Bacillus producing transglutaminase as a template, and the specific DNA extraction method was referred to Promega instructions, according to the gene sequence of Bacillus subtilis transglutaminase reported in Genbank ( The accession number is: E13095) designed and synthesized primers as follows: upstream primer TgP1:

[0024] 5′-AGG GGATCC GATTATTGTATCAGGACAATTGCTCCGT-3′,

[0025] TgP2: 5′-GAT TCTAGA TCATTAGCGGACGATGCGGAAAAG-3′; BamHI and XbaI restriction sites and corresponding protective bases were added to its 5-terminus to amplify the full-length transglutaminase gene. PCR amplification conditions: 95°C pre-denaturation for 3 minutes, 95°C Denaturation for 30 seconds, renaturation at 59°C for 1 minute, extension at 72°C for 1 minute, 30 cycles; extension reaction at 72°C for 10 minutes; prepare 0.8% TBE agarose gel, take 5 μL of PCR amplification product and mix with 6× ...

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Abstract

The invention relates to a recombined microorganism glutamine transaminase gene of a bacillus and expression thereof, belonging to the field of food industrial biotechnology. After the glutamine transaminase gene from the bacillus is cloned and separated, a lactococcus lactis recombinant expression vector spNZ8048-Tgase containing the gene is established and expressed in a lactococcus lactis N9800 strain; the recombined lactococcus lactis is named L.lactis; after an expression product is separated and purified, a recombined glutamine transaminase with a purity of about 90% is obtained; the activity of the recombined glutamine transaminase reaches 2.2U/mL which is greater than twice the activity of the original strain. Due to the adoption of the lactococcus lactis in production of the glutamine transaminase, the production method of the recombined microorganism glutamine transaminase gene has the advantages of high enzyme-producing activity, low production cost and good safety and is simple to operate, and the expressed enzyme is easy to separate and purify, so that a feasible manner for production of the glutamine transaminase is provided.

Description

technical field [0001] The present invention relates to the field of genetic engineering and protein expression, in particular to the soluble expression of transglutaminase zymogen gene in a lactic acid bacteria expression system, specifically a method for preparing recombinant Bacillus microbial transglutaminase. Background technique [0002] The animal product processing industry is an important industry that connects animal husbandry production with people's living needs. On the one hand, with the rapid development of animal husbandry, my country's animal products show a balance between supply and demand and surplus characteristics, but the processing rate of animal products is only about 5%, which is far lower than 60% to 70% in developed countries. Seriously lagging behind, restricting the rapid and efficient development of animal husbandry. On the other hand, with the increase of people's economic income, the improvement of living standards, the optimization of dietary...

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Application Information

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IPC IPC(8): C12N15/54C12N15/10C12N15/74C12N9/10
Inventor 胡春霞
Owner SHAOXING UNIV YUANPEI COLLEGE
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