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Strain capable of producing transglutaminase

A technology of glutamine and transaminase, applied in the field of genetic engineering, can solve the problems of low fermentation enzyme activity, limited application value, and limited industrial production capacity, and achieve the effect of simplifying the extraction process and shortening the fermentation time

Inactive Publication Date: 2020-06-30
NANJING BESTZYME BIO ENG CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But on the one hand, its fermentation enzyme activity output is low, and the fermented liquid has only 2U / ml, has limited its industrial production capacity; It is banned in the fermentation industry, which greatly limits its application value

Method used

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  • Strain capable of producing transglutaminase
  • Strain capable of producing transglutaminase
  • Strain capable of producing transglutaminase

Examples

Experimental program
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Effect test

Embodiment 1

[0035] Embodiment 1: the construction of pksB plasmid

[0036] pksB( figure 1 shown) is a thermosensitive E.coli / B.subtilis shuttle plasmid. The plasmid includes a temperature-sensitive origin of replication (active at 30°C) and an erythromycin resistance gene (ermC). The resistance of the resistance gene in E.coli is 300ug / ml, and in B.subtilis The resistance in is 5ug / ml. At 37°C, the origin of replication on the plasmid is inactivated, and the plasmid is integrated into the designated site of the host bacterial genome, and is screened with a certain concentration of erythromycin.

[0037] The construction method of pksb plasmid is as follows. Plasmid pUC57-KS-erm (synthesized by Genscript Company, sequence SEQ ID NO: 1) was double-digested with BglII, and the 3.8kbp fragment was recovered and purified, self-ligated with T4 ligase (purchased from New England Biolabs), and the cloned plasmid was It is pksB. This plasmid was transformed into E. coli TOP10 for propagation ...

Embodiment 2

[0038] Embodiment 2: the construction of protease-deficient Bacillus subtilis bacterial strain

[0039] The subtilisin E (AprE), neutral protease E (NprE) and sigF genes were knocked out using a single-crossover Campbell-type mechanism in a sequential manner. The details are as follows: use BglII endonuclease to treat the plasmid pksB, and then use CIP enzyme to prevent self-ligation after digestion and recovery to obtain the linear fragment of the vector; respectively amplify the homology of about 500 bp flanking each gene to be knocked out by PCR method area. Using a single colony of Bacillus subtilis as a template, PCR reaction primers were synthesized by Genscript, wherein pksb-apr_R1 / pksb-apr_F2, pksb-npr_R1 / pksb-npr_F2 and pksb-sig_R1 / pksb-sig_F2 were used for PCR reaction amplification apr, The flanking sequences of npr and sigF genes, the primer sequences are as follows:

[0040] The primers for amplifying the upstream sequence of the apr gene are:

[0041] pksb-apr...

Embodiment 3

[0069] Embodiment 3: the construction of MTG production bacterial strain

[0070] The fragments of 500 bp upstream and downstream of the amyE gene were integrated into the plasmid pksB to construct the plasmid pksB-amyFR. The specific method is as follows: the plasmid pksB( figure 1 ) was digested with KpnI-EcoRI to recover and purify the 3711bp fragment pksB-KpnI-EcoRI; use primers pksb-amyF-F / pksb-amyF-R and pksb-amyR-F / pksb-amyR-R to amplify the insert respectively The upstream and downstream homology arm sequences of amyE are amyF and amyR; then using overlapping PCR technology, the upper and lower primers pksb-amyF-F / pksb-amyR-R are amplified to connect the fragments to obtain the gene fragment amyFR; use KpnI-EcoRI to digest The amyFR fragment was then ligated with the linearized vector pksB-KpnI-EcoRI through T4 ligase (purchased from New England Biolabs), and the cloned plasmid was pksB-amyFR. The plasmid was transferred into E.coli TOP10 for propagation and served a...

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Abstract

The invention discloses a strain capable of producing transglutaminase. The strain capable of producing the transglutaminase provided by the invention can be obtained by the steps of knocking out geneAprE of subtilisin of bacillus subtilis, gene NprE of neutral protease and sigF gene, and constructing a host strain; and integrating an expression cassette of a MTG zymogen gene into the host strainto obtain the strain capable of producing the transglutaminase. The engineering strain constructed by the invention is used for MTG fermentation, MTG zymogen is activated by endogenous extracellularprotease of the host cell in the fermentation process, so that other exogenous proteases do not need to be integrated for a subsequent activation process, MTG having activity can be directly recoveredin a fermentation supernatant, and the extraction process is simplified; and the method has the advantages of short fermentation time and no need of adding antibiotics in the process, and has great industrial value.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and specifically relates to a bacterial strain capable of producing glutamine transaminase and its application in producing glutamine transaminase. Background technique [0002] Transglutaminase (Transglutaminase, EC 2.3.2.13, TGase) is a kind of protein with amido transfer catalytic function, which can catalyze the acyl transfer reaction of converting glutamine residues in peptide chains into γ-carboxamide groups , especially form ε-(γ-Gln)-Lys cross-links with ε-acyl groups of lysine residues in protein molecules and intermolecularly. The special catalytic function of TGase makes it widely used in many fields, especially in the field of food processing. As a safe food cross-linking agent, TGase is used in industries such as noodle products, baked products, meat products and soybean products. In addition, TGase enzyme has a wide range of applications in textile and leather processing, tissue ...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/75C12N15/54C12N9/10C12R1/125
CPCC12N9/1044C12N9/54C12N15/75C12Y203/02013
Inventor 曹国强白挨玺严婷孙艳徐红
Owner NANJING BESTZYME BIO ENG CO LTD
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