Transglutaminase mutants as well as genes, engineering bacteria and preparation method thereof

A glutamine, mutant technology, applied in the field of bioengineering, can solve problems such as difficulty in obtaining satisfactory results

Active Publication Date: 2018-01-16
TIANJIN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it is generally difficult to obtain satisfactory results for a gene mutated once, so a sequential error-prone PCR (Sequential Error-prone PCR) strategy has been developed.

Method used

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  • Transglutaminase mutants as well as genes, engineering bacteria and preparation method thereof
  • Transglutaminase mutants as well as genes, engineering bacteria and preparation method thereof
  • Transglutaminase mutants as well as genes, engineering bacteria and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment 1 The acquisition of wild-type transglutaminase gene promtg

[0044]1. Grinding Streptomyces mobaraense CICC 11018 cells in liquid nitrogen, and extracting the genome of Streptomyces mobaraense according to the instructions of the genome extraction kit.

[0045] 2. Take the extracted Streptomyces Maoyuan genome as a template, and design a pair of primers at the upstream and downstream of the ORF frame according to the transglutaminase sequence registered in Genbank sequence number AY241675.1, and introduce restriction enzyme sites BamH I, HindⅢ, the amplification primer of transglutaminase gene of the present invention is as follows:

[0046] Upstream primer P1 (SEQ ID NO.1):

[0047] 5'-CGCGGATCCGGGCAGCGGCACCGGGGAAG-3'

[0048] Downstream primer P2 (SEQ ID NO.2):

[0049] 5'-CCCAAGCTTTCACGGCCAGCCCTGTGTCACCT-3'

[0050] Using P1 and P2 as upstream and downstream primers, amplified with Streptomyces Maoyuan transglutaminase genome as a template;

[0051] ...

Embodiment 2

[0054] Obtaining of embodiment 2 transglutaminase mutant gene

[0055] 1. Random mutation based on error-prone PCR technology to construct a new type of transglutaminase, and design primers as follows:

[0056] Upstream primer P1 (SEQ ID NO.1):

[0057] 5'-CGCGGATCCGGGCAGCGGCACCGGGGAAG-3'

[0058] Downstream primer P2 (SEQ ID NO.2):

[0059] 5'-CCCAAGCTTTCACGGCCAGCCCTGTGTCACCT-3'

[0060] In the error-prone PCR reaction system, P1 and P2 were used as upstream and downstream primers, and the mature peptide gene of wild-type transglutaminase was used as a template to perform error-prone PCR.

[0061] The reaction conditions for its amplification are:

[0062]

[0063]

[0064] The amplification conditions were: pre-denaturation at 95°C for 10 min; denaturation at 94°C for 30 s, annealing at 54°C for 45 s, 30 cycles of extension at 72°C for 1 min and 30 s; extension at 72°C for 10 min.

[0065] 2. Clone the transglutaminase mutant gene into the expression vector pET22b...

Embodiment 3

[0078] Example 3 Construction of Bacillus subtilis Novel Transglutaminase Recombinant Bacteria

[0079] 1. Construction of expression vector pBSA43

[0080] pBSA43 is obtained by using the E. coli-Bacillus subtilis shuttle cloning vector pBE2 as the backbone, cloning into a strong Bacillus constitutive promoter P43, and the fructan sucrase signal sequence sacB that can directly secrete the recombinant protein into the medium . it comes with amp r Gene that can use ampicillin resistance as a selectable marker in E. coli; also has Km r Gene, kanamycin resistance can be used as a selection marker in Bacillus subtilis and Bacillus licheniformis. For the construction of the pBSA43 plasmid, refer to the Chinese invention patent "Production Process of Bacillus amyloliquefaciens Adding Precursor Fermentation to Produce Antiviral Drug Ribavirin" CN 103146785 B.

[0081] 2. Construction of transglutaminase expression vectors pBSA43-promtgm1 and pBSA43-promtgm2

[0082] The transglu...

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Abstract

The invention belongs to the technical field of bioengineering and particularly relates to transglutaminase mutants as well as genes, engineering bacteria and a preparation method thereof. The mutantsare obtained from target genes through mutation with a sequential error-prone PCR method and screening, specific enzyme activity of the mutants expressed in a Bacillus subtillis expression system isincreased by 19% and 27% respectively compared with that of wild type transglutaminase, and the application prospect is broad.

Description

Technical field: [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a transglutaminase mutant, its gene, engineering bacteria and a preparation method. technical background: [0002] Transglutaminase (Transglutaminase, EC 2.3.2.13, TGase) is an enzyme that can catalyze the acyl transfer reaction, which can make the ε-amino group on the lysine residue and the glutamine residue on the protein molecule The γ-hydroxyl amido group interacts to form an ε-(γ-glutamyl) lysine bond, enabling covalent cross-linking between proteins (or polypeptides). Transglutaminase can change the structural and functional properties of protein, thereby improving the nutritional value of protein. Therefore, transglutaminase has broad application prospects in food, textile, biopharmaceutical and other fields. [0003] In the food industry, transglutaminase can improve the appearance, flavor and texture of meat products to increase the added value of p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12N1/21C12R1/125
Inventor 路福平刘逸寒贾雷博
Owner TIANJIN UNIV OF SCI & TECH
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