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Bacillus subtilis of secretory expression proline aminopeptidase and application thereof

A Bacillus subtilis, proline aminopeptidase technology, applied in the field of biological enzyme engineering, can solve the problems of low proline aminopeptidase expression efficiency, weak extracellular secretion ability and the like

Inactive Publication Date: 2015-11-04
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there has been no report on the expression of proline aminopeptidase in Bacillus subtilis. The present invention focuses on this and aims at the shortcomings of low proline aminopeptidase expression efficiency and weak extracellular secretion ability by using Bacillus subtilis The expression system can efficiently express proline aminopeptidase, and make more extracellular secretion through appropriate secretion strategy

Method used

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  • Bacillus subtilis of secretory expression proline aminopeptidase and application thereof
  • Bacillus subtilis of secretory expression proline aminopeptidase and application thereof
  • Bacillus subtilis of secretory expression proline aminopeptidase and application thereof

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Experimental program
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Effect test

Embodiment 1

[0028] The construction method of embodiment 1 proline aminopeptidase Bacillus subtilis

[0029] Primers were designed according to the proline aminopeptidase cDNA sequence (SEQ ID NO.1): upstream primer P1: 5'CG GGATCC ATGGCTGCCAAAC 3'(BamHI); downstream primer P2: 5'CGCG ACGCGT CTAATCAATAGAGTC 3' (MluI). Amplify the target gene with BamHI and MluI restriction sites, double-digest the target gene and plasmid pMA5 with BamHI and MluI, recover and purify the gel, connect overnight at 16°C, transform Escherichia coli JM109 and coat ampicillin-containing LB plate, after the transformants grow out, colony PCR verification is carried out, the positive transformants are cultured and the plasmids are extracted for double-enzyme digestion verification, and the verified recombinant plasmid pMA5-pap is sent to Shanghai Sangon for sequencing.

[0030] The constructed recombinant plasmid pMA5-pap was electrotransformed into Bacillus subtilis WB600, and positive recombinants were scree...

Embodiment 2

[0039] Embodiment 2 The method for secreting and expressing proline aminopeptidase from recombinant Bacillus subtilis

[0040] Pick a single colony of recombinant Bacillus subtilis on the LB plate (containing 50 μg / mL kanamycin) and culture it in 50 mL LB liquid medium (containing 50 μg / mL kanamycin) at 37 ° C for 14 hours at 220 r, then press 1% The inoculum was inoculated in 50mL STB medium (containing 50μg / mL kanamycin) at 37°C and cultured at 220r for 24h. After the fermentation, centrifuge at 8000r, 4°C for 10min, and the obtained fermentation supernatant is the proline aminopeptidase crude enzyme solution, and the measured enzyme activity is 36.0U / mL.

Embodiment 3

[0041] Example 3 Characterization of Proline Aminopeptidase Secreted by Recombinant Bacillus subtilis

[0042]The crude enzyme solution obtained in Example 2 was subjected to two Hitrap Q HP ion-exchange chromatography to obtain proline aminopeptidase that was basically electrophoretic pure, and it was characterized. The results showed that: the optimum reaction temperature was 50°C, It exhibits good temperature stability at and below 50°C; the optimum reaction pH is 7.5, and has good pH stability between pH 6-11; it has strict substrate specificity for proline-p-nitroaniline ; The Michaelis constant Km and the maximum reaction rate Vmax are 0.171mmol L respectively -1 and 55.99 μmol min -1 . The characteristics of the intracellular proline aminopeptidase expressed in recombinant Escherichia coli are basically consistent, indicating that the structure and characteristics of the enzyme have not been changed after secreted expression in Bacillus subtilis. In addition, the sal...

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Abstract

The invention discloses bacillus subtilis of secretory expression proline aminopeptidase and application thereof and belongs to the technical field of biological enzyme engineering. PMA5 serves as an expression vector of genes of coding proline aminopeptidase, the bacillus subtilis WB600 is imported, recombinant bacteria are obtained, the secretory expression is achieved, and the extracellular enzyme activity reaches 36.0 U / mL. The bacillus subtilis and the application thereof lay a good foundation for the industrial production of the proline aminopeptidase and application in the food industry.

Description

technical field [0001] The invention relates to a bacillus subtilis that secretes and expresses proline aminopeptidase and an application thereof, belonging to the technical field of biological enzyme engineering. Background technique [0002] Aminopeptidases (Aps for short, EC3.4.11) are a class of exopeptidases that hydrolyze amino acids one by one from the N segment of proteins or polypeptides, debittering of protein hydrolysates, N-terminal sequencing of proteins and polypeptides, deep processing of protein raw materials and biological Active peptide preparation and other aspects play an important role. Prolyl aminopeptidase (PAP for short, EC3.4.11.5) is an aminopeptidase with strict substrate specificity, which specifically hydrolyzes proline residues at the N-terminal of polypeptides or proteins. The removal of bitterness of hydrolyzed processed products and the hydrolysis of collagen have good application prospects. [0003] At present, the research on aminopeptida...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/75C12N9/48C12R1/125
Inventor 田亚平汪克红
Owner JIANGNAN UNIV