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[Cloning and expression of protease PT121 from Pseudomonas aeruginosa and application in peptide synthesis].
- Source :
-
Wei sheng wu xue bao = Acta microbiologica Sinica [Wei Sheng Wu Xue Bao] 2015 Jan 04; Vol. 55 (1), pp. 67-72. - Publication Year :
- 2015
-
Abstract
- Objective: We studied the cloning and expression of lasB encoding solvent-resistant protease from Pseudomonas aeruginosa PT121. The recombinant protease was then characterized and applied in peptide synthesis.<br />Methods: The PCR primers were designed to acquire the open read frame (ORF) of lasB according to similar protease gene (pseudolysin) reported in the literature. Inducible expression plasmid pET22b-lasB' was constructed and expressed in E. coli BL21 (DE3). The recombinant protease was then characterized and applied in peptide synthesis.<br />Results: The protease PT121 was defined as metalloproteinase M4 family according to sequence blast. Gene sequence analysis shows that lasB encodes signal peptide, pro-peptide and mature peptide. Mature protein contains 301 residues with molecular weight of 33 kDa. One-step preparation of the recombinant proteases PT121 was optimized by breaking cell wall. The specific activity of protease PT121 reached up to 7700U/mg, and it was stable similar with wild type PT121 from P. aeruginosa PT121 in temperature, pH and organic solvent. The synthesis rate of various dipeptides in 50% DMSO was effective, especially productivity of aspartame precursor reached up to 91%.<br />Conclusions: Successful hetero-expression of protease PT121 lays the foundation of studying mechanism of catalysis and molecular evolution.
- Subjects :
- Bacterial Proteins chemistry
Biocatalysis
Enzyme Stability
Escherichia coli genetics
Escherichia coli metabolism
Kinetics
Peptide Hydrolases chemistry
Pseudomonas aeruginosa chemistry
Pseudomonas aeruginosa genetics
Sequence Homology, Amino Acid
Temperature
Bacterial Proteins genetics
Bacterial Proteins metabolism
Cloning, Molecular
Gene Expression
Peptide Hydrolases genetics
Peptide Hydrolases metabolism
Peptides metabolism
Pseudomonas aeruginosa enzymology
Subjects
Details
- Language :
- Chinese
- ISSN :
- 0001-6209
- Volume :
- 55
- Issue :
- 1
- Database :
- MEDLINE
- Journal :
- Wei sheng wu xue bao = Acta microbiologica Sinica
- Publication Type :
- Academic Journal
- Accession number :
- 25958684