Cloning, sequencing and extracellular expression of the alkaline protease gene from Bacillus stearothermophilus F1
The gene of a highly thermostable alkaline protease was amplified from Bacillus stearothermophilus F1 by polymerase chain reaction using consensus primers based on the sequences of serine protease genes from related species. Nucleotide sequence analysis of the gene revealed an open reading frame...
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| Format: | Thesis |
| Language: | English English |
| Published: |
2001
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| Subjects: | |
| Online Access: | http://psasir.upm.edu.my/id/eprint/9210/1/FSAS_2001_6_IR.pdf |
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| Summary: | The gene of a highly thermostable alkaline protease was amplified from
Bacillus stearothermophilus F1 by polymerase chain reaction using
consensus primers based on the sequences of serine protease genes from
related species. Nucleotide sequence analysis of the gene revealed an open
reading frame containing 1,206 bp which encodes for a polypeptide of 401
amino acid residues. The polypeptide composed of a signal peptide (25
amino acid residues), a prosequence (97 amino acid residues), and a
mature protein of 279 amino acid residues. Amino acid sequence
comparison revealed that it shared high homology with those of other serine
proteases from a number of Bacillus spp. The recombinant F1 protease was
efficiently excreted into culture medium using E.coli XL1-Blue harbouring
two vectors: pTrcHis bearing the protease gene and pJL3 containing the
bacteriocin-release-protein (BRP). Both vectors contain the E.coli lac
promoter-operator system. In the presence of 40 uM isopropyl-β-D thiogalactopyranoside (IPTG), the recombinant F1 protease and the BRP
were expressed and the mature F1 protease was released into the culture
medium. The enzyme was purified through a one-step heat treatment at
70°C for 3 h, and this method purified the protease to near homogeneity.
The purified enzyme showed a pH optimum of 9.0, temperature optimum of
80°C, and was stable at 70°C for 24 h in pH ranges from 8.0 to 10.0. The
enzyme exhibited a high degree of thermostability with half-lives of 3.5 h at
85°C, 25 min at 90°C, and was inhibited by the serine protease inhibitor,
phenylmethanesulfonyl fluoride (PMSF). |
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