Cloning and expression of thermostable protease gene isolated from Bacillus SP.

Mohd Zairi Salleh, (2011) Cloning and expression of thermostable protease gene isolated from Bacillus SP. Masters thesis, Universiti Malaysia Kelantan.

Abstract

The first objective of this study was to identify Bacillus species for the production of thermostable protease which were isolated from hot spring habitat. Additionally, the goal was also to analyze the characteristic properties of the crude protease for both Bacillus species. Then, ptotease gene from the isolates were then cloned and recombinant protease was expressed and subjected for comparison of the characteristics with native protease. Screening and isolation of proteolytic bacteria were carried out from water and soil samples of hot spring area at Besut (Northern part of Terengganu, Malaysia). Two isolates gave positive result on skim milk agar (SMA) and identified as Bacillus subtilis A1 and Bacillus licheniformis A2 through morphological and 16 S rDNA gene identification. B. licheniformis A2 was found to produce the highest protease production. The activities of crude protease of both isolates were further characterized based on the effect of temperature, pH and inhibitors. Maximum enzyme activity was observed between 37 °C and 65 °C and pH 5 to 10. Ethylenediamine tetraacetic acid (EDTA) showed 90% inhibition for protease activity of both species and thus strongly proved that this enzyme belongs to the alkaline metalloprotease family. From the results showed that protease of both Bacillus species have optimum temperature at 60 °C and also indicates that protease produce would be thermostable. The gene encoding protease of B. licheniformis A2 was successfully amplified via polymerase chain reaction (PCR) using newly designed primers based on the sequence of alkaline metalloprotease gene from related species. To exploit the potential of high temperature activity and stability, the interest gene of thermostable protease from B. lichenoformis A2 was cloned into 3.9 kbp pCR®2.1-TOPO vector. The sequence analysis showed an open reading frame (ORF) starting from the initation codon (ATG) consists of 1503 bases coding for 501 amino acid residues. The amino acid sequence contained the sequence motif His-Glu-X-X-His, which is considered as an active site (zinc-binding sequence) for zinc-dependent metalloprotease. The gene product was succesfully expressed in E. coli intra and extracellularly by induction with IPTG and the expressed protein was detected by SDS-PAGE analysis with the molecular weight of ~27 kDa. The optimum temperature of the recombinant protease activity was found to be at 60 °C similar as nature protease. The recombinant protease had the half-lives of 2 hours and 30 minutes at 55 C and 2 hours at 60 C, and was stable for 35 minutes at 65 C. The activity of recombinant protease displayed a pH optimum of 9.0 and less than 15% of its maximum activity was observed at pH 10.

Item Type: UMK Thesis (Masters)
Subject Heading: Microbial biotechnology
Subject Heading: Microbial biotechnology - Industrial microbiology
Faculty: Faculty of Agro - Based Industry
Call Number: QH 442 .M64 2011 tes
Supervisor: Dr. Noor Azlina Ibrahim
Deposited By: En. Pahmi Abdullah
Date Deposited: 09 Dec 2012 14:49
Last Modified: 08 Feb 2017 01:49
URI: http://umkeprints.umk.edu.my/id/eprint/1049

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