Chiang Mai Journal of Science

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Protease gene from thermophilic bacterium Bacillus stearothermophilus TLS33 was cloned into Escherichia coli DH5a by using pUC19 as a vector. Colony that produced clear zone on skim milk agar plate and had highest protease activity was selected for characteristic study of the cloned protease. The molecular weight of protease from Cloned TLS33 determined by SDS-PAGE and SDS-Zymography were estimated to be 65 and 118 kDa, respectively. The size of inserted DNA from Cloned TLS33 was estimated to be 3,120 bp which coded for many genes. Subcloning was used to lineate the protease gene in Cloned TLS33. The recombinant plasmid of the Cloned TLS33 was digested with restriction enzyme HindIII and EcoRI and the inserted DNA was extracted and digested with Sau3AI. Digested DNA were ligated to pUC19 that was cut with BamHI and transformed into E.coli DH5a . The recombinant cells were grown on skim milk agar plate and two colonies were found to produce clear zone (Subclone I and Subclone II). Protease products from Cloned TLS33, Subclone I and Subclone II had optimum temperature for activity and thermostability at 50-65⁰C and 50⁰C for 1 h, respectively which was different from the native strain that had optimum temperature for protease activity and thermostability at 70-80⁰C and 75⁰C for 1 h, respectively. Proteases from Cloned TLS33, Subclone I, Subclone II and the native strain had the same optimum pH for activity at 7. The molecular weights of the proteases from Subclone I and Subclone II were the same and estimated to be 14 and 93 kDa by SDS-PAGE and Zymography, respectively. It was suggested that Subclone I and Subclone II contained protease gene of the same size.

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