Journal
GENES & GENETIC SYSTEMS
Volume 88, Issue 2, Pages 85-91Publisher
GENETICS SOC JAPAN
DOI: 10.1266/ggs.88.85
Keywords
Bacillus stearothermophilus; Pichia pastoris; secretory expression; thermostable alkaline protease
Funding
- Malaysia Genome Institute project from the Ministry of Science, Technology and Innovation, Malaysia [07-05-MGI-GMB003]
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The thermostable alkaline protease from Bacillus stearothermophilus F1 has high potential for industrial applications, and attempt to produce the enzyme in yeast for higher yield was undertaken. Secretory expression of F1 protease through yeast system could improve enzyme's capability, thus simplifying the purification steps. Mature and full genes of F1 protease were cloned into Pichia pastoris expression vectors (pGAPZ alpha B and pPICZ alpha B) and transformed into P. pastons strains (GS115 and SMD1168H) via electroporation method. Recombinant F1 protease under regulation constitutive GAP promoter revealed that the highest expression was achieved after 72 h cultivation. While inducible AOX promoter showed that 0.5% (v/v) methanol was the best to induce expression. It was proven that constitutive expression strategy was better than inducible system. The a-secretion signal from the plasmid demonstrated higher secretory expression level of F1 protease as compared to native Open Reading Frame (ORF) in G5115 strain (GE6GS). Production medium YPTD was found to be the best for F1 protease expression with the highest yield of 4.13 U/mL. The protein was expressed as His-tagged fusion protein with a size about 34 kDa.
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