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Minerva Biotecnologica 2016 Settembre;28(3):136-45

lingua: Inglese

Gene cloning, heterologous expression, characterization and optimization of fermentation conditions of keratinase from Bacillus sp. MKR1

Maryam SHAHBAZI 1, 2, Fatemeh DABBAGH 1, 2, Mohammad B. GHOSHOON 1, 2, Aboozar KAZEMI 1, 2, Younes GHASEMI 1, 2

1 Department of Pharmaceutical Biotechnology, School of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran; 2 Pharmaceutical Sciences Research Center, Shiraz University of Medical Sciences, Shiraz, Iran


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BACKGROUND: In this study we cloned a novel gene (ker SM) encoding keratinase from the chromosomal DNA of Bacillus sp. MKR1 (HQ141583).
METHODS: This gene was amplified by PCR and cloned into pET-15b and transferred to Escherichia coli BL21 under IPTG inducible promoter.
RESULTS: The recombinant strain produced an intracellular keratinase with activity of 209 U ml-1 using keratin azure as substrate. A ~39kDa band was detected in SDS-PAGE. The obtained recombinant keratinase was a thermostable serine alkaline protease which was active in the pH range of 5.0-11.0 (with optimum at 8.0) and temperature of 30-90° C (with optimum at 70° C). This enzyme was stable over a wide pH range of 4-11 for 4 h. In order to improve the level of keratinase production and optimization of fermentation conditions, response surface methodology (RSM) was employed. Maximum keratinolytic activity (410 U ml-1) was observed within 4 h of fermentation using initial inoculums concentration of 0.5 OD600 nm and incubation temperature of 25° C under 1mM IPTG induction, indicating more than four-fold rise comparing to the wild type strain, Bacillus sp. MKR1.
CONCLUSIONS: This study provides the evidence that recombinant keratinase production could be increased significantly using RSM. The commercial availability of keratinase is of great importance for industrial application and also pharmaceutical purposes.

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