Optimization of Keratinase Production by Amycolatopsis sp. Strain MBRL 40 from a Limestone Habitat
Background: The class Actinobacteria accounts for a high proportion of soil microbial biomass and more than half of the bioactive compounds including antibiotics, immunosuppressive agents, antitumor agents and industrial enzymes such as keratinases. Keratinases are modern proteases that can valorise poultry and leather industry wastes and may find applications in prion degradation and treatment of neurodegenerative diseases. A wide diversity of bacteria, actinomycetes and fungi are known to be keratin degraders. Manipur, being a part of Indo Burma Hotspot, might harbor a rich diversity of bioactive actinomycetes. The objectives of the study are: a) Isolation of new keratinolytic actinomycetes. b) Medium optimization by statistical approaches for keratinase production. Methods: A protease producing actinomycete strain from Hundung limestone quarry at Ukhrul, Manipur, India, was investigated for its keratinolytic activities. Optimization of keratinase production was done using statistical methods namely, Plackett Burman Design (PBD) and Response Surface Methodology (RSM) Eleven (11) variables were screened using PBD. Results: The strain showed keratinolytic activity with keratin azure and chicken feather as substrates. Maximum keratinolytic activity was observed at 40degC, pH 7 and 48 h of incubation. Yeast extract, MgSO4, and corn flour were found to affect the response signal positively whereas glucose, CaCO3, K2HPO4, NaCl and soyabean meal had negative effects. Yeast extract, cornflour, and soyabean meal were further studied using RSM. A 2.3 fold increase in keratinase production was achieved in submerged fermentation after the use of statistical optimization methods. Conclusion: On the basis of biochemical properties and 16S rRNA gene sequence analysis, the strain was identified as Amycolatopsis sp. strain MBRL 40. Novelty of the work: There are meagre reports of keratinolytic bacteria from limestone biotopes. In addition, keratinolytic Amycolatopsis species are scanty in the literature. This possibly is the first report of a keratinolytic Amycolatopsis strain from a limestone habitat.