Studies on Isolation and Identification of Alkaline Protease Producers from Selected Alkaline Habitats in Maharashtra

Abstract

Soda lakes are one of the natural primary habitats and sea water, sea sand, garden soil, fermented food, poultry waste and manure are the some secondary habitats for the occurrence of industrially important alkaline protease producers. In this scenario, we have selected Lonar soda lake, Buldhana, poultry farm, Parbhani and Arabian sea, Mumbai for collection of samples. Various alkaline and saline media were used for the isolation of different morphotypes. Primary and secondary screening techniques were performed for the qualitative and quantitative estimation of the alkaline proteases produced by isolates. 43, 5 and 4 efficient alkaline protease producers were isolated from Lonar lake, poultry farm waste and Arabian seawater samples respectively. Isolates were observed for morphological, microscopic and physiological characters, enzyme profile, sugar utilization and antibiotic sensitivity pattern etc. Out of these 26 fast growing alkaline protease producers were further identified by 16S rRNA partial gene sequencing method and the deduced sequences were submitted to Genebank for public access. The identified isolates were belonged to the different phyla viz. firmicutes, proteobacteria and actinobacteria and various genera viz. Alcaligenes, Alteromonas, Alkalibacterium, Bacillus, Brachybacterium, Brevibacterium, Halobacillus, Halomonas, Micrococcus, Marinobacter, Pseudomonas, Planococcus and Paracoccus. The evolutionary analyses were conducted by constructing phylogenetic trees using MEGA version 6.06 freeware. Seven alkaline protease producers have been deposited at Microbial Culture Collection, NCCS, Pune (India) for public access. Production of alkaline proteases from the selected 6 isolates was optimized by adopting a one-variable-at-a-time (OVAT) approach in submerged fermentation system by using modified fermentation medium (MFM) and more than 3 fold enhancement in the alkaline protease production was recorded. The partially purified alkaline proteases from the selected isola