Research Journal of Biotechnology Vol. 10 (4) April (2015) Res. J. Biotech 33 Bioprospecting for extracellular hydrolytic enzymes from culturable thermotolerant bacteria isolated from Manikaran thermal springs Suman Archna*, Verma Priyank, Yadav Ajar Nath and Saxena Anil Kumar Division of Microbiology, Indian Agricultural Research Institute, New Delhi- 110012, INDIA *archsuman@yahoo.com Abstract Microbial enzymes play a major role in hydrolysis of lignocellulosic compounds to fermentable sugars. In order to isolate thermotolerant bacterial isolates in Manikaran hot springs having potential to produce extracellular hydrolytic enzymes were analyzed using different nutrient combinations. Of 120 isolates, twenty strains showed hydrolytic enzymes production at >70 C. Phylogenetic analysis of positive strains, based on 16S rDNA sequences indicated that isolates were clustered within 48% Proteobacteria, 33% Firmicutes and 19% Actinobacteria. Evaluation of hydrolytic enzymes production under submerged and solid state fermentation was done using paddy straw as sole carbon source. Out of twenty, seven bacterial isolates were found to be novel and efficient for the production of hydrolytic enzymes. A large variation was observed among different isolates for hydrolytic enzymes production. Seven isolates Lysinibacillus sp., Enterobacter cloacae, Rhodococcus qingshengii, Paenibacillus pabuli, Bacillus pumilus, Micrococcus indicus and Pseudomonas fragi based on their maximum production on all different temperature especially 70°C were evaluated in terms of enzyme properties and kinetics. The enzyme of above seven isolates is active over broad range of high temperature. Such thermo stable isolates have potential to be used to develop as consortia for bioconversion of lignocellulosic residue to fermentable sugars preferably at high temperature. Keywords: Hydrolytic enzymes, Thermotolerant bacteria, Manikaran, 16S rRNA gene. Introduction Cellulose is the most abundant and renewable biological carbon resource on earth. In nature, cellulases from bacteria and fungi hydrolyze crystalline cellulose into oligosaccharides which are ultimately hydrolyzed into glucose by the synergistic action of at least three types of cellulolytic enzymes, namely, endo-1,4-b-D-glucanase, cellobiohydrolases and β-glucosidase 45 while hemicellulase mainly includes xylan which is the second most abundant natural polysaccharide on earth. Xylanase refers to a class of enzymes that specifically degrade xylan into oligosaccharides and xylose 5,9 . Hydrolysis of xylan is undoubtedly an important step toward proper utilization of abundantly available lignocellulosic material in nature. Xylan hydrolysis using enzymes such as xylanases provides a viable alternative to chemical hydrolysis as it is highly specific in nature apart from being an environment friendly process 20 . Cellulase and xylanase have been widely used in energy, food, animal feed, medical, papermaking and textile industries 10 . In all such industries thermostable enzymes active at alkaline pH are of great importance. Exotic niches such as thermal springs, harbor populations of microorganisms can be source of commercially important products like enzymes, sugars, compatible solutes and antibiotics 35,32 . Thermal springs are manifestation of geological activity and represent aquatic microcosms that are produced by the emergence of geothermally heated groundwater from the Earth’s crust. Prokaryotes are the major component of most ecosystems, being ubiquitous in nature because of their small size, easy dispersal, metabolic versatility, ability to utilize a broad range of nutrients and tolerance to unfavorable and extreme conditions. Thermal springs are, therefore, no exception to colonization by prokaryotes. Diversity analysis of such extreme environments has grown in significance because of their diverse and unusual chemistry and the opportunity they provide to identify rare compounds and genes 19 . In recent years, interest in production of cellulases has increased due to thrust on production of second generation biofuel from lignocellulosic biomass. Besides their use for biofuel production, cellulases are being used in textile industry for cotton softening and denim finishing and in detergent markets for colour care, cleaning and anti-redeposition in washing powders 8 . Since then researchers worldwide have focussed their attention toward newer microbial isolates, the xylanases from which can be used in the pulp and paper industries. The scientific interest in this field is reflected by the number of research papers published during recent years describing numerous xylanases from newer sources, as well as bleaching experiments reported using various hemicellulases, pulps and bleaching sequence. The xylanases have been reported mainly from bacteria 38 , fungi 38 , actinomycetes 4 and yeast 25 . Bacteria, having high growth rate as compared to fungi have a better potential to replace later for commercial cellulase production. Although