Laccase production by a temperature and pH tolerant fungal strain (GBPI-CDF-03) isolated from a glacial site in Indian Himalayan Region (IHR) has been investigated. The fungus developed white cottony mass on potato dextrose agar and revealed thread-like mycelium under microscope. ITS region analysis of fungus showed its 100% similarity with Trametes hirsuta. The fungus tolerated temperature from 4 to 48°C？±？2 (25°C opt.) and pH 3–13 (5–7 opt.). Molecular weight of laccase was determined approximately 45？kDa by native PAGE. Amplification of laccase gene fragment (corresponding to the copper-binding conserved domain) contained 200？bp. The optimum pH for laccase production, at optimum growth temperature, was determined between 5.5 and 7.5. In optimization experiments, fructose and ammonium sulfate were found to be the best carbon and nitrogen sources, respectively, for enhancing the laccase production. Production of laccase was favored by high carbon/nitrogen ratio. Addition of CuSO4 (up to 1.0？mM) induced laccase production up to 2-fold, in case of 0.4？mM concentration. Addition of organic solvents also induced the production of laccase; acetone showed the highest (2-fold) induction. The study has implications in bioprospecting of ecologically resilient microbial strains. 1. Introduction Laccases (phenol oxidases; E.C. 188.8.131.52.), also known as multicopper blue oxidases, belong to the oxidoreductase group of enzymes. Biochemically, they are glycoproteins carrying molecular mass between 50？kDa and 130？kDa . Fungi, belonging to ascomycetes, deuteromycetes, and basidiomycetes, are known to produce laccases of ecological as well as biotechnological importance, such as biodegradation and bioremediation [1–4]. In addition, laccases are also responsible for various physiological functions in fungi . Due to their broad specificity toward substrate, they can oxidize a range of chemical compounds leading to various industrial applications . Enhancement of laccase production, by modifying the nutritional and physiological conditions during cultivation of promising fungi, is a prerequisite for their optimum utilization at industrial scale. Besides nutritional supplements, inducers like organic solvents and metal ions also play important role in production of laccases [3, 7, 8]. Isolation of new microbial strains of biotechnological applications from various ecological habitats is a prerequisite for industrial growth. The fungi capable of producing laccase at wider temperature and pH range are likely to play important role in biodegradation under low
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