Microorganisms producing laccases may be used for the pretreatment of lignocellulosic biomass to recover fermentable sugar. Very few fungi and other microbes growing in high altitudes have been tested for this purpose. As part of this study, we have collected soil samples from different parts of the Kathmandu Valley and the Rautah at district of Nepal (1600 to 2303 m above sea level) and successfully cultured 53 different isolates of microorganisms. Among the 53 isolates obtained 30 were Actinomycetes, 20 were Streptomycetes, and three were fungi). These isolates were tested for laccase expression using guaiacol, tannic acid, and 1-naphthol as substrates. Twelve of the 53 isolates tested positive for the expression of laccase. Among the laccase- positive isolates, a fungal species designated as CDBT-F-G1was found to produce high levels of laccase. This isolate was identified as Pestalotiopsis species based on 18S rRNA sequencing. Pestalotiopsis spp. CDBT-F-G1 isolate grows efficiently in PDB media containing 1% Kraft lignin at pH 5 and 30 °C and secretes 20 ± 2 U/mL laccase in culture medium. Further optimization of growth conditions reveled that addition of (i) metal salts, e.g., 1 mM magnesium sulfate (51 ± 25 U/mL); (ii) agitation of cultures at 200 rpm (51 ± 9U/mL); (iii) surfactants, e.g., 0.75 mM Tween 80 (54 ± 14 U/mL); (iv) 40% dissolved O2 (57 ± 2 U/mL) and inducers, e.g., 1 mM gallic acid (69 ± 11 U/mL), further promote laccase production by Pestalotiopsis spp. CDBT-F-G1 isolate. On the other hand, 0.1 mM cysteine inhibited laccase production. The secretory laccase obtained from fermentation broth of CDBT-F-G1 was partially purified by ammonium sulfate (13-fold purification with specific activity 26,200 U/mg) and acetone (14-fold purification with specific activity 31,700 U/mg) precipitation methods. The enzyme has an approximate molecular mass of 43 kDa, pH and temperature optima werepH6 and 60 °C, respectively. Vmax and Km were 100 μmol/min and 0.10 mM, respectively, with ABTS as the substrate. Given the above characteristics, we believe Pestalotiopsis spp. CDBT-F-G1 strain native to high altitudes of Nepal could be used to pretreat lignocellulosic biomass to efficiently recover fermentable sugars.

Cellulases catalyze the hydrolysis of 1,4-β-D-glycosidic linkages in cellulose and play a significant role in nature by recycling polysaccharide debris. This enzyme has enormous potential in industries such as textile wet processing, bio-stoning of denim fabric, biopolishing of textile fibres, softening of garments and removal of excess dye from the fabrics. Therefore, the research focused on obtaining new cellulose-producing microorganisms with higher specific activities and greater efficiency. By identifying a good strain, improving the production medium and using an alternative carbon source such as waste residue, this study aimed to lower the manufacturing cost of cellulase. This study was designed to assess the cellulase production by fungi isolated from water, soil, straw, dung, leaf and goat manure. In the present research, cellulase-producing fungal isolates obtained from waste samples were identified by morphological and microscopic features. On Congo red test, the largest zone of hydrolysis was found as 1.2 cm. From the morphological and microscopic test, the fungal strain was expected as Aspergillus sp. The assay of the enzyme cellulase was performed by measuring the release amount of reducing sugar. Optimization of process parameters was carried out for the isolate to maximize enzyme yield. On optimization, isolated fungal species showed maximum enzyme activity at a temperature of 30 °C and pH 6. Under optimized conditions of temperature and pH, agitation at 200 rpm with a 1 L/m air flow rate showed better cellulase activity. Cellulase production from Aspergillus sp. can be an advantage as the enzyme production rate is normally higher as compared to other fungi.