Secretory Laccase from Pestalotiopsis Species CDBT-F-G1 Fungal Strain Isolated from High Altitude: Optimization of Its Production and Characterization

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 Rautahat 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-G1 was 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 degrees C and secretes 20 +/- 2 U/mL laccase in culture medium. Further optimization of growth conditions revealed that addition of (i) metal salts, e.g., 1 mM magnesium sulfate (51 +/- 25 U/mL); (ii) agitation of cultures at 200 rpm (51 +/- 9 U/mL); (iii) surfactants, e.g., 0.75 mM Tween 80 (54 +/- 14 U/mL); (iv) 40% dissolved O-2 (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 were pH 6 and 60 degrees C, respectively. V-max and K-m were 100 mu mol/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.