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

Abstract

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.

Isolation and Screening of Microorganisms for the Effective Pretreatment of Lignocellulosic Agricultural Wastes

“…During our screening process, 15 bacteria, 7 basidiomycete fungi, and 5 lower fungi tested positive for laccase activity, and all these isolates were also positive for peroxidases activity. Rich supplements of specific substrates like phenolic substrates indicate secretion of ligninolytic enzymes was proven in other studies [ 34 – 36 ]. All the ligninolytic enzyme-positive strains tested for cellulases activity by congo red-CMC produced variable zones (CMC clearance) around their colony, and the decolorized zones of congo red-CMC demonstrated the ability to degrade CMC [ 37 ].…”

Laccase Production Under Optimized Parameters by Aspergillus oryzae, an Endophytic Fungus and their Application to Waste Water Treatment

“…With the addition of inducer Tannic Acid, the laccase gene's expression rises, and laccase production increases in the growth medium (41) . It has previously been observed that binding of sodium azide to the types 2 and 3 copper sites affects internal electron transfer, decreasing laccase activity and that formaldehyde, as an organic solvent, alters the pH of an aqueous solution, resulting in lower enzyme output (42) .…”

Isolation and Screening of Dye Degrading Lignocellulolytic Bacteria from Sundarban Mangrove Ecosystem, West Bengal, India

“…Supplements with specific phenolic substrates indicate extracellular secretion of ligninolytic enzymes which was proven in different research. [44][45][46] All the cellulolytic isolates were tested for cellulase activity by congo red-CMC agar and malachite green CMC-agar plate assay that produced non-homogenous clear zones (CMC clearance) and the decolorized zones of congo red CMC-agar (Figure 3b) and malachite green CMC-agar (Figure 3a) demonstrated the cellulosedegrading capacity 47 and dye decolorization ability. Cellulase plays a crucial role in the breakdown of lignocellulosic biomass to release the fermentable sugar monomers that are then used in enzymatic saccharification for the production of biofuel.…”