Nocardiopsis synnemataformans NBRM9, an extremophilic actinomycete producing extremozyme cellulase, using lignocellulosic agro-wastes and its biotechnological applications.

Abstract

Actinomycetes are an attractive source of lignocellulose-degrading enzymes. The search for actinomycetes producing extremozyme cellulase using cheap lignocellulosic waste remains a priority goal of enzyme research. In this context, the extremophilic actinomycete NBRM9 showed promising cellulolytic activity in solid and liquid assays. This actinomycete was identified as Nocardiopsis synnemataformans based on its phenotypic characteristics alongside phylogenetic analyses of 16S rRNA gene sequencing (OQ380604.1). Using bean straw as the best agro-waste, the production of cellulase from this strain was statistically optimized using a response surface methodology, with the maximum activity (13.20 U/mL) achieved at an incubation temperature of 40 °C, a pH of 9, an incubation time of 7 days, and a 2% substrate concentration. The partially purified cellulase (PPC) showed promising activity and stability over a wide range of temperatures (20-90 °C), pH values (3-11), and NaCl concentrations (1-19%), with optimal activity at 50 °C, pH 9.0, and 10% salinity. Under these conditions, the enzyme retained >95% of its activity, thus indicating its extremozyme nature. The kinetics of cellulase showed that it has a V_max of 20.19 ± 1.88 U/mL and a Km of 0.25 ± 0.07 mM. The immobilized PPC had a relative activity of 69.58 ± 0.13%. In the in vitro microtiter assay, the PPC was found to have a concentration-dependent anti-biofilm activity (up to 85.15 ± 1.60%). Additionally, the fermentative conversion of the hydrolyzed bean straw by Saccharomyces cerevisiae (KM504287.1) amounted to 65.80 ± 0.52% of the theoretical ethanol yield. Overall, for the first time, the present work reports the production of extremozymatic (thermo, alkali-, and halo-stable) cellulase from N. synnemataformans NBRM9. Therefore, this strain is recommended for use as a biotool in many lignocellulosic-based applications operating under harsh conditions.