Biochemical, structural insights of newly isolated AA16 family of Lytic Polysaccharide Monooxygenase (LPMO) from Aspergillus fumigatus and investigation of its synergistic effect using biomass.

Abstract

The efficient conversion of lignocellulosic biomass into fermentable sugar is a bottleneck for the cheap production of bio-ethanol. The recently identified enzyme Lytic Polysaccharide Monooxygenase (LPMO) family has brought new hope because of its boosting capabilities of cellulose hydrolysis. In this report, we have identified and characterized a new class of auxiliary (AA16) oxidative enzyme LPMO from the genome of a locally isolated thermophilic fungus Aspergillus fumigatus (NITDGPKA3) and evaluated its boosting capacity of biomass hydrolysis. The AfLPMO16 is an intronless gene and encodes the 29kDa protein. While Sequence-wise, it is close to the C1 type of AaAA16 and cellulose-active AA10 family of LPMOs, but the predicted three-dimensional structure shows the resemblance with the AA11 family of LPMO (PDB Id: 4MAH). The gene was expressed under an inducible promoter (AOX1) with C-terminal His tag in the Pichia pastoris. The protein was purified using Ni-NTA affinity chromatography, and we studied the enzyme kinetics with 2,6-dimethoxyphenol. We observed polysaccharides depolymerization activity with Carboxymethyl cellulose (CMC) and Phosphoric acid swollen cellulose (PASC). Moreover, the simultaneous use of cellulase cocktail (commercial) and AfLPMO16 enhances lignocellulosic biomass hydrolysis by 2-fold, which is highest so far reported in the LPMO family. ImportanceThe auxiliary enzymes, such as LPMOs, have industrial importance. These enzymes are used in cellulolytic enzyme cocktail due to their synergistic effect along with cellulases. In our study, we have biochemically and functionally characterized the new AA16 family of LPMO from Aspergillus fumigatus (NITDGPKA3). The biochemical characterization is the fundamental scientific elucidation of the newly isolated enzyme. The functional characterization, biomass degradation activity of AfLPMO16, and cellulase cocktail (commercial) combination enhancing the activity by 2-fold. This enhancement is the highest reported so far, which gives the enzyme AfLPMO16 enormous potential for industrial use.