Biology subjects

Aikat, K.

Publications and source records attributed to Aikat, K..

2 recordsLinked to original sources

bioRxiv2023

Intercalation of the disulfide bond between the A2 and A4 loop of Cellobiohydrolase (Cel7A) of Aspergillus fumigatus enhances catalytic activity and thermostability

Disulfide bond is important for maintaining the structural conformation and stability of the protein. Introducing new disulfide bond is a promising strategy for rational protein design. In this report, disulfide bond engineering has been applied to improve the stability of an industrially important enzyme, Glycoside Hydrolase family GH 7 cellobiohydrolase (GH7 CBHs) or Cel7A of A fumigatus origin. Disulfide by Design 2.0 (DbD2), an online tool, was used for the detection of the mutation sites and created four mutations (D276C-G279C; DSB1, D322C-G327C; DSB2, T416C-I432C; DSB3, G460C-S465C; DSB4) both inside and outside of the peripheral loops but, not in the catalytic region. The disulfide bond formed between the A2 and A4 loop of DSB3 showed higher thermostability (70% activity at 700C), higher substrate affinity (Km= 0.081mM) and higher catalytic activity (Kcat =9.75 min-1; Kcat/Km = 120.37 mM min-1) compared to wild type AfCel7A (50% activity at 700C; Km= 0.128mM; Kcat = 4.833 min-1; Kcat/Km = 37.75 mM min -1). The other three mutants with high B factor showed loss of thermostability and catalytic activity. Molecular dynamic simulations revealed that the mutation T416C-I432C makes the tunnel wider (DSB3:13.6 [A]; Wt: 5.3 [A]) at the product exit site; giving flexibility in the entrance region and mobility of the substrate. It may facilitate substrate entry into the catalytic tunnel and releases the product faster than the wild type. Whereas in other mutants, the tunnel is not prominent (DSB4), the exit is lost (DSB1), and the ligand binding site is absent (DSB2). This is the first report of the gain of function of both thermostability and enzyme activity of cellobiohydrolase Cel7A by disulfide bond engineering in the loop. ImportanceCellulases are important for their role in the production of bioethanol, the cleanest renewable replacement of fossil fuels. Engineering of the cellulases is a chalange to increase their catalytic activity and thermostability for production of cheap ethanol. In this report we have introduced disulfide bond and successfully increased the both thermostabilty and catalytic activity of AfCel7A.

biochemistry↗

bioRxiv2020

Investigation the biomass conversion efficiency, biochemical characterisation and structural insights of the newly isolated AA16 family of Lytic Polysaccharide Monooxygenase (LPMO) from Aspergillus fumigatus

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.

biochemistry↗