Comparative insights into the saccharification potentials of a relatively unexplored but robustPenicillium funiculosumglycoside hydrolase 7 cellobiohydrolase.
BACKGROUND
GH7 cellobiohydrolases (CBH1) are vital for the breakdown of cellulose. We had previously observed the enzyme as the most dominant protein in the active cellulose-hydrolyzing secretome of the hypercellulolytic ascomycete-Penicillium funiculosum(NCIM1228). To understand its contributions to cellulosic biomass saccharification in comparison with GH7 cellobiohydrolase from the industrial workhorse-Trichoderma reesei, we natively purified and functionally characterized the only GH7 cellobiohydrolase identified and present in the genome of the fungus.


RESULTS
There were marginal differences observed in the stability of both enzymes, withP. funiculosum(PfCBH1) showing an optimal thermal midpoint (Tm) of 68 °C at pH 4.4 as against an optimalTmof 65 °C at pH 4.7 forT. reesei(TrCBH1). Nevertheless, PfCBH1 had an approximate threefold lower binding affinity (Km), an 18-fold higher turnover rate (kcat), a sixfold higher catalytic efficiency as well as a 26-fold higher enzyme-inhibitor complex equilibrium dissociation constant (Ki) than TrCBH1 onp-nitrophenyl-β-d-lactopyranoside (pNPL). Although both enzymes hydrolyzed cellooligomers (G2-G6) and microcrystalline cellulose, releasing cellobiose and glucose as the major products, the propensity was more with PfCBH1. We equally observed this trend during the hydrolysis of pretreated wheat straws in tandem with other core cellulases under the same conditions. Molecular dynamic simulations conducted on a homology model built using the TrCBH1 structure (PDB ID: 8CEL) as a template enabled us to directly examine the effects of substrate and products on the protein dynamics. While the catalytic triads-EXDXXE motifs-were conserved between the two enzymes, subtle variations in regions enclosing the catalytic path were observed, and relations to functionality highlighted.


CONCLUSIONS
To the best of our knowledge, this is the first report about a comprehensive and comparative description of CBH1 from hypercellulolytic ascomycete-P. funiculosumNCIM1228, against the backdrop of the same enzyme from the industrial workhorse-T. reesei. Our study reveals PfCBH1 as a viable alternative for CBH1 fromT. reeseiin industrial cellulase cocktails.
Microbial Engineering Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, 110067 India.
Funso Emmanuel
FE
Ogunmolu
Microbial Engineering Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, 110067 India.
Navya Bhatt Kammachi
NBK
Jagadeesha
Translational Bioinformatics Group, International Centre for Genetic Engineering and Biotechnology, New Delhi, India.
Rakesh
R
Kumar
Translational Bioinformatics Group, International Centre for Genetic Engineering and Biotechnology, New Delhi, India.
Pawan
P
Kumar
Translational Bioinformatics Group, International Centre for Genetic Engineering and Biotechnology, New Delhi, India.
Dinesh
D
Gupta
Microbial Engineering Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, 110067 India.|DBT-ICGEB Centre for Advanced Bioenergy Research, International Centre for Genetic Engineering and Biotechnology, New Delhi, India.
Syed Shams
0000-0002-6558-4612
SS
Yazdani
Biomass degradation; CAZymes: glycoside hydrolases: Penicillium funiculosum; Cellobiohydrolase 1; Molecular dynamics