Functional genomics and statistical optimization of cellulase production by Exiguobacterium sp. M4 isolated from the Axis axis faeces and its application in Typha angustifolia biomass hydrolysis.

Abstract

The sustainable hydrolysis of lignocellulosic biomass (LCB) into fermentable sugars through cellulolytic microorganisms represents an essential step toward advancing bio-based industries. In this investigation, Exiguobacterium sp. M4, a novel cellulolytic bacterium, was obtained from the excrement of an Axis axis (chital deer) and characterized through morpho-biochemical and molecular characterization. The genome of Exiguobacterium sp. M4 was 2,724,915 bp and has a 52.37% of G + C content. It encodes 2,836 protein-coding genes, among which 118 were assigned to carbohydrate-active enzyme (CAZyme) families, representing 22 glycoside hydrolase (GH) families. The carboxymethyl cellulase (CMCase) synthesis was enhanced using the one-variable-at-a-time (OVAT) approach, succeeded by central composite design-based response surface methodology (RSM-CCD), resulting in 43.09 ± 2.60 U/mL of CMCase enzyme activity after incubation of 51 h at 40℃ and a pH of 6.0, a 3.82-fold improvement over the unoptimized activity of 11.28 ± 0.14 U/mL. Crude enzymes exhibited substantial specificity towards carboxymethyl cellulose (CMC) in addition to p-nitrophenyl-β-glucosidase, indicating the existence of β-D-glucoside glucohydrolase and glycoside hydrolase components. The extracellular crude enzyme released a maximum of 21.67 ± 0.20 mg/g total reducing sugars (TRS) from pre-treated Typha angustifolia leaves during the hydrolysis of various pre-treated lignocellulosic biomasses, surpassing the intracellular extract (15.70 ± 0.43 mg/g). The exceptional hydrolytic efficacy of the extracellular enzyme indicates that Exiguobacterium sp. M4 could become a viable candidate for the various LCB conversion processes and allied industries.

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