Bioprospecting novel hemicellulases from the gut microbiota of the common black slug (Arion ater)

Madubuike, HK

Abstract

The attendant effect of continuous fossil use on the environment and energy security has prompted the search for a suitable energy alternative devoid of negative characteristics. Lignocellulose-biomass (LCB) is a renewable source of carbon for the development of fuels and high-value chemicals in the concept of biorefinery. Biocatalysts that can drive sustainable biorefining are of huge interest to mitigate the hazards resulting from the use of inorganic chemicals in LCB pretreatment. Screening of under-studied environments has greatly expanded the repertoire of novel enzymes with putative carbohydrate-active activities able to degrade LCB. Of particular interest, metagenomic exploration of gut microbiota from invertebrates has yielded a significant resource. In this study, the output from the metagenomic analysis of the slug-gut microbiota was explored in search of novel enzymes targeting the hemicellulose fraction of LCB. Genes from the metagenomic library (designated as Gene_id_(2151, 10239, 35667, 40363, and 5461)) and predicted to belong to the glycosyhydrolase family 43, abhydrolase_3, acetyl xylan esterase, abhydrolase_2, and an esterase family respectively were selected for characterisation. The conserved domains and related proteins were identified using various bioinformatic tools. The protein structure was developed with the homology modelling tool hosted in I-Tasser. Candidate genes were PCR amplified and cloned into expression vectors for recombinant protein expression. Enzyme activity testing was achieved with suitable synthetic and natural substrates using purified proteins. The enhancement of enzyme properties targeting an improved performance in organic solvent was explored with protein engineering. NCBI Blast search confirmed that candidate proteins contained conserved domain function relating to LCB degradation, and catalytic residues of candidate proteins were inferred by sequence homology to characterised proteins. The candidate genes were PCR amplified and estimated gene sizes were confirmed as expected on an agarose gel. Gene_id_40363 matched closely to an uncharacterised esterase from Buttiauxella agrestis and hence designated as BaAXE. The conserved domain of BaAXE returned functions described as putative hydrolase and carboxylesterase/phospholipase. BaAXE showed activity on short acyl chains of 4-Nitrophenyl with a specific activity of 78.11 U/mg and Km value of 0.4 mM with 4-Nitrophenyl acetate. BaAXE showed optimum activity at pH 8 and 40 oC. The enzyme showed high thermostability and was moderately tolerant to all the assayed organic solvents and metal additives. BaAXE released acetic acid from acetylated xylan and β-D-glucose pentaacetate confirming its designation as an acetyl xylan esterase and mutant variants of BaAXE showed enhanced activity and stability in hydrophilic organic solvents. This study proves the potential of biodiversity in mining novel enzymes with unique characteristics needed in biotechnological applications, and supports the prospect of enzyme engineering in improving enzyme properties.

Citation

Madubuike, H. Bioprospecting novel hemicellulases from the gut microbiota of the common black slug (Arion ater). (Thesis). University of Salford

• Thesis Type: Thesis
• Deposit Date: Oct 5, 2022
• Award Date: May 25, 2022