Insight into CAZymes of Alicyclobacillus mali FL18: Characterization of a New Multifunctional GH9 Enzyme

Abstract

In the bio-based era, cellulolytic and hemicellulolytic enzymes are biocatalysts used in many industrial processes, playing a key role in the conversion of recalcitrant lignocellulosic waste biomasses. In this context, many thermophilic microorganisms are considered as convenient sources of carbohydrate-active enzymes (CAZymes). In this work, a functional genomic annotation of Alicyclobacillus mali FL18, a recently discovered thermo-acidophilic microorganism, showed a wide reservoir of putative CAZymes. Among them, a novel enzyme belonging to the family 9 of glycosyl hydrolases (GHs), named AmCel9, was identified; in-depth in silico analyses highlighted that AmCel9 shares general features with other GH9 members. The synthetic gene was expressed in Escherichia coli and the recombinant protein was purified and characterized. The monomeric enzyme has an optimal catalytic activity at pH 6.0 and has comparable activity at temperatures ranging from 40 °C to 70 °C. It also has a broad substrate specificity, a typical behavior of multifunctional cellulases; the best activity is displayed on β-1,4 linked glucans. Very interestingly, AmCel9 also hydrolyses filter paper and microcrystalline cellulose. This work gives new insights into the properties of a new thermophilic multifunctional GH9 enzyme, that looks a promising biocatalyst for the deconstruction of lignocellulose.

Thermophilic biocatalysts for one-step conversion of citrus waste into lactic acid

“…Supernatants and crude extract were prepared according to Aulitto et al ( 2021 ). Briefly, 30 μL of the supernatants and 20 μg of crude extract were tested on (i) PNP-alpha-gal following the procedure described in Aulitto et al ( 2021 ); (ii) on arabino-xylan, xylan, konjac, and CMC according to the 3,5-dinitrosalicylic acid (DNS) method (Carbonaro et al 2022 ); and (iii) on ABTS (Ing et al 2021 ; Salzano et al 2022 ). The reaction activities were measured spectrophotometrically through Synergy H4 Plate Reader (BioTek Instruments Inc., Winooski, VT, USA).…”

Taxonomy, Ecology, and Cellulolytic Properties of the Genus Bacillus and Related Genera

“…The β-glucosidases encoded by these genes belong to the GH1 and GH73 families, and the endoglucanase belongs to the GH5 family [99]. Moreover, Carbonaro et al [100] analyzed the genome of Alicyclobacillus mali FL18 to find new cellulose-degrading enzymes. The analysis revealed four genes belonging to the GH1, GH9, GH51, and GH94 families, of which GH1 and GH94 legitimately hydrolyse short oligosaccharides, and a gene from GH1 encodes a β-glucosidase.…”