Abstract
PurposeGracilibacillus dipsosauri strain DD1 is a salt-tolerant Gram-positive bacterium that can hydrolyze the synthetic substrates o-nitrophenyl-β-d-galactopyranoside (β-ONP-galactose) and p-nitrophenyl-α-d-galactopyranoside (α-PNP-galactose). The goals of this project were to characterize the enzymes responsible for these activities and to identify the genes encoding them.MethodsG. dipsosauri strain DD1 was grown in tryptic soy broth containing various carbohydrates at 37 °C with aeration. Enzyme activities in cell extracts and whole cells were measured colorimetrically by hydrolysis of synthetic substrates containing nitrophenyl moieties. Two enzymes with β-galactosidase activity and one with α-galactosidase activity were partially purified by ammonium sulfate fractionation, ion-exchange chromatography, and gel-filtration chromatography from G. dipsosauri. Coomassie Blue-stained bands corresponding to each activity were excised from nondenaturing polyacrylamide gels and subjected to peptide sequencing after trypsin digestion and HPLC/MS analysis.ResultFormation of β-galactosidase and α-galactosidase activities was repressed by d-glucose and not induced by lactose or d-melibiose. β-Galactosidase I had hydrolytic and transgalactosylation activity with lactose as the substrate but β-galactosidase II showed no activity towards lactose. The α-galactosidase had hydrolytic and transgalactosylation activity with d-melibiose but not with d-raffinose. β-Galactosidase I had a lower Km with β-ONP-galactose as the substrate (0.693 mmol l−1) than β-galactosidase II (1.662 mmol l−1), was active at more alkaline pH, and was inhibited by the product d-galactose. β-Galactosidase II was active at more acidic pH, was partially inhibited by ammonium salts, and showed higher activity with α-PNP-arabinose as a substrate. The α-galactosidase had a low Km with α-PNP-galactose as the substrate (0.338 mmol l−1), a pH optimum of about 7, and was inhibited by chloride-containing salts. β-Galactosidase I activity was found to be due to the protein A0A317L6F0 (encoded by gene DLJ74_04930), β-galactosidase II activity to the protein A0A317KZG3 (encoded by gene DLJ74_12640), and the α-galactosidase activity to the protein A0A317KU47 (encoded by gene DLJ74_17745).ConclusionsG. dipsosauri forms three intracellular enzymes with different physiological properties which are responsible for the hydrolysis of β-ONP-galactose and α-PNP-galactose. BLAST analysis indicated that similar β-galactosidases may be formed by G. ureilyticus, G. orientalis, and G. kekensis and similar α-galactosidases by these bacteria and G. halophilus.
Highlights
The genus Gracilibacillus includes a diverse group of halotolerant Gram-positive bacteria, which usually form thin motile rods with terminal spherical or ellipsoidal endospores (Editorial Board 2015)
G. dipsosauri forms three intracellular enzymes with different physiological properties which are responsible for the hydrolysis of β-ONP-galactose and α-PNP-galactose
Deutch et al Annals of Microbiology (2021) 71:46 β-galactosidases may be formed by G. ureilyticus, G. orientalis, and G. kekensis and similar α-galactosidases by these bacteria and G. halophilus
Summary
The genus Gracilibacillus includes a diverse group of halotolerant Gram-positive bacteria, which usually form thin motile rods with terminal spherical or ellipsoidal endospores (Editorial Board 2015). Others were isolated from animals (G. dipsosauri (Deutch 1994), G. timonensis (Diop et al 2018), G. phocaeensis (Senghor et al 2017)) or from food products (G. thailandensis (Chamroensaksri et al 2010), G. kimchii (Oh et al 2016), G. massiliensis (Diop et al 2016), and G. oryzae (He et al 2020)). As part of their initial characterization and identification, most of these bacteria were subjected to a standard set of physiological tests which included hydrolysis of the synthetic substrate β-ONPG (β-ONP-galactose, o-nitrophenyl-β-d-galactopyranoside, 2-nitrophenyl-βd-galactopyranoside). A partial summary of the species of Gracilibacillus with positive or negative results for these activities is included in the papers by Kim et al (2012) and Diop et al (2016). β-Galactosidases and α-galactosidases from other sources have been found to be industrially significant due to their ability to hydrolyze a wide range of carbohydrates or to form prebiotics and probiotics (Husain 2010; Saqib et al 2017; Vera et al 2020; Katrolia et al 2014; Bhatia et al 2020)
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