First Glycoside Hydrolase Family 2 Enzymes from Thermus antranikianii and Thermus brockianus with β-Glucosidase Activity

Abstract

Two glycoside hydrolase encoding genes (tagh2 and tbgh2) were identified from different Thermus species using functional screening. Based on amino acid similarities, the enzymes were predicted to belong to glycoside hydrolase (GH) family 2. Surprisingly, both enzymes (TaGH2 and TbGH2) showed twofold higher activities for the hydrolysis of nitrophenol-linked β-D-glucopyranoside than of -galactopyranoside. Specific activities of 3,966 U/mg for TaGH2 and 660 U/mg for TbGH2 were observed. In accordance, Km values for both enzymes were significantly lower when β-D-glucopyranoside was used as substrate. Furthermore, TaGH2 was able to hydrolyze cellobiose. TaGH2 and TbGH2 exhibited highest activity at 95 and 90°C at pH 6.5. Both enzymes were extremely thermostable and showed thermal activation up to 250% relative activity at temperatures of 50 and 60°C. Especially, TaGH2 displayed high tolerance toward numerous metal ions (Cu2+, Co2+, Zn2+), which are known as glycoside hydrolase inhibitors. In this study, the first thermoactive GH family 2 enzymes with β-glucosidase activity have been identified and characterized. The hydrolysis of cellobiose is a unique property of TaGH2 when compared to other enzymes of GH family 2. Our work contributes to a broader knowledge of substrate specificities in GH family 2.

Zinc binding proteome of a phytopathogen Xanthomonas translucens pv. undulosa

“…Five secretory Zn-binding proteins were found in the class of metabolic process having glycoside hydrolase (4) and carboxylesterase (1) domain. It was stated previously that some glycoside hydrolases play role in host–microbe interactions and the enzymatic activity of some glycoside hydrolases was inhibited by Zn 2+ metal ion [70–72]. The carboxylesterase protein also has affinity for Zn ion [87] and its role to hydrolyse ester bonds and virulence of bacteria was also known earlier [88,142].…”

Genome-Wide Analysis of β-Galactosidases in Xanthomonas campestris pv. campestris 8004

“…The other possibility is that these proteins have a novel activity to cope with environmental change during the Xcc -environment interaction. In addition to β-galactosidase, GH2 were described to function as β-mannosidase, β-glucuronidase, endo-β-mannosidase, β-glucosidase, or exo-β-glucosaminidase (Schröder et al, 2015 ). For example, the GH2 protein, NixH, was found to have, β-mannosidase activity (Dupoiron et al, 2015 ).…”

Recombinant expression of insoluble enzymes in Escherichia coli: a systematic review of experimental design and its manufacturing implications

“…Allows greater control over expression [ 73 ] Arylamine N -acetyltransferase [ 14 ] Beta-glucosidase One Shot™ BL21 Star™ (DE3) (B) Thermo Fisher Scientific High expression of non-toxic proteins. Promotes high expression of protein product [ 74 ] N -Acyl- d -glucosamine 2-epimerase Tuner™(DE3) (B) Merck KGaA Allows slower protein synthesis to promote solubility using adjustable inducer concentrations [ 71 ] Sphingomyelinase Rosetta/Rosetta 2 (DE3) (B) Merck KGaA Promote correct synthesis and folding for proteins (i.e. eukaryotic proteins) with rare codons (AGA, AGG, AUA, CUA, GGA, CCC, and CGG) in E .…”

“…These strains are marketed to have superior performance that are well-suited to mitigating issues concerning: High disulfide bond formation—BL21 Origami B [ 66 ] Codon Bias BL21 CodonPlus [ 67 ]; BL21 Rosetta Temperature instability—BL21 ArcticExpress [ 14 , 31 ] Toxic proteins—BL21 AI [ 68 , 69 ], BL21 Tuner [ 70 – 72 ]. Low expression yield—BL21 pLysS [ 14 , 73 ] and BL21 Star [ 74 , 75 ]. …”