Cloning, purification, and characterization of GH3 β-glucosidase, MtBgl85, from Microbulbifer thermotolerans DAU221

Background β-Glucosidases have attracted considerable attention due to their important roles in various biotechnological processes such as cellulose degradation to make energy and hydrolysis of isoflavone. Microbulbifer thermotolerans (M. thermotolerans) is isolated from deep-sea sediment and has not been researched much yet. As a potential candidate for a variety of biotechnological industries, β-glucosidases from the novel bacterial species should be researched extensively. Methods β-Glucosidase, MtBgl85, from M. thermotolerans DAU221 was purified by His-tag affinity chromatography and confirmed by SDS-PAGE and zymogram. Its biochemical and physiological properties, such as effects of temperature, pH, metal ions, and organic solvents, substrate specificity, and isoflavone hydrolysis, were investigated. Results M. thermotolerans DAU221 showed β-glucosidase activity in a marine broth plate containing 0.1% esculin and 0.25% ammonium iron (III) citrate. The β-glucosidase gene, mtbgl85, was isolated from the whole genome sequence of M. thermotolerans DAU221. The β-glucosidase gene was 2,319 bp and encoded 772 amino acids. The deduced amino acid sequence had a 43% identity with OaBGL84 from Olleya aquimaris and 35% and 32% identity with to CfBgl3A and CfBgl3C from Cellulomonas fimi among bacterial glycosyl hydrolase family 3, respectively. The optimal temperature of MtBgl85 was 50 °C and the optimum pH was 7.0. MtBgl85 activity was strongly reduced in the presence of Hg2+ and Cu2+ ions. As a result of measuring the activity at various concentrations of NaCl, it was confirmed that the activity was maintained up to the concentration of 1 M, but gradually decreased with increasing concentration. MtBgl85 showed higher enzyme stability at non-polar solvents (high Log Pow) than polar solvents (low Log Pow). The hydrolyzed products of isoflavone glycosides and arbutin were analyzed by HPLC.

Presence of β-glucosidase (bgl1) gene in Phaeosphaeria nodorum and Phaeosphaeria avenaria f.sp. triticea

Phaeosphaeria avenaria f.sp. avenaria (Paa), the causal agent of stagonospora leaf blotch in oats, produces a glycosyl hydrolase family 3 enzyme, β-glucosidase, which is responsible for detoxification of steroidal avenacosides in oat leaves, but is not essential for pathogenicity. For a comparative genetic relatedness study, a Paa-like β-glucosidase gene (bgl1) was PCR-amplified from Phaeosphaeria nodorum and P. avenaria f.sp. triticea, and Phaeosphaeria spp. from dallis grass (Paspalum dilatatum) (isolate S-93-48) and rye (Secale cereale) (isolate Sn48-1). Different sizes of bgl1 gene coding sequences ranging from 3018 to 3023 bp were determined. The bgl1 gene structure in these Phaeosphaeria species was identical to that of Paa and contained four exons and three introns. Nucleotide variations occurring in introns 1 and 2 of the bgl1 gene divided wheat-biotype P. nodorum into four groups. Two 12-bp-long direct sequence repeats (5'-TCA/G ACT GGT TT/CA/G) were found in the promoter region of the bgl1 gene in Phaeosphaeria species; only one repeat was present in the two P. avenaria f.sp. triticea isolates ATCC26370 and ATCC26377 (Pat2) from foxtail barley (Hordeum jubatum) and some homothallic P. avenaria f.sp. triticea isolates (Pat1). With sequence similarities in the noncoding internal transcribed spacer region of nuclear rDNA, the partial gpd gene fragment containing the intron 4, and the full-length bgl1 gene, five Phaeosphaeria isolates (5413, 1919WRS, 1920WRS, 1921WRS, Sa37-2) from oat (Avena sativa) were molecularly determined to be Paa. Two oat isolates (Sa38-1 and Sa39-2) from Poland appeared to be Pat1. The results suggest that classification of two Phaeosphaeria avenaria formae speciales based on host specificity should be re-evaluated.Key words: wheat, oat, Phaeosphaeria, β-glucosidase gene.

The celA Gene, Encoding a Glycosyl Hydrolase Family 3 β-Glucosidase in Azospirillum irakense, Is Required for Optimal Growth on Cellobiosides

ABSTRACT The CelA β-glucosidase of Azospirillum irakense,belonging to glycosyl hydrolase family 3 (GHF3), preferentially hydrolyzes cellobiose and releases glucose units from the C3, C4, and C5oligosaccharides. The growth of a ΔcelA mutant on these cellobiosides was affected. In A. irakense, the GHF3 β-glucosidases appear to be functional alternatives for the GHF1 β-glucosidases in the assimilation of β-glucosides by other bacteria.

Growth of Azospirillum irakense KBC1 on the Aryl β-Glucoside Salicin Requires either salA or salB

ABSTRACT The rhizosphere nitrogen-fixing bacteriumAzospirillum irakense KBC1 is able to grow on pectin and β-glucosides such as cellobiose, arbutin, and salicin. Two adjacent genes, salA and salB, conferring β-glucosidase activity to Escherichia coli, have been identified in a cosmid library of A. irakense DNA. The SalA and SalB enzymes preferentially hydrolyzed aryl β-glucosides. A Δ(salA-salB) A. irakense mutant was not able to grow on salicin but could still utilize arbutin, cellobiose, and glucose for growth. This mutant could be complemented by either salA or salB, suggesting functional redundancy of these genes in salicin utilization. In contrast to this functional homology, the SalA and SalB proteins, members of family 3 of the glycosyl hydrolases, show a low degree of amino acid similarity. Unlike SalA, the SalB protein exhibits an atypical truncated C-terminal region. We propose that SalA and SalB are representatives of the AB and AB′ subfamilies, respectively, in glycosyl hydrolase family 3. This is the first genetic implication of this β-glucosidase family in the utilization of β-glucosides for microbial growth.