Cloning, biochemical characterization and molecular docking of novel thermostable ß-glucosidase BglA9 from Anoxybacillus ayderensis A9 and its application in de-glycosylation of Polydatin.

This study reports a novel BglA9 gene of 1345 bp encoding ß-glucosidase from Anoxybacillus ayderensis A9, which was amplified and expressed in E. coli BL21 (DE3): pLysS cells, purified with Ni-NTA column having molecular weight of 52.6 kDa and was used in the bioconversion of polydatin to resveratrol. The kinetic parameters values using pNPG as substrate were Km (0.28 mM), Vmax (43.8 µmol/min/mg), kcat (38.43 s-1) and kcat/Km (135.5 s-1 mM-1). The BglA9 was active in a broad pH range and had an activity half-life around 24 h at 50 °C. The de-glycosylation efficiency of BglA9 for polydatin was determined by estimating the amount of glucose released after enzymatic reaction by a dinitrosalicylic acid (DNS) assay. The kinetic parameters of BglA9 for polydatin were 5.5 mM, 20.84 µmol/min/mg, 18.28 s-1and 3.27 s-1 mM-1 for Km, Vmax, kcat, and kcat/Km values, respectively. The Ki value for glucose was determined to be 1.7 M. The residues Gln19, His120, Glu355, Glu409, Glu178, Asn222 may play a crucial role in the deglycosylation as revealed by the 3D structure of enzyme docked with polydatin.

Cloning, expression and biochemical characterization of lignin-degrading DyP-type peroxidase from Bacillus sp. Strain BL5.

Lignin is a major byproduct of pulp and paper industries, which is resistant to depolymerization due to its heterogeneous structure. The enzymes peroxidases can be utilized as potent bio-catalysts to degrade lignin. In the current study, an Efeb gene of 1251bp encoding DyP-type peroxidase from Bacillus sp. strain BL5 (DyPBL5) was amplified, cloned into a pET-28a (+) vector and expressed in Escherichia coli BL21 (DE3) cells. A 46 kDa protein of DyPBL5 was purified through ion-exchange chromatography. Purified DyPBL5 was active at wide temperature (25-50 °C) and pH (3.0-8.0) range with optimum activity at 35 °C and pH 5.0. Effects of different chemicals on DyPBL5 were determined. The enzyme activity was strongly inhibited by SDS, DDT and ß-mercaptoethanol, whereas stimulated in the presence of organic solvents such as methanol and ethanol. The kinetic parameters were determined and Km, Vmax and Kcat values were 1.06 mM, 519.75 µmol/min/mg and 395 S̶ 1, respectively. Docking of DyPBL5 with ABTS revealed that, Asn 244, Arg 339, Asp 383 and Thr 389 are putative amino acids, taking part in the oxidation of ABTS. The recombinant DyPBL5 resulted in the reduction of lignin contents up to 26.04 %. The SEM and FT-IR analysis of test samples gave some indications about degradation of lignin by DyPBL5. Various low molecular weight lignin degradation products were detected by analyzing the samples through gas chromatography mass spectrometry. High catalytic efficiency and lignin degradation rate make DyPBL5 an ideal bio-catalyst for remediation of lignin-contaminated sites.