Thermal inactivation and conformational lock studies on glucose oxidase

In this study, the dissociative thermal inactivation and conformational lock theories are applied for the homodimeric enzyme glucose oxidase (GOD) in order to analyze its structure. For this purpose, the rate of activity reduction of glucose oxidase is studied at various temperatures using b-D-glucose as the substrate by incubation of enzyme at various temperatures in the wide range between 40 and 70 �C using UV–Vis spectrophotometry. It was observed that in the two ranges of temperatures, the enzyme has two different forms. In relatively low temperatures, the enzyme is in its dimeric state and has normal activity. In high temperatures, the activity almost disappears and it aggregates. The above achievements are confirmed by dynamic light scattering. The experimental parameter ‘‘n’’ as the obvious number of conformational locks at the dimer interface of glucose oxidase is obtained by kinetic data, and the value is near to two. To confirm the above results, the X-ray crystallography structure of the enzyme, GOD (pdb, 1gal), was also studied. The secondary and tertiary structures of the enzyme to track the thermal inactivation were studied by circular dichroism and fluorescence spectroscopy, respectively. We proposed a mechanism model for thermal inactivation of GOD based on the absence of the monomeric form of the enzyme by circular dichroism and fluorescence spectroscopy

Unraveling the Lipolytic Activity of Thermophilic Bacteria Isolated from a Volcanic Environment

In a bioprospecting effort towards novel thermostable lipases, we assessed the lipolytic profile of 101 bacterial strains isolated from the volcanic area of Santorini, Aegean Sea, Greece. Screening of lipase activity was performed both in agar plates and liquid cultures using olive oil as carbon source. Significant differences were observed between the two screening methods with no clear correlation between them. While the percentage of lipase producing strains identified in agar plates was only 17%, lipolytic activity in liquid culture supernatants was detected for 74% of them. Nine strains exhibiting elevated extracellular lipase activities were selected for lipase production and biochemical characterization. The majority of lipase producers revealed high phylogenetic similarity with Geobacillus species and related genera, whilst one of them was identified as Aneurinibacillus sp. Lipase biosynthesis strongly depended on the carbon source that supplemented the culture medium. Olive oil induced lipase production in all strains, but maximum enzyme yields for some of the strains were also obtained with Tween-80, mineral oil, and glycerol. Partially purified lipases revealed optimal activity at 70–80°C and pH 8-9. Extensive thermal stability studies revealed marked thermostability for the majority of the lipases as well as a two-step thermal deactivation pattern

Biochemical and Thermodynamic Studies on a Novel Thermotolerant GH10 Xylanase from Bacillus safensis

Xylanases have a broad range of applications in agro-industrial processes. In this study, we report on the discovery and characterization of a new thermotolerant GH10 xylanase from Bacillus safensis, designated as BsXyn10. The xylanase gene (bsxyn10) was cloned from Bacillus safensis and expressed in Escherichia coli. The reduced molecular mass of BsXyn10 was 48 kDa upon SDS-PAGE. Bsxyn10 was optimally active at pH 7.0 and 60 °C, stable over a broad range of pH (5.0–8.0), and also revealed tolerance toward different modulators (metal cations, EDTA). The enzyme was active toward various xylans with no activity on the glucose-based polysaccharides. KM, vmax, and kcat for oat spelt xylan hydrolysis were found to be 1.96 g·L−1, 58.6 μmole·min−1·(mg protein)−1, and 49 s−1, respectively. Thermodynamic parameters for oat spelt xylan hydrolysis at 60 °C were ΔS* = −61.9 J·mol−1·K−1, ΔH* = 37.0 kJ·mol−1 and ΔG* = 57.6 kJ·mol−1. BsXyn10 retained high levels of activity at temperatures up to 60 °C. The thermodynamic parameters (ΔH*D, ΔG*D, ΔS*D) for the thermal deactivation of BsXyn10 at a temperature range of 40–80 °C were: 192.5 ≤ ΔH*D ≤ 192.8 kJ·mol−1, 262.1 ≤ ΔS*D ≤ 265.8 J·mol−1·K−1, and 99.9 ≤ ΔG*D ≤ 109.6 kJ·mol−1. The BsXyn10-treated oat spelt xylan manifested the catalytic release of xylooligosaccharides of 2–6 DP, suggesting that BsXyn10 represents a promising candidate biocatalyst appropriate for several biotechnological applications