Cloning, expression, purification and characterisation of a thermostable chitinase from Bacillus licheniformis A1

The chitinase B gene (ch/B65) of Bacillus licheniformis A1 (BlicA1) isolated from Diyadin hotspring in Turkey was cloned and sequenced. The gene is 1779 bp long and encodes a protein 592 amino acids with a 35-amino acid signal peptide at N-terminal. The gene has 99% percent similarity to chiB gene of Bacillus licheniformis under the GenBank number AY205293. The gene without signal peptide was overexpressed in Escherichia coli and the recombinant protein purified by nickel affinity chromatography. The activity of enzyme was shown on SDS-PAGE with the flourogenic substrate 4-methylumbelliferyl ß-D-N,N?-diacetylchitobioside. Kinetic characterisation of the enzyme was performed at 65 °C by using chromogenic substrate p-nitrophenyl N,N?-diacetyl-ß-D-chitobioside, and Km and V max were found to be 0.02 ?M and 1017 U/mg protein, respectively. Enzyme has maximal activity at pH 6.0 and was stable over a broad pH range of 5.0-9.0 for 24 h at room temperature and 4 h at 65 °C. Enzyme was 60% stable at 65 °C for 1.5 h. The inhibition or activation of some substances on the activity of enzyme was determined. High kinetic properties of enzyme open the possibility of an extensive structural and enzyme-substrate interaction studies

Characterization of a xylanase from a thermophilic strain of Anoxybacillus pushchinoensis A8

A facultatively anaerobic, thermophilic, xylanolytic bacterium was isolated from a sample collected from the Diyadin Hot Springs, Turkey. According to morphological, biochemical and molecular identification, this new strain was suggested to be representative of the Anoxybacillus pushchinoensis and it was designated as Anoxybacillus pushchinoensis strain A8. It exhibited 97% similarity to 16S rRNA gene sequence of A. pushchinoensis and 77% DNA homology by DNA-DNA hybridization studies. Q-sepharose and CM-sepharose chromatography was used to purify an extracellular xylanase to >90% purity from this species. The enzyme had a molecular mass of approximately 83 kDa. The enzyme showed optimum activity at pH 6.5 and it was 96% stable over a broad pH range of 6.5-11 for 24 hours. The enzyme had optimum activity at 55°C and it was 100% stable at temperature between 50-60°C up to 24 hours. Kinetic characterization of the enzyme was performed at temperature optima (55°C) and Vmax and K m were found to be 59.88 U/mg protein and 0.909 mg/mL, respectively. Oat spelt xylan but not xylooligosaccharides was degraded by the enzyme and xylose was the only product detected from oat xylan degradation. This suggested that the enzyme was an exo-acting xylanase. © 2008 Versita Warsaw and Springer-Verlag Berlin Heidelberg