Characterization and gene cloning of cyclodextrinase from Paenibacillus sp.A11

Abstract

Bacillus circulans A11 isolated from South-East Asian soil was reclassified as Paenibacillus sp. A11 using 16S rRNA gene sequence, G+C content and cellular fatty acid composition analyses. Levels of similarity of 16S rRNA gene between strain A11 and the Paenibacillus species were 90-99%, while similarity with Bacillus circulans was only 86%. The major cellular fatty acid was anteiso-C15:0 which accounted for 59.3% of total cellular fatty acids and the G+C content was 50.3 mol%. This bacterium was proved to possess cyclodextrinase (CDase) activity which could be detected on a specific screening medium containing β-CD and phenolphthalein. The CDase was purified approximately 22 folds with 28% recovery to a specific activity of 133 units/mg protein by 40-60% ammonium sulfate precipitation, DEAE Sephadex A-50, and Phenyl Sepharose CL-4B chromatography. It was proved to be homogeneous by non-denaturing and SDS-PAGE. The enzyme was a single polypeptide with a molecular weight of 80 kDa as determined by gel filtration and SDS-PAGE. The optimum pH and temperature for activity of the purified enzyme were pH 7.0 and 40 degree celsius. The enzyme had isoelectric point of 5.4. N-Terminal sequence was M F L E A V Y H R P R K N W S. When relative hydrolytic activity of the CDase on different substrates were compared, it was found that high specificity was exerted by β-CD. The enzyme recognizes γ-1,4-glucose units and the hydrolysis depends on the size of oligosaccharides. The major product from γ-1,4-glucan substrates, either cyclic or linear, was maltose. The k[subscript cat]/Km values for γ-, β- and γ-CD were 1.41x10⁵, 8.28x10⁵ and 3.73x10⁵ M⁻¹min⁻¹. The enzyme activity was completely inactivated by 1 mM N-bromosuccinimide and diethylpyrocarbonate suggesting the crucial importance of Trp and His for its catalytic activity. Essential Trp was confirmed to be at enzyme active site by substrate protection experiment. In this study, the CDase gene coding for this enzyme was cloned into E. coli. The open reading frame of CDase gene was 1,959 bp encoding CDase of 653 amino acid residues. Expression of Paenibacillus sp. A11 CDase gene using pUC 18 vector in Escherichia coli JM 109 was induced by adding 0.5 M sorbitol as an osmolyte in the culturing medium. After 24 h induction, the formation of insoluble CDase was prevented while a three-fold increase in cytoplasmic CDase activity was achieved in parallel with increase in cell growth. The recombinant CDase protein was successfully purified and characterized. Biochemical properties of the CDases from Paenibacillus sp. A11 and E. coli transformant (pJK 555) were almost identical.