Purification and characterizaton of cyclodextrin glycosyltransferase from bacteria isolated from hot spring soil

Abstract

Hot spring soils from northern Thailand was screened for cyclodextrin glycosyltransferase (CGTase) producing bacteria. A bacterial isolate from Mae ka sa, Tak province was selected and identified, using biochemical characteristics and 16S rRNA gene analysis, to be Gram positive bacterium of the genus Paenibacillus, possibly P. macerans. The optimum conditions for bacterial growth and CGTase production were in Horikoshi liquid medium, pH 10.0 with 0.05 mM CaCl2 at 37ํC for 84 hours . CGTase production was initiated as cells entered log phase but the enzyme was highest at stationary phase. CGTase was purified by starch adsorption and b-CD affinity column to 100 folds with 21.2% yield. The dilution limit of the enzyme by CD-TCE precipitation was 1:211. Its molecular weight on sodium dodecyl sulfate-gel electrophoresis (SDS-PAGE) was 76,000 daltons. Its isoelectric point was estimated by isoelectrofocusing gel to be 5.20. It was proved to be a glycoprotein by Periodic acid-Schiffʼs staining on SDS-PAGE. The optimum conditions for dextrinizing and CD-forming (phenolphthalein method) activities were in Tris-HCl buffer (pH 8.0) with 10mM CaCl2 at 65 ํC for 10 minutes and in Tris-HCl buffer (pH 8.0) with 15mM CaCl2 at 75 ํC for 15 minutes, respectively. The thermostability of the enzyme was 60 ํC but could be enhanced to 70 ํC in the presence of 10mM CaCl2 and was stable at pH 4.0-11.0. Analysis of its CD products by HPLC demonstrated that the ratio of a-, b-, and g-cyclodextrin was 0.74 : 1 : 0.27 using 2% starch as substrate. Calcium chloride had stimulated and stabilizing effects on the enzyme. Mercuric ion, PMSF and EDTA inhibited the enzyme considerably.