Cyclodextrin glycosyltransferase from thermotolerant bacteria: screening, optimization, partial purification and characterization

Abstract

Screening of thermotolerant bacteria producing cyclodextrin glycosyltransferase (CGTase) was performed. Soil and water samples were collected from hot spring areas in different parts of Thailand. Primary screening for amylase using Medium I, which contained starch as enzyme inducer was carried out. Thirty-eight isolates exhibited clear zone when staining with 0.02% I2 in 0.2% KI were found. Isolates with amylase activity were further screened for CGTase. Selection medium was Horikoshi medium to which dyes system to follow CGTase activity was incorporated. Two isolates (RB01 and KB01) with yellowish orange clear zone on an intense pink background were observed. These isolates were checked for growth and enzyme activity in liquid culture in the temperature range of 30-55 degree celcius for selection of the best thermotolerant strain. It was found that RB01 was grown and was able to produce CGTase at temperature range of 30-45 degree celcius, while grown best at 37 degree celcius but exerted highest activity at 40 degree celcius. Biochemical and physiological characterization indicated that RB01 was Bacillus circulans. Characterization by 16S rRNA gene demonstrated that RB01 was closely related to Paenibacillus campinasensis (strain 324) with 99% homology. The optimum conditions for cell growth and enzyme production were culturing RB01 in Horikoshi medium with 1.0% soluble starch at 40 degree celcius, pH 10.0 for 60 hours. The enzyme was partially purified by starch adsorption, the % recovery and purification fold were 57.3 and 26.8, respectively. The final specific activity was 3,568 U/mg. The optimum pH and temperature of the enzyme were 6.0 and 55 degree celcius, the pH and temperature stability of the enzyme were 7.0 and 40 degree celcius, respectively. The molecular weight was estimated to be 65,000 by SDS-PAGE. The enzyme formed mainly beta-cyclodextrin with small amounts of alpha- and gramma- cyclodextrin. The ratio of alpha-, beta- and gramma-cyclodextrins was 1.0: 5.4: 1.2. The best condition for storing enzyme was -20 degree celcius.