Purification Of Thermostable Glucoamylase From Chaetomium Thermophilum And Cloning And Expression Of The Encoding Gene

Glucoamylase is an exo-acting enzyme that removes the glucose unites from the non-reducing ends of amylose, amylopectin and glycogen by hydrolyzingα-1.4 linkages in a consecutive manner, producing-D-glucose as the sole product. It also hydrolysesα-1.6 and the rareα-1.3 linkages although at a much slower rate. Glucoamylases are an important group of enzymes in starch-processing. It is second to protease in worldwide distribution and sales among industrial enzymes. Glucoamylases find many applications in industry. This enzyme is used in dextrose production, in the baking industry, in the brewing of low calorie beer and in whole grain hydrolysis for the alcohol industry. The primary commercial application of glucoamylases is the production of glucose syrups from starch. A number of glucoamylases from cultures of various microorganisms, including Aspergillus, Rhizopus, Saccharomyces spieces have been screened for potential industrial application. In recent years glucoamylases from thermophilic fungi, which are expected to be thermostable, have aroused increasing attention among researchers. Among all the glucoamylases, only few thermostable glucoamylases have been described, including the enzymes of Myrothecium, Aspergillus fumigatus, Humicola grisea, Humicola grisea var. thermoidea and Thermomyces lanuginosus. C. thermophilum is a widely distributed soil-inhibiting fungus of considerable interest producer of thermostable enzymes, including cellucose, endocellulase, xylanase, laccase which have been purified and well studied. However, glucoamylse from the genus C. thermophilum has not yet been studied. During growth in a medium containing soluble starch,C. thermophilum also produced glucoamylase. The enzyme was purified from the culture filtrate of the strain by fractional ammonium sulphate precipitation, DEAE-Sepharose chromatography and Phenyl-Sepharose chromatography. The purified enzyme was shown to be homogeneous by SDS-PAGE, and the molecular weight was estimated to be 64kDa.The optimum pH and temperature obtained for the purified enzyme was 4.0 and 65 oC.