Isolation, characterization and regulation of amylases from Clostridium perfringens
An $\alpha$-amylase (EC 220.127.116.11) secreted by Clostridium perfringens NCTC 8679 Type A was purified to homogeneity and characterized. It was isolated from concentrated cell-free culture medium by ion exchange and gel permeation chromatography. The pH optimum and pI of the enzyme were 6.5 and 4.75 respectively. The estimated molecular weight of the purified enzyme was 76 000. Calcium (5 mM) increased the optimal temperature for activity of the purified enzyme from 30 to 40$\sp\circ$C. The purified enzyme was inactivated between 35 and 40$\sp\circ$C which increased to between 45 and 50$\sp\circ$C in the presence of calcium. The purified enzyme produced a mixture of oligosaccharides as major endproducts of starch hydrolysis indicating $\alpha$-amylase activity.
The effect of glucose and other sugars on sporulation and extracellular amylase production by Clostridium perfringens NCTC 8679 type A in a defined medium was also studied. Cells grown in the presence of glucose and mannose yielded the highest levels of amylase activity while disaccharides such as lactose, maltose and sucrose resulted in moderate amylase production. Little amylase activity was detected in the medium in the presence of ribose or galactose. The concentration of each sugar resulting in highest amylase production was between 6 and 10 mM except for fructose (25 mM). Levels of heat resistant spores decreased as sugar concentrations increased.
The addition of even small amounts of glucose to the medium before exponential growth suppressed sporulation but maximized amylase activity. The addition of glucose after the initiation of sporulation did not inhibit spore formation. However, its addition to 3-h amylase-producing cells did inhibit subsequent sporulation but promoted the continued excretion of amylase. The extracellular amylase produced by C. perfringens exhibited eight major extracellular amylolytic activity bands when examined by activity staining following polyacrylamide gel electrophoresis (PAGE). Extracellular amylolytic enzymes released in sporulation process were different from those released during vegetative growth (extracellular amylase accumulation) whereas their intracellular amylolytic activities remained identical. A significant increase of an endo-acting amylolytic activity which cleaved both $\alpha$-1,4- and $\alpha$-1,6-glucosidic linkage was observed when C. perfringens sporulated. The different response to glucose between sporulating cells and amylase-producing cells suggests that the mechanisms of catabolite repression of extracellular amylase production and sporulation are distinct in C. perfringens whereas regulation of amylolytic activity during sporulation is possibly mediated by sporulation-related, amylolytic enzyme modification.