AMINO-ACID SUBSTITUTIONS ENHANCING THERMOSTABILITY OF BACILLUS-POLYMYXA BETA-GLUCOSIDASE-A

Mutations enhancing the thermostability of beta-glucosidase A of Bacil lus polymyxa, a family 1 glycosyl hydrolase, have been obtained after hydroxylamine mutagenesis of a plasmid containing the bglA gene, trans formation of Escherichia coli with the mutagenized plasmid, and identi fication of transformant colonies that showed beta-glucosidase activit y after a thermal treatment that inactivated the wild-type enzyme. Two additive mutations have been characterized that cause replacement of glutamate at position 96 by lysine and of methionine at position 416 b y isoleucine respectively. The thermoresistant mutant enzymes showed i ncreased resistance to other denaturing agents, such as pH and urea, w hile their kinetic parameters did not change. CD spectra indicated tha t the E96K replacement caused an increase in alpha-helix content, The observed effect of the M416I mutation is consistent with the lower con tent of cysteine and methionine found in family 1 enzymes of thermophi lic species compared with similar ones from mesophilic organisms.