Mutational modulation of substrate bond-type specificity and thermostability of glucoamylase from Aspergillus awamori by replacement with short homologue active site sequences and thiol/disulfide engineering

Rational protein engineering based on three-dimensional structure, sequence alignment, and previous mutational analysis served to increase thermostability and modulate bond-type specificity in glucoamylase from Aspergillus awamori. The single free cysteine, Cys320, became disulfide bonded in the Ala246-->Cys mutant, thus enhancing T-50 by 4 degrees C to 73 degrees C. Compared to wild-type, Ala246-->Cys was roughly twice as active at 66 degrees C, but half as active at 45 degrees C. The alternative, elimination of the thiol group in Cys320-->Ala, barely improved thermostability or altered activity. Secondly, to acquire exceptionally high specificity toward alpha-1,6 glucosidic linkages, characteristic of Hormoconis resinae glucoamylase, two short sequential mutants, Val181-->Thr/Asn182-->Tyr/Gly183-->Ala (L3 glucoamylase) and Pro307-->Ala/Thr310-->Val/Tyr312-->Met/Asn313-->Gly (L5 glucoamylase), were made. These homologue mutants are located in the (alpha/alpha)(6)-fold of the catalytic domain in segments that connect alpha-helices 5 and 6 and alpha-helices 9 and 10, respectively. The kinetics of malto- and isomaltooligosaccharides hydrolysis clearly demonstrated that combination of the mutations in L3L5 compensated adverse effects of the single replacements in L3 or L5 glucoamylases to yield wild-type or higher activity. On alpha-1,4-linked substrates, typically K-m increased 2-fold for L3, and k(cat) decreased up to 15-fold for L5 glucoamylase. In contrast, on alpha-1,6-linked substrates L3 showed both a 2-fold increase in K-m and a 3-fold decrease in k(cat), while L5 GA caused a similar k(cat) reduction, but up to 9-fold increase in k(m). L3L5 glucoamylase had remarkably Low K-m for isomaltotriose through isomaltoheptaose and elevated k(cat) on isomaltose, resulting in an approximately 2-fold improved catalytic efficiency (k(cat)/K-m). Rational loop replacement thus proved powerful in achieving variants with enhanced properties of a highly evolved enzyme.