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

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Abstract
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 T50 by 4 °C to 73 °C. Compared to wild-type, Ala246→Cys was roughly twice as active at 66 °C, but half as active at 45 °C. The alternative, elimination of the thiol group in Cys320→Ala, barely improved thermostability or altered activity. Secondly, to acquire exceptionally high specificity toward α-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 (α/α)6-fold of the catalytic domain in segments that connect α-helices 5 and 6 and α-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 α-1,4-linked substrates, typically Km increased 2-fold for L3, and kcat decreased up to 15-fold for L5 glucoamylase. In contrast, on α-1,6-linked substrates L3 showed both a 2-fold increase in Km and a 3-fold decrease in kcat, while L5 GA caused a similar kcat reduction, but up to 9-fold increase in Km. L3L5 glucoamylase had remarkably low Km for isomaltotriose through isomaltoheptaose and elevated kcat on isomaltose, resulting in an approximately 2-fold improved catalytic efficiency (kcat/Km). Rational loop replacement thus proved powerful in achieving variants with enhanced properties of a highly evolved enzyme.

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