title: 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.
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 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 Km increased 2-fold for L3, and Kcat decreased up to 15-fold for L5 glucoamylase. In contrast, on alpha-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.
authors.affiliation: Department of Chemistry, Carlsberg Laboratory, Copenhagen Valby, Denmark.
authors.forename: H P
authors.initials: HP
authors.lastname: Fierobe
authors.forename: B B
authors.initials: BB
authors.lastname: Stoffer
authors.forename: T P
authors.initials: TP
authors.lastname: Frandsen
authors.forename: B
authors.initials: B
authors.lastname: Svensson
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D017931:DNA Primers; D004795:Enzyme Stability; D005087:Glucan 1,4-alpha-Glucosidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009844:Oligosaccharides / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: D017931:DNA Primers; D009844:Oligosaccharides; D005087:Glucan 1,4-alpha-Glucosidase