Analysis of the raw starch-binding domain by mutation of a glucoamylase from Aspergillus awamori var. kawachi expressed in Saccharomyces cerevisiae.
AUTOR(ES)
Goto, M
RESUMO
Carboxy-terminal deletions were introduced into the raw starch-binding domain (A-515 to R-615) encoded by the gene for glucoamylase I (GAI) from Aspergillus awamori var. kawachi. Genes coding for proteins designated GA596 (A-1 to E-596), GA570 (A-1 to A-570), and GA559 (A-1 to N-559) were constructed and resulted in truncated proteins. All of the mutant genes were expressed heterologously in Saccharomyces cerevisiae. GA596 adsorbed to raw starch and digested it. GA570 and GA559 did not adsorb to raw starch or to an alpha-cyclodextrin-Sepharose CL-4B gel under our experimental conditions. However, GA570 was able to digest raw starch, and the digestion of raw starch by GA570 was inhibited by beta-cyclodextrin. Residue Trp-562 of GAI, which was suggested previously to contribute to formation of an inclusion complex with raw starch, was replaced by Leu (GAW562L), Phe (GAW562F), and Gly (GAW562G). GAW562L and GAW562F adsorbed to raw starch and an alpha-cyclodextrin gel, but GAW562G did not. Although GAW562L digested raw starch to the same extent as wild-type GAI (designated GAY), GAW562F and GAW562G exhibited less ability to digest raw starch. On the basis of our results, it appears that the sequence around Trp-562, PL(W-562)YVTVTLPA, is the minimal sequence necessary for digestion of raw starch and that hydrophobic residue Trp-562 contributes to formation of an inclusion complex. The sequence near Trp-589, which has abundant hydrogen bond-forming residues and the charged amino acid residues needed for stable adsorption to raw starch, probably assists in the formation of the inclusion complex.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=201917Documentos Relacionados
- Production and Characteristics of Raw Starch-Digesting Glucoamylase O from a Protease-Negative, Glycosidase-Negative Aspergillus awamori var. kawachi Mutant
- Production and Characteristics of Raw Starch-Digesting Glucoamylase O from a Protease-Negative, Glycosidase-Negative Aspergillus awamori var. kawachi Mutant
- Functional analysis of the threonine- and serine-rich Gp-I domain of glucoamylase I from Aspergillus awamori var. kawachi.
- Expression and functional analysis of a hyperglycosylated glucoamylase in a parental host, Aspergillus awamori var. kawachi.
- Introduction of Raw Starch-Binding Domains into Bacillus subtilis α-Amylase by Fusion with the Starch-Binding Domain of Bacillus Cyclomaltodextrin Glucanotransferase