Construction and characterization of mutant glucoamylases from the yeast Saccharomycopsis fibuligera.

Abstract

To find amino acid residues which are required for glucoamylase activity, mutant glucoamylase genes were constructed by in vitro mutations of GLUlDNAencoding Saccharomycopsis fibuligera glucoamylase and introduced into Saccharomyces cerevisiae, and the resulting mutant proteins were assayed for enzymatic activities. Eighteen mutant proteins were obtained by random insertions of a BamHllinker DNA.Six out of 7 proteins with mutations in conserved regions among divergent glucoamylases showed no activities, while 8 out of ll proteins with mutations in unconserved regions had similar specific activities to a wild-type value, suggesting that the conserved regions are important to the activity. Aseries of amino-terminal deletion mutants were also constructed. A mutant protein with a deletion of only two amino acid residues from the amino terminus had a significant reduction in the activity, suggesting an essential role for the amino-terminal peptide. Ten mutant proteins with single amino acid replacements were produced by site-directed mutagenesis. Analyses for thermal stability and temperature dependency of these mutant proteins revealed that Ala81, Asp89, Trp94, Arg96, Asp97, and Trpl66 are required for wild-type levels of activities, and that at least Ala81 and Asp89 are not essential to catalytic activities, but act in thermal stability. Glucoamylase catalyzes the release of glucose from the non-reducing ends of starchy materials and other a-glucopyranosides. The enzyme is widely distributed from microorganisms to vertebrates and is important in starch fermentation industries. Recently, we cloned the glucoamylase gene GLU1from a genomic library of Saccharomycopsis fibuligera by screening Saccharomyces cerevisiae transformants capable of starch fermentation1} and determined the nucleotide

Structure of a bacterial glycoside hydrolase family 63 enzyme in complex with its glycosynthase product, and insights into the substrate specificity

“…Therefore, Asp55 was thought to be involved in the hydrolytic transition state rather than ground‐state binding . Similarly, Sf GA D70N lost hydrolytic activity for soluble starch . These results indicate that Asp324 of YgjK plays a similar role in substrate binding and the hydrolytic reaction.…”

Glucoamylases: Microbial Sources, Industrial Applications and Molecular Biology ? A Review

“…Glucoamylase has been identified in Saccharomyces cerevisiae (Pugh et al 1989), Saccharomyces cerevisiae var. diastaticus (Kleinman et al 1988;Pretorius et al 1991), Saccharomycopsis $buligera (Itoh et al 1989), Schwanniomyces castellii (Sills et al 1984), Schwanniomyces occidentalis (Gellissen et al 1991), Pichia burlonii and Talaromyces sp. The expression of glucoamylase activity in S. cerevisiae is confined only to the sporulation phase of the life cycle, and is not regulated directly by composition of the growth medium (Pugh et al 1989).…”

Purification of Glucoamylase from Lactobacillus amylovorus ATCC 33621

“…niveus and R. delemar (Ohnishi 1990 ;Ohnishi et al 1990). Glucoamylase has also been demonstrated in sporulating yeast strains of Saccharomyces cerevisiae (Kleinman et al 1988;Pugh et al 1989), S. diastaticus (Lambrechts et al 1991) and S.Jibuligera (Itoh et al 1989). Many ofthese strains also produce transglucosidase which affects the efficiency of saccharification with the formation of oligosaccharides from glucose.…”