Expression of an α-galactosidase from Saccharomyces cerevisiae in Aspergillus awamori and Aspergillus oryzae

Genome of an early-diverged yeast Blastobotrys (Arxula) adeninivorans (Ba) encodes 88 glycoside hydrolases (GHs) including two α-glucosidases of GH13 family. One of those, the rna_ARAD1D20130g-encoded protein (BaAG2; 581 aa) was overexpressed in Escherichia coli, purified and characterized. We showed that maltose, other maltose-like substrates (maltulose, turanose, maltotriose, melezitose, malto-oligosaccharides of DP 4‒7) and sucrose were hydrolyzed by BaAG2, whereas isomaltose and isomaltose-like substrates (palatinose, α-methylglucoside) were not, confirming that BaAG2 is a maltase. BaAG2 was competitively inhibited by a diabetes drug acarbose (Ki = 0.8 µM) and Tris (Ki = 70.5 µM). BaAG2 was competitively inhibited also by isomaltose-like sugars and a hydrolysis product—glucose. At high maltose concentrations, BaAG2 exhibited transglycosylating ability producing potentially prebiotic di- and trisaccharides. Atypically for yeast maltases, a low but clearly recordable exo-hydrolytic activity on amylose, amylopectin and glycogen was detected. Saccharomyces cerevisiae maltase MAL62, studied for comparison, had only minimal ability to hydrolyze these polymers, and its transglycosylating activity was about three times lower compared to BaAG2. Sequence identity of BaAG2 with other maltases was only moderate being the highest (51%) with the maltase MalT of Aspergillus oryzae.

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The effect of Saccharomyces cerevisiae and Aspergillus oryzae on the digestion of the cell wall fraction of a mixed diet in defaunated and refaunated sheep rumen

Reproduction Nutrition Development ◽

10.1051/rnd:19980405 ◽

1998 ◽

Vol 38 (4) ◽

pp. 401-416 ◽

Cited By ~ 24

Author(s):

Jean-Pierre Jouany ◽

Frédérique Mathieu ◽

Jean Senaud ◽

Jacques Bohatier ◽

Gérard Bertin ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Wall ◽

Aspergillus Oryzae ◽

Mixed Diet ◽

Cell Wall Fraction ◽

Sheep Rumen

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Substitution of asparagine residues in Aspergillus awamori glucoamylase by site-directed mutagenesis to eliminate N-glycosylation and inactivation by deamidation

Biochemical Journal ◽

10.1042/bj3010275 ◽

1994 ◽

Vol 301 (1) ◽

pp. 275-281 ◽

Cited By ~ 35

Author(s):

H M Chen ◽

C Ford ◽

P J Reilly

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Membrane ◽

Specific Activity ◽

Site Directed Mutagenesis ◽

Aspergillus Awamori ◽

Directed Mutagenesis ◽

Wild Type ◽

Wild Type Enzyme ◽

Recognition Sites ◽

Membrane Components

Aspergillus awamori glucoamylase is a secreted glycoprotein containing N-linked carbohydrate recognition sites at Asn-171, Asn-182 and Asn-395. Site-directed mutagenesis was performed at Asn-182 and Asn-395 to determine whether these residues were N-glycosylated by Saccharomyces cerevisiae, to investigate the function of any glycans linked to them, and to determine the effect of their deamidation on glucoamylase thermostability. Asn-171 and Asn-395, but not Asn-182, were N-glycosylated. Deletion of the glycan N-linked to Asn-395 did not affect specific activity, but greatly decreased enzyme secretion and thermostability. The mutant lacking the N-glycan linked to Asn-395 was synthesized very slowly, and was more associated with cell membrane components and susceptible to proteinase degradation than were wild-type or other mutant glucoamylases. Its secreted form was 30-fold less thermostable than wild-type enzyme at pH 4.5. Replacement of Asn-182 by Gln to eliminate deamidation at this site did not change glucoamylase specific activity or thermostability, while replacement by Asp decreased specific activity about 25%, but increased thermostability moderately at pH 4.5 below 70 degrees C. Both mutations of Asn-182 increased glucoamylase production.

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