Extensive N-glycosylation reduces the thermal stability of a recombinant alkalophilic Bacillus alpha-amylase produced in Pichia pastoris

Tull, D; Gottschalk, TE; Svendsen, I; Kramhoft, B; Phillipson, BA; Bisgard-Frantzen, H; Olsen, O; Svensson, B

Author(s): Tull, D; Gottschalk, TE; Svendsen, I; Kramhoft, B; Phillipson, BA; Bisgard-Frantzen, H; Olsen, O; Svensson, B;
Details: Publication Year 2001-02,Volume 21,Issue #1,Page 13-23
Journal Title: PROTEIN EXPRESSION AND PURIFICATION
Publication Type: Journal Article
Abstract: Alkalophilic Bacillus alpha -amylase (ABA) was produced in the yeast Pichia pastoris with a yield of 50 mg L-1 of culture supernatant. The recombinant protein, rABA, was glycosylated at seven of the nine sites for potential N-glycosylation as identified by automated peptide sequencing and MALDI-TOF MS of tryptic fragments. The number of hexose units within each glycan chain was found to vary from 8 to 18 as calculated from the masses of glycosylated peptide fragments. Temperature stability measurements in the absence of substrate showed that the T-50 of glycosylated rABA and its endoglycosidase H-deglycosylated form was 76 degreesC while that of ABA purified from Bacillus was 89 degreesC thus demonstrating that the original temperature stability of ABA was not retained by rABA. The relative thermoperformance, i.e., the activity at 80 degreesC relative to that at 37 degreesC was 0.9 +/- 0.3 for rABA, Removal of all seven N-linked glycans by endoglycosidase H increased the relative thermoperformance to 2.4 +/- 0.6, compared to the value of 3.5 +/- 1.1 for ABA. Thus, removal of the N-linked glycans did not improve the thermostability of rABA but modified its thermoperformance to approach that of the original Bacillus enzyme. rABA had the highest activity around pH 6. Treatment of rABA with endoglycosidase H shifted the pH activity profile in a more alkaline direction approaching the pH activity profile of ABA. (C) 2001 Academic Press.
Publisher: ACADEMIC PRESS INC
Keywords: LINKED OLIGOSACCHARIDE STRUCTURES; AMINO-ACID; BARLEY ALPHA-AMYLASE-1; METHYLOTROPHIC YEAST; IRREVERSIBLE THERMOINACTIVATION; HYPERTHERMOSTABLE MUTANTS; SUBSTRATE-BINDING; EXPRESSION; PROTEINS; THERMOSTABILITY
Publisher's Version: https://doi.org/10.1006/prep.2000.1348
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Creation Date: 2001-02-01 12:00:00