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

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 chai n was found to vary from 8 to 18 as calculated from the masses of glycosyla ted peptide fragments. Temperature stability measurements in the absence of substrate showed that the T-50 of glycosylated rABA and its endoglycosidas e H-deglycosylated form was 76 degreesC while that of ABA purified from Bac illus was 89 degreesC thus demonstrating that the original temperature stab ility 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 inc reased 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 impr ove the thermostability of rABA but modified its thermoperformance to appro ach that of the original Bacillus enzyme. rABA had the highest activity aro und 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.