Journal article

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

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

Protein Expression and Purification | ACADEMIC PRESS INC | Published : 2001


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 degrees C while that of ABA purified from Bacillus was 89 degrees C..

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University of Melbourne Researchers