Extensive N-glycosylation reduces the thermal stability of a recombinant alkalophilic bacillus alpha-amylase produced in Pichia pastoris
| Autor: | Dedreia Tull, Birte Svensson, Birte Kramhøft, Henrik Bisgard-Frantzen, Ib Svendsen, Ole Olsen, Tine E. Gottschalk, Belinda A. Phillipson |
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| Rok vydání: | 2001 |
| Předmět: |
Glycan
Glycosylation Hot Temperature Molecular Sequence Data Bacillus Polymerase Chain Reaction Endoglycosidase Pichia law.invention Pichia pastoris Endoglycosidase H N-linked glycosylation law Enzyme Stability Hexose Trypsin Amino Acid Sequence Cloning Molecular Thermostability chemistry.chemical_classification biology Sequence Homology Amino Acid fungi Glycopeptides biology.organism_classification Molecular biology Peptide Fragments Recombinant Proteins Molecular Weight Kinetics chemistry Biochemistry Spectrometry Mass Matrix-Assisted Laser Desorption-Ionization biology.protein Recombinant DNA alpha-Amylases Sequence Alignment Biotechnology |
| Zdroj: | Protein expression and purification. 21(1) |
| ISSN: | 1046-5928 |
| Popis: | 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 thus demonstrating that the original temperature stability of ABA was not retained by rABA. The relative thermoperformance, i.e., the activity at 80 degrees C relative to that at 37 degrees C 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. |
| Databáze: | OpenAIRE |
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