Regular Article
Extensive N-Glycosylation Reduces the Thermal Stability of a Recombinant Alkalophilic Bacillus α-Amylase Produced in Pichia pastoris

https://doi.org/10.1006/prep.2000.1348Get rights and content

Abstract

Alkalophilic Bacillus α-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 T50 of glycosylated rABA and its endoglycosidase H-deglycosylated form was 76°C while that of ABA purified from Bacillus was 89°C thus demonstrating that the original temperature stability of ABA was not retained by rABA. The relative thermoperformance, i.e., the activity at 80°C relative to that at 37°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.

References (36)

  • T.R. Gemmill et al.

    Overview of N- and O-linked oligosaccharide structures found in various yeast species

    Biochim. Biophys. Acta

    (1999)
  • S.H. Shakin-Eshleman et al.

    The amino acid at the X position of an Asn-X-Ser sequon is an important determinant of N-linked core-glycosylation efficiency

    J. Biol. Chem.

    (1996)
  • S.J. Tomazic et al.

    Why is one Bacillus α-amylase more resistant against irreversible thermal inactivation than another?

    J. Biol. Chem.

    (1988)
  • M. Vihinen et al.

    C-terminal truncations of a thermostable Bacillus stearothermophilus α-amylase

    Protein Eng.

    (1994)
  • B. Conrad et al.

    Hybrid Bacillus amyloliquefaciens × Bacillus licheniformis α-amylases: Construction, properties and sequence determinants

    Eur. J. Biochem.

    (1995)
  • N. Declerck et al.

    Hyperthermostable mutants of Bacillus licheniformis α-amylase: Multiple amino acid replacements and molecular modelling

    Protein Eng.

    (1995)
  • N. Declerck et al.

    Hyperthermostable mutants of Bacillus licheniformis α-amylase: Thermodynamic studies and structural interpretation

    Protein Eng.

    (1997)
  • Cited by (32)

    • Characteristics of a recombinant Lentinula edodes endoglucanase and its potential for application in silage of rape straw

      2019, International Journal of Biological Macromolecules
      Citation Excerpt :

      These results were slight different from the report by Takeda et al. (2013) [16], in which the LeCel12A activity was maximum at 40 °C and pH 4.5. Different expression system used and the presence or absence of native signal peptide [27] and glycosylation [28] may be responsible for these discrepancy. The LeCel12A activity was enhanced by 10.5%, 14.1%, and 31.6% in the presence of 5 mM of KCl, ZnCl2, and MnCl2, respectively, but was reduced by other salts, especially CuCl2 (Fig. 3c).

    • The influence of N-glycosylation on biochemical properties of Amy1, an α-amylase from the yeast Cryptococcus flavus

      2009, Carbohydrate Research
      Citation Excerpt :

      The native enzyme treated at 50 °C for 60 min retained 97.8% of its maximum activity, whereas the activity of the non-glycosylated form under the same conditions was lowered to about 94.5% (Table 3). The difference in the behavior of the enzyme in terms of activity and stability after the addition of tunicamycin during synthesis may be due to the decreased carbohydrate content, as previously reported in the literature.2,19,20 These results suggest that the function of the N-linked carbohydrate may be to stabilize the structure of the α-amylase produced by C. flavus and so protect it from physical and proteolytic attack after secretion.

    View all citing articles on Scopus
    1

    Permanent address: The Walter and Eliza Hall Institute of Medical Research, Post Office, The Royal Melbourne Hospital, Victoria 3050, Australia.

    2

    Permanent address: M & E Biotech A/S, Kogle Allé 6, DK-2970 Hørsholm, Denmark.

    3

    To whom correspondence and reprint requests should be addressed. Fax: +45 33 27 47 08. E-mail: [email protected].

    View full text