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Effects of N224 Glycosylation in Saccharomycopsis fibuligera R64 α-Amylase on Enzyme Activity and Stability

Author(s):

Yovin Sugijo, Tina Dewi Rosahdi, Fernita Puspasari, Wangsa Tirta Ismaya, Khomaini Hasan, Ihsanawati, Toto Subroto and Dessy Natalia*   Pages 1 - 9 ( 9 )

Abstract:


Background: The amino acid sequence of an α-amylase of the yeast Saccharomycopsis fibuligera R64 (SfamyR64) contains two putative N-linked glycosylation sites N153 and N224. N224 is hypothetically responsible for the binding of starch substrate because it is highly conserved among SfamyR64 homologs.

Objective: To test whether N224 plays a key role in enzyme activity and stability.

Methods: N224Q substitution was introduced by site-directed mutagenesis. The wild type and the mutant were independently over-produced in Pichia pastoris KM71. Activities of the wild type and of the mutant were compared, and their thermal-stability was assessed using heat treatments. The evolutionary relationship of SfamyR64 with its structural homologs with different glycosylation patterns was examined.

Results: Activity of the N224Q mutant was approximately 80% lower than that of the wild type. The mutant showed no activity after 10 min of pre-incubation at 50 °C, whereas the wild type SfamyR64 showed activity until 30 min of treatment. Sfamy appeared to have evolved earlier than its structural homolog.

Conclusion: SfamyR64 N224 is crucial for enzyme activity and thermal stability. This glycosylation site is unique for fungal and bacterial α-amylases.

Keywords:

α-amylase, SfamyR64, N-linked glycosylation, Saccharomycopsis fibuligera R64, enzyme activity, Pichia pastoris KM71.

Affiliation:

Biochemistry Division, Faculty of Mathematics and Natural Sciences, Institut Teknologi Bandung, Bandung, Biochemistry Division, Faculty of Mathematics and Natural Sciences, Institut Teknologi Bandung, Bandung, Biochemistry Division, Faculty of Mathematics and Natural Sciences, Institut Teknologi Bandung, Bandung, Dexa Laboratories of Biomolecular Sciences, Cikarang, Faculty of Medicine, Universitas Jenderal Ahmad Yani, Cimahi, Biochemistry Division, Faculty of Mathematics and Natural Sciences, Institut Teknologi Bandung, Bandung, Biochemistry Laboratory, Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Padjajaran, Bandung, Biochemistry Division, Faculty of Mathematics and Natural Sciences, Institut Teknologi Bandung, Bandung



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