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Effect of introducing a disulphide bond between the A and C domains on the activity and stability of Saccharomycopsis fibuligera R64 α-amylase

Paper ID Volume ID Publish Year Pages File Format Full-Text
22930 43401 2015 7 PDF Available
Title
Effect of introducing a disulphide bond between the A and C domains on the activity and stability of Saccharomycopsis fibuligera R64 α-amylase
Abstract

•α-Amylase mutant with additional disulphide bridge has higher melting temperature.•α-Amylase mutant with additional disulphide bridge less resistant toward EDTA.•Additional disulphide bridge alters the active site environment.•Introduction of a new disulphide bridge has no effect on catalytic efficiency.

Native enzyme and a mutant containing an extra disulphide bridge of recombinant Saccharomycopsis fibuligera R64 α-amylase, designated as Sfamy01 and Sfamy02, respectively, have successfully been overexpressed in the yeast Pichia pastoris KM71H. The purified α-amylase variants demonstrated starch hydrolysis resulting in a mixture of maltose, maltotriose, and glucose, similar to the wild type enzyme. Introduction of the disulphide bridge shifted the melting temperature (TM) from 54.5 to 56 °C and nearly tripled the enzyme half-life time at 65 °C. The two variants have similar kcat/KM values. Similarly, inhibition by acarbose was only slightly affected, with the IC50 of Sfamy02 for acarbose being 40 ± 3.4 μM, while that of Sfamy01 was 31 ± 3.9 μM. On the other hand, the IC50 of Sfamy02 for EDTA was 0.45 mM, nearly two times lower than that of Sfamy01 at 0.77 mM. These results show that the introduction of a disulphide bridge had little effect on the enzyme activity, but made the enzyme more susceptible to calcium ion extraction. Altogether, the new disulphide bridge improved the enzyme stability without affecting its activity, although minor changes in the active site environment cannot be excluded.

Keywords
Aotamy, Aspergillus oryzae Taka-amylase; Anamy, Aspergillus niger α-amylase; DNS, 3,5-Dinitrosalicylic acid; EDTA, ethylene diamine tetra-acetate; MES, 2-(N-morpholino) ethanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; PEG, polyethylene glyco
First Page Preview
Effect of introducing a disulphide bond between the A and C domains on the activity and stability of Saccharomycopsis fibuligera R64 α-amylase
Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 195, 10 February 2015, Pages 8–14
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering