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

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.

Chemical modification of Saccharomycopsis fibuligera R64 α-amylase to improve its stability against thermal, chelator, and proteolytic inactivation.

α-Amylase catalyzes hydrolysis of starch to oligosaccharides, which are further degraded to simple sugars. The enzyme has been widely used in food and textile industries and recently, in generation of renewable energy. An α-amylase from yeast Saccharomycopsis fibuligera R64 (Sfamy) is active at 50 °C and capable of degrading raw starch, making it attractive for the aforementioned applications. To improve its characteristics as well as to provide information for structural study ab initio, the enzyme was chemically modified by acid anhydrides (nonpolar groups), glyoxylic acid (GA) (polar group), dimethyl adipimidate (DMA) (cross-linking), and polyethylene glycol (PEG) (hydrophilization). Introduction of nonpolar groups increased enzyme stability up to 18 times, while modification by a cross-linking agent resulted in protection of the calcium ion, which is essential for enzyme activity and integrity. The hydrophilization with PEG resulted in protection against tryptic digestion. The chemical modification of Sfamy by various modifiers has thereby resulted in improvement of its characteristics and provided systematic information beneficial for structural study of the enzyme. An in silico structural study of the enzyme improved the interpretation of the results.