Chemical Modification of Saccharomycopsis fibuligera R64 α-Amylase to Improve its Stability Against Thermal, Chelator, and Proteolytic Inactivation

Abstract

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.

Raw starch degrading α-amylases: an unsolved riddle

“…Multipoint covalent binding on polyglutaraldehydeactivated chitosan beads of the same RSDA exhibited improved thermal and storage stability [86]. On the other hand, a raw starch degrading α-amylase Sfamy from yeast Saccharomycopsis fibuligera R64 was chemically modified by various modifiers, which resulted in improvement of its properties and enabled more thorough information for enzyme structural study [87].…”

Chromatography as the Major Tool in the Identification and the Structure-Function Relationship Study of Amylolytic Enzymes from Saccharomycopsis Fibuligera R64

“…The six deviating residues in AMY comprise an additional predicted glycosylation site (Asn153), which according to a structural modelling, is highly plausible because it resides in a long surface loop (33).…”

“…Benefiting from the availability of amino acid sequences of AMY and GLL1, structural study in silico was performed (33). The structural model for AMY was prepared using the online model building program from the EMBL-EBI (56).…”

“…Further, AMY was pre-treated under various conditions that resulted in denatured and partially denatured enzymes prior to proteolysis. Similar experiments were also carried out using a chemically modified AMY (33). The results were employed to assess the domain organization and assignment of AMY as well as to predict the precise location of trypsin cleavage and the nature of the catalytic domain.…”

Combination of Genetic Manipulation Improved <i>Saccharomycopsis fibuligera</i> α-Amylase Secretion by <i>Pichia pastoris</i>

“…Saccharomycopsis fibuligera R64 α-amylase (Sfamy) is an extracellular enzyme that has the potential to be applied in the industry because it is known to degrade raw starch although it cannot absorb row starch [1][2]. Sfamy R64 (GenBank accession no.…”