Citraconylation and maleylation on the catalytic and thermodynamic properties of raw starch saccharifying amylase from Aspergillus carbonarius

Abstract

Amylase capable of raw starch digestion presents a cheap and easier means of reducing sugar generation from various starch sources. Unfortunately, its potential for use in numerous industrial processes is hindered by poor stability. In this work, chemical modification by acylation using citraconic anhydride (CA) and maleic anhydride (MA) was used to stabilize the raw starch saccharifying amylase from A. carbonarius . The effect of the anhydrides on the pH and thermal stability of the free amylase was investigated. Enzyme kinetics and thermodynamic studies of the free and modified amylase were also carried out. Blue shifts in fluorescent spectra were observed after modification with both anhydrides. Citraconylation led to increased affinity of the enzyme for raw potato starch, unlike maleylation. The activation energy (kJ mol −1 ) for enzyme inactivation was increased by 94.8% after modification with CA while only 17.9% increase was noted after modification with MA. Acylation led to an increase in Gibb's free energy and enthalpy while a reduction in entropy was observed. At 80 °C the half-life (h) was 5.92, 11.18 and 14.74 for free, MA and CA enzyme samples, respectively. These findings have potential value in all industries interested in starch conversion to sugars.

Distribution of Aromatic Amino Acid Residues in Substrate-Binding Regions Modulates Substrate Specificity of Microbial Debranching Enzymes

“…1−3 In the sugar industry, starch is also a pivotal raw material to produce sugars by diverse amylolytic enzymes. 4,5 For example, starch can be enzymatically hydrolyzed into glucose and maltose by glucoamylase (EC 3.2.1.3) and β-amylase (EC 3.2.1.2), respectively. 6 Besides, starch can be also applied to produce cyclodextrin and oligosaccharides through the action of cyclodextrin glycosyltransferase (EC 2.4.…”

“…Starch is the second largest renewable resource in nature and is considered one of the most important raw materials for the manufacture of foods, chemicals, and medicines. − In the sugar industry, starch is also a pivotal raw material to produce sugars by diverse amylolytic enzymes. , For example, starch can be enzymatically hydrolyzed into glucose and maltose by glucoamylase (EC 3.2.1.3) and β-amylase (EC 3.2.1.2), respectively . Besides, starch can be also applied to produce cyclodextrin and oligosaccharides through the action of cyclodextrin glycosyltransferase (EC 2.4.1.19) and maltooligosaccharide-forming amylases (EC 3.2.1.-), respectively. − Although various amylolytic enzymes have been used in starch processing, most of them are very expensive or inefficient in starch conversion .…”

Recent Advances in Biocatalysis with Chemical Modification and Expanded Amino Acid Alphabet

“…148 The modified RSDA with PA showed a pH shift from 5.0 to 7.0 for optimum activity and an increase in half-life at 80 °C from 6.1 to 25.7 h. On the other hand, RSDA modified with chitosan showed a shift in optimum temperature from 30 to 60 °C and in half-life at 80 °C from 6.1 to 138.6 h. Likewise, modification of raw starch saccharifying amylase from A. carbonarius using CA and MA enhanced the half-life by 5.3 and 8.8 h, respectively. 140 Chen and co-workers 124 reported a kinetically controlled method for the site-selective modification of a single Lys residue exposed on the surfaces of proteins such as RNase A, lysozyme C, and somatostatin. The activated biotinylating reagents could conjugate with the Lys residue at mild conditions and resulted in the high labeling yield of over 90%.…”

“…Some of the oldest approaches, such as treatment with acid anhydride reagents, are the preferred choice in modifying enzymes for industrial use . The reaction between anhydride and the ε-amino group of Lys residues introduces extra-hydrophilic groups, neutralizing their cationic nature, and may change the overall charge of the protein from positive to negative. − Introducing these hydrophilic groups has been shown to improve protein stability (Figure ), for instance in the modification of α-amylase from Bacillus lichenformis using acetic anhydride (AcA)-mediated acetylation of Lys . This generates highly negatively charged variants that exhibit higher thermostability and resistance to irreversible inactivation in denaturing conditions (1 h at 90 °C in aqueous solutions containing 100 mM sodium dodecyl sulfate).…”

Protein Modifications: From Chemoselective Probes to Novel Biocatalysts

“…The effects on enzyme activity vary among different protein scaffolds, however an improvement in overall thermal stability and resistance to detergents and solvents is well documented. This approach has been employed to enzymes with industrial and biotechnological application, namely amylases [118][119][120][121]; bromelain [122]; α-chimotrypsin [123]; horseradish peroxidase [124]; and lypases [123,125,126].…”