CHANGES IN THE STABILITY AND KINETIC PARAMETERS UP ON GLYCATION OF THERMOSTABLE Α-Amylase FROM BACILLUS SUBTILIS

Abstract

Glycation of the thermostable α‐amylase, KLE, from Bacillus subtilis occurred during incubation with maltodextrin at 95C. This was revealed by the release of 5‐hydroxymethyl‐2‐furfuraldehyde from the acid hydrolysis of glycated KLE (gKLE), the differences in the protein band patterns on SDS and Native‐PAGE, and the shifting of the pI value from the range of 5.6–6.5 to that of 5.2–6.5. After glycation, the activity of gKLE was still retained. Furthermore, gKLE was more resistant to heat and pH compared with the nonglycated enzyme. The Km, reaction rate and efficiency to convert gelatinized cornstarch into maltodextrin of KLE were remained unchanged after glycation. This was different from the result obtained for BAN, another thermostable α‐amylase produced by B. amyloliquefaciens. Glycation in BAN decreased the activity in converting gelatinized cornstarch into maltodextrin. Moreover, the stability and kinetic parameters of BAN were found to be negatively affected by glycation. PRACTICAL APPLICATIONS One of the major applications of starch is for the production of glucose, either in a form of crystalline or syrup which can be further processed into high‐fructose syrup. This is done by starch hydrolysis which is composed of two major enzymatic steps, i.e., liquefaction and saccharification. Liquefaction, converting gelatinized starch to maltodextrin, is carried out by the action of thermostable α‐amylase, while saccharification is by glucoamylase. During the process under the conditions of high concentration of reducing sugar and high temperature, thermostable α‐amylase can be glycated. This non‐enzymatic process occurs when reducing sugar and free amino groups are coexisted in the system. However, the glycated enzyme was found to be more stable than the native form. Hence, industrial thermostable α‐amylase with a suitable degree of glycation, can be more efficient in hydrolytic process.

Neoglycoenzymes

“…177−183 A wet modification of this method has been also reported for the glycosylation of enzymes with maltodextrin at 95 °C in buffered solutions of pH 6.5. 184 Maillard reaction is a complex process involving many transformations and pathways, leading to advanced glycation end products with undefined structure (Maillard browning reaction), which makes it difficult to predict the structure of the artificial glycoenzymes prepared in this way. It can be assumed that this transformation started with the formation of a Schiff base between the reducing end sugar in the polysaccharide and the primary amino groups of the enzyme with further rearrangement to the corresponding N-substituted 1-amino-1-deoxy-ketose derivative called the Amadori compound (Figure 10).…”

“…In general, as anticipated, carbohydrate−enzyme conjugates show higher molecular weight than their native counterparts. 147,158,184 This mass increase depends on the molecular weight of the modifying carbohydrate and the degree of modification. 153,184,251 A broad molecular weight distribution is often shown by neoglycoenzymes due to formation of enzyme−carbohydrate conjugates of different composition.…”

“…147,158,184 This mass increase depends on the molecular weight of the modifying carbohydrate and the degree of modification. 153,184,251 A broad molecular weight distribution is often shown by neoglycoenzymes due to formation of enzyme−carbohydrate conjugates of different composition. 137,162,167 Group-specific glycosylation and the use of polysaccharides as modifying agents contribute to this high molecular weight dispersion for the conjugates.…”

Impact of Residual Impurities and Contaminants on Protein Stability

“…Incubation of a highly glycation‐sensitive monoclonal antibody (mAb1), with 5% glucose at 37°C for 5 days, increased the level of glycation product to 80% of the total protein and the majority of glycation was localized to lysine 98 of an unique sequence in the heavy chain complementarity determining region 3 . The glycation products can retain or lose activity with greater or less stability, depending on the protein and site of glycation . Because of the electrostatic and/or steric effects, the glycated protein can have a higher or lower tendency for aggregation …”

“…36 The glycation products can retain or lose activity with greater or less stability, depending on the protein and site of glycation. [149][150][151] Because of the electrostatic and/or steric effects, the glycated protein can have a higher or lower tendency for aggregation. 37,38 Sucrose is a widely used stabilizing and/or bulking agent for proteins.…”