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

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.