Crosslinked enzyme crystals of glucoamylase as a potent catalyst for biotransformations Original Research Article

Glucoamylase (E.C: 3.2.1.3, α-(1 → 4)-glucan glucohydrolase) mainly hydrolyzes starch and has been extensively used in the starch, glucose (dextrose), and fermentation industries. Immobilized glucoamylase has an inherent disadvantage of lower conversion rates and low thermostability of less than 55 °C when used in continuous operations. We have developed crosslinked enzyme crystals (CLEC) of glucoamylase that overcome the above disadvantages, possess good thermal stability and retain 98.6% of their original activity at 70 °C for 1 h, 77% activity at 80 °C for 1 h, and 51.4% activity at 90 °C for 0.5 h. CLEC glucoamylase has a specific activity of 0.0687 IU/mg and a yield of 50.7% of the original activity of the enzyme under optimum conditions with starch as the substrate. The crystals obtained are rhombohedral in shape having a size ∼10–100 μm, a density of 1.8926 g/cm3 and a surface area of 0.7867 m2/g. The pH optimum of the glucoamylase crystals was sharp at pH 4.5, unlike the soluble enzyme. The kinetic constants Vmax and Km exhibited a 10-fold increase as a consequence of crystallization and crosslinking. The continuous production of glucose from 10% soluble starch and 10% maltodextrin (12.5 DE) by a packed-bed reactor at 60 °C had a productivity of 110.58 g/L/h at a residence time of 7.6 min and 714.1 g/L/h at a residence time of 3.4 min, respectively. The CLEC glucoamylase had a half-life of 10 h with 4% starch substrate at 60 °C.