Cunninghamella echinulata PA3S12MM invertase: Biochemical characterization of a promiscuous enzyme

Abstract

The Cunninghamella echinulata PA3S12MM fungus is a great producer of invertases in a growth medium supplemented by apple peels. The enzyme was purified 4.5 times after two chromatographic processes, and it presented a relative molecular mass of 89.2 kDa. The invertase reached maximum activity at pH of 6 and at 60°C, in addition to presenting stability in alkaline pH and thermal activation at 50°C. The enzymatic activity increased in the presence of Mn²⁺ and dithiothreitol (DTT), while Cu²⁺ and Z²⁺ ions inhibited it. Also, DTT showed to protect enzymatic activity. The apparent values for K_m , V_máx , and K_cat for the sucrose hydrolysis were, respectively, 173.8 mmol/L, 908.7 mmol/L min^-1 , and 1,388.79 s^-1 . The carbohydrate content was of 83.13%. The invertase presented hydrolytic activity over different types of glycosidic bonds, such as α1 ↔ 2β (sucrose), α1 → 4 (polygalacturonic acid), α1 → 4 and α1 → 2 (pectin), and α1 ↔ 1 (trehalose), indicating that the enzyme is multifunctional. Thus, the biochemical properties showed by the C. echinulata PA3S12MM suggest a broad industrial application, such as in the biomass hydrolysis or in the food industry. PRACTICAL APPLICATIONS: Invertases are hydrolytic enzymes employed in several industrial sectors. Given their great importance for the economy and several industrial sectors, there is a growing interest in microorganisms producing this enzyme. The analysis of the biochemical properties of invertase in C. echinulata PA3S12MM suggest applications in the food industry. Due to its increased hydrolytic activity, the hydrolysis process of the sucrose may employ invertase for the production of invert sugar. The stability at alkaline pH suggests an application in the development of enzymatic electrodes for the quantification of sucrose in food and beverage. The multifunctional activity may work in the biomass hydrolysis or saccharification of by-products for the extraction of fermentable sugars. The high level of invertase N-linked glycosylation of invertase grants this enzyme thermal stability at high temperatures, in addition to resistance against the action of proteases, which are desirable characteristics for the application of this enzyme in industrial processes.

Keywords: Cunninghamella echinulata; apple peel; invertase; promiscuous enzyme; sucrose hydrolysis; β-fructofuranosidase.