Purification, biochemical characterization, and biotechnological applications of a multifunctional enzyme from the Thermoascus aurantiacus PI3S3 strain.

The filamentous Thermoascus aurantiacus fungus characterized by its thermophilic nature, is recognized as an exceptional producer of various enzymes with biotechnological applications. This study aimed to explore biotechnological applications using polygalacturonase (PG) derived from the Thermoascus aurantiacus PI3S3 strain. PG production was achieved through submerged fermentation and subsequent purification via ion-exchange chromatography and gel filtration methods. The crude extract exhibited a diverse spectrum of enzymatic activities including amylase, cellulase, invertase, pectinase, and xylanase. Notably, it demonstrated the ability to hydrolyze sugarcane bagasse biomass, corn residue, and animal feed. The purified PG had a molecular mass of 36 kDa, with optimal activity observed at pH 4.5 and 70 °C. The activation energy (Ea) was calculated as 0.513 kJ mol-1, highlighting activation in the presence of Ca2+. Additionally, it displayed apparent Km, Vmax, and Kcat values of at 0.19 mg mL-1, 273.10 U mL-1, and 168.52 s-1, respectively, for hydrolyzing polygalacturonic acid. This multifunctional PG exhibited activities such as denim biopolishing, apple juice clarification, and demonstrated both endo- and exo-polygalacturonase activities. Furthermore, it displayed versatility by hydrolyzing polygalacturonic acid, carboxymethylcellulose, and xylan. The T. aurantiacus PI3S3 multifunctional polygalacturonase showed heightened activity under acidic pH, elevated temperatures, and in the presence of calcium. Its multifunctional nature distinguished it from other PGs, significantly expanding its potential for diverse biotechnological applications.

Production, immobilization and application of invertase from new wild strain Cunninghamella echinulata PA3S12MM.

AIMS: The objective of this study was to determine the best conditions to produce invertase by Cunninghamella echinulata PA3S12MM and to immobilize and apply the enzyme. METHODS AND RESULTS: The maximum production was verified in 8 days of cultivation at 28°C supplemented with 10 g L-1 apple peel, reaching 1054.85 U ml-1 . The invertase was purified from the DEAE-Sephadex column. The derivative immobilized in alginate-gelatin-calcium phosphate showed reusability >50% for 19 cycles. The derivative immobilized in glutaraldehyde-chitosan showed greater thermostability and at a different pH. The hydrolysis of 15 ml of sucrose 500 g L-1 in a fixed bed reactor (total volume of 31 ml) produced 24.44 µmol min-1 of glucose and fructose at a residence time of 30 min and a conversion factor of 0.5. CONCLUSIONS: The new wild strain C. echinulata PA3S12MM presents high invertase production in medium supplemented with an agro-industrial residue and the immobilized enzyme showed high thermal stability and resistance at a different pH. SIGNIFICANCE AND IMPACT OF THE STUDY: The fungus C. echinulata PA3S12MM is an excellent producer of invertases in Vogel medium supplemented with apple peel. The enzyme is promising for industrial application since it has good performance in reusability and inverted sugar production.