Purification and characterization of an invertase and a transfructosylase from Aspergillus terreus

Abstract

Two intracellular invertases from Aspergillus terreus were purified and characterized. Invertase 1 has been shown maximal activity at pH 3.0 and optimum temperature at 60°C, while invertase 2 presented maximal activity at pH 4.6 and optimum temperature at 55°C. Km of invertase 1 and 2 for sucrose were 9.8 and 6.2 mM, respectively. Invertase 2 showed high thermostability presenting half‐life of 44, 14.9, and 2 hr at 40, 55, and 60°C, respectively. Gel filtration and SDS‐PAGE analysis revealed that invertase 2 has a dimeric structure composed by two monomers of 32 kDa. Fructose was a competitive inhibitor for invertase 2 presenting Ki of 61.5 mM. Besides hydrolytic activity, invertase 2 also showed transfructosylating activity producing fructooligosaccharides. The enzymes have potential to be applied in food industry since its product, inverted sugar, is used in candies and syrup production, while fructooligosacharides are prebiotics, low calorie and noncariogenic sweeteners. Practical applications Enzymes invertases hydrolyze sucrose in glucose and fructose producing inverted sugar. This product is widely used in food industry to produce jams, syrups, and fondant fillings for chocolates. Besides hydrolysis activity, some invertases can also present transfructosylating activity producing fructooligosaccharides. These oligosaccharides can act as prebiotic, providing beneficial health effects and can be used as low calorie and noncariogenic sweeteners. Aspergillus terreus produced two intracellular enzymes, one exclusively invertase, and the other, invertase and transfructosylase. The enzymes were purified and characterized in this study. This is an important step on screening new enzymes able to produce inverted sugar and fructooligosaccharides from sucrose.

Cunninghamella echinulata PA3S12MM invertase: Biochemical characterization of a promiscuous enzyme

“…The assays of optimum temperature showed a result close to that found in the A. niger (Rubio & Maldonado, 1995), A. ochraceus (Guimarães et al, 2007), and A. terreus (Almeida et al, 2018) enzymes. Regarding the thermal stability, even though the C. echinulata PA3S12MM invertase had presented thermal activation at 50°, 60°, and 70°, it showed a similar behavior to that reported for the A. phoenicis (Rustiguel et al, 2015) enzyme.…”

“…The value calculated for the Michaelis constant (K m ) in the invertase of C. echinulata PA3S12MM was higher than the value found for the invertases of A. terreus (K m = 6.2 mmol/L) (Almeida et al, 2018);…”

“…The value calculated for the Michaelis constant ( K m ) in the invertase of C. echinulata PA3S12MM was higher than the value found for the invertases of A. terreus ( K m = 6.2 mmol/L) (Almeida et al., 2018); A. ochraceus ( K m = 13.4 mmol/L) (Guimarães et al., 2007); A. oryzae (K m = 37.19 mmol/L) (Xu et al., 2014); and A. sojae ( K m = 10.17 mmol/L) (Lincoln & More, 2018). However, the V max value was nearly 2.5 times higher than the invertase of Candida utilis ( V máx = 355 mmol/L min ‐1 ) (Chávez et al., 1997), and the value of the catalytic constant ( K cat ) was nearly 3.6 times higher than that of the Bifidobacterium longum ( K cat = 383.30 s −1 ) enzyme (Ávila‐Fernández et al., 2016).…”

“…The invertase is ubiquitous in plants, considered as a key enzyme in the metabolism of sucrose (Roitsch & González, 2004), and this enzyme is widely distributed among different organisms (Manoochehri et al., 2020). The invertases are also produced by bacteria (Ávila‐Fernández et al., 2016; Pek et al., 2017), yeasts (Bhalla et al., 2017; Jiang et al., 2016), and filamentous fungi such as of the genera Aspergillus, Cladosporium, Fusarium, Trichoderma, and Paecilomyces (Almeida et al., 2018; Fernandes et al, 2018; Manoochehri et al., 2020; Nascimento et al., 2019; Oliveira et al., 2020). Despite the great knowledge about invertase produced by different microbial species, so far, there are no reports of the production of this enzyme by Cunninghamella echinulata .…”

Sucrose Hydrolysis in a Continuous Packed-Bed Reactor with Auto-Immobilised Aspergillus Niger Biocatalyst Obtained by Solid-State Fermentation

“…In the case of invertase produced by Aspergillus strains, the temperature at which they present their highest activity is lower than that reported in this work; for example, the temperature for the highest invertase activity for A. terreus is 60°C [24], similar to that reported for the invertase produced by A. niger IBK1 [26]. A temperature above 60ºC is preferable for industrial invert sugar production to reduce the viscosity of the solutions and decrease the risk of contamination [5].…”