Purification and biochemical characterization of β-d-fructofuranosidase fromBacillussubtilisLYN12

Abstract

The purification and biochemical characterization of extracellular β‐d‐fructofuranosidase from Bacillus subtilis LYN12 was carried out. The enzyme was purified 6.94 folds over the crude extract by gel filtration chromatography with recovery of 15.58%. The molecular mass of ∼66 kDa estimated by SDS‐PAGE was confirmed by LC‐MS as 64512.31 Da. Bacterial β‐d‐fructofuranosidase was found to be a glycoprotein with 62.64% carbohydrate content, and exhibited enhanced activities at broad pH, temperature and stable at pH 7.0, 40°C, respectively. The enzyme showed high affinity for d‐sucrose. Kinetic parameters Km and Vmax were 41.98 mM and 1.184 µmol/min, respectively. β‐d‐fructofuranosidase activity was inhibited by the divalent metal ions Cu2+ and Hg2+, whereas improved by Mg2+, Fe2+ and few sulphydryl group reagents. β‐d‐fructofuranosidase demonstrated ethanol tolerance up to 15% with 76.4% of activity. B. subtilis LYN12 invertase is suggested as a potential enzyme with suitable characteristics for numerous industrial applications. Practical applications β‐d‐fructofuranosidases are one of the industrially important carbohydrases utilized in many applications such as beverages, baking, confectionaries, nutraceuticals and also medicinal formulations. The production of β‐d‐fructofuranosidase from Bacillus subtilis LYN12 by solid‐state fermentation demonstrates the utilization of agro‐industrial wastes, such as wheat bran and molasses. The bacterial β‐d‐fructofuranosidase was produced extracellularly which aids in down‐streaming processes. Most of the industrial alcohol fermentations generally operate at 10%–14% (v/v) of ethanol at the end of fermentation. Interestingly, β‐d‐fructofuranosidase exhibited a significant tolerance towards ethanol, the tolerance level of the bacterial invertase indicates its potentiality in the alcoholic fermentation processes. On the other hand, B. subtilis LYN12 β‐d‐fructofuranosidase was found to be active at a broad pH and temperature range possessing high affinity for d‐sucrose. The biochemical characterization of the bacterial β‐d‐fructofuranosidase is crucial to understand the enzymic nature and properties.

Cunninghamella echinulata PA3S12MM invertase: Biochemical characterization of a promiscuous enzyme

“…Similarly, the purification after two phases was also reported for the A. niveus (Fernandes et al, 2018), A. ochraceus (Guimarães et al., 2007), and A. phoenicis (Rustiguel et al., 2015) invertases. The apparent molecular mass of 89.2 kDa found in the C. echinulata PA3S12MM invertase was greater than the A. sojae (35 kDa) (Lincoln & More, 2018), A. niveus (37 kDa) (Fernandes et al, 2018), Bacillus subtilis LYN12 (66 kDa) (Lincoln et al., 2018), and A. versicolor (75 kDa) (Dapper et al., 2016) enzymes and quite close to the invertase of A. oryzae FS4, which is 95 kDa (Xu et al., 2014).…”

“…The invertase (β‐D‐fructofuranoside fructohydrolase), also called β‐ fructosidase (EC 3.2.1.26) is an enzyme belonging to the family GH32 of the glycosyl hydrolases that catalyzes the hydrolysis of the glycosidic bond α1 ↔ 2β in sucrose (Oliveira et al., 2020). Invertase consolidated in the food industry as one of the most important carbohydrase for the sector (Lincoln et al., 2018). It presents numerous applications, such as the production of artificial honey (Nascimento et al., 2019), the synthesis of fructooligosaccharides (FOS) (Aung et al., 2019), and the development of enzymatic electrodes for the detection of sucrose in food and beverage (Park et al., 1991), in the production of invert sugar (Ozcan et al., 2019).…”

Efficient Degradation for Raffinose and Stachyose of a β-D-Fructofuranosidase and Its New Function to Improve Gel Properties of Coagulated Fermented-Soymilk

“…Upon analysis, it was determined that the LcFFase1s protein belongs to the class of typical bacterial β-D-fructofuranosidases. These enzymes typically exhibit molecular weights ranging from 50 kDa to 100 kDa, as exemplified by Bifidobacteriaceae lactis DSM10140T (59.4 kDa) [ 28 ], Bacillus subtilis LYN12 (66 kDa) [ 19 , 29 ], and Microbacterium trichothecenolyticum (approximately 66.2 kDa) [ 3 ]. In contrast, certain fungal β-D-fructofuranosidases display higher protein sizes, such as Aspergillus niger (116 kDa) [ 30 ] and A. oryzae S719 (95 kDa) [ 31 ].…”

“…For instance, β-D-fructofuranosidases from B. subtilis , Microbacterium trichothecenolyticum , and Synechocystis spp. exhibit optimal temperatures of 40 °C, 35 °C, and 30 °C, respectively [ 3 , 29 , 32 ]. However, certain β-D-fructofuranosidases secreted by microorganisms, such as those from A. thermomutatus [ 24 ] and A. terreus [ 33 ], have optimal temperatures as high as 60 °C, indicating that they are thermophilic enzymes that require more heat to maintain their enzymatic activity.…”

“…w5 [ 17 ], and β-D-fructofuranosidases from B. breve UCC2003 [ 36 ]. However, studies have also shown that Mn 2+ and Ca 2+ can enhance enzyme activity, as observed in InvDz13 [ 3 ], β-D-fructofuranosidases from A. thermomutatus (122.8%) [ 24 ], and B. subtilis (127.2%) [ 29 ]. Fe 3+ has also been found to effectively enhance the catalytic activity of LcFFase1s, similar to the characteristics of β-D-fructofuranosidase from A. sojae [ 38 ].…”