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Purification and biochemical characterization of β‐ d ‐fructofuranosidase from Bacillus subtilis LYN12
Author(s) -
Lincoln Lynette,
More Sunil S.,
Reddy Shwetha V.
Publication year - 2018
Publication title -
journal of food biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.507
H-Index - 47
eISSN - 1745-4514
pISSN - 0145-8884
DOI - 10.1111/jfbc.12592
Subject(s) - bacillus subtilis , invertase , chemistry , fermentation , enzyme , solid state fermentation , biochemistry , ethanol , sucrose , food science , substrate (aquarium) , chromatography , bacillales , divalent , enzyme assay , extracellular , bacteria , biology , genetics , ecology , organic chemistry
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 K m and V max were 41.98 mM and 1.184 µmol/min, respectively. β‐ d ‐fructofuranosidase activity was inhibited by the divalent metal ions Cu 2+ and Hg 2+ , whereas improved by Mg 2+ , Fe 2+ 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.