Purification and biochemical characterization of an extracellular β-d-fructofuranosidase from Aspergillus sp.

Abstract

This study focused on the purification and characterization of an extracellular β-d-fructofuranosidase or invertase from JU12. The protein was purified by size exclusion chromatography with 5.41 fold and 10.87% recovery. The apparent molecular mass of the enzyme was estimated to be ~ 35 kDa using SDS-PAGE and confirmed by deconvoluted mass spectrometry. The fungal β-d-fructofuranosidase was suggested to be a monomer by native PAGE and zymography, and was found to be a glycoprotein possessing 68.92% carbohydrate content. The products of enzyme hydrolysis were detected by thin layer chromatography and revealed the monosaccharide units, d-glucose and d-fructose. β-d-fructofuranosidase showed enhanced activity at broad pH 4.0-9.0 and activity at a temperature range from 30 to 70 °C, while the enzyme was stable at pH 8.0 and 40 °C, respectively. The β-d-fructofuranosidase activity was lowered by metal ion inhibitors Ag and Hg whereas elevated by SDS and β-ME. The fungal β-d-fructofuranosidase was capable of hydrolyzing d-sucrose and the kinetics were determined by Lineweaver-Burk plot with of 10.17 mM and of 0.7801 µmol min. Additionally, the extracellular β-d-fructofuranosidase demonstrated tolerance to high ethanol concentrations indicating its applicability in the production of alcoholic fermentation processes.

Purification and biochemical characterization of β-d-fructofuranosidase fromBacillussubtilisLYN12

“…True invertases are required for the production of invert sugar syrups, alcoholic beverages, sucrose fermentation and hydrolysis of sugarcane molasses (Kulshrestha et al, ; Nadeem et al, ). The substrate specificity of β‐ d ‐fructofuranosidase towards d ‐sucrose have been reported in bacterial invertases (De Gines et al, ; Ryan et al, ; Win et al, ; Yoon et al, ) and fungal invertases (Alves et al, ; Lincoln & More, ). Contrastingly, β‐ d ‐fructofuranosidase from B. infantis ATCC 15697 has shown exo‐inulinase activity along with invertase activity (Warchol et al, ).…”

“…Similarly, hydrolysed products of invertase depicted similar spots in case of Aspergillus sp. (Lincoln & More, ) as well as in Leishmania major (BfrA) which was observed by TLC UV‐densitometry (Ferey et al, ).…”

Partial purification of invertase from Momordica charantia (bitter melon) by three phase partitioning (TPP) method

“…The highest invertase activty (1.23 U/mL) was observed at 70% (w/v) ammonium sulfate saturation. Optimum ammonium sulphate saturation for invertase purification is variable based on the source of the enzyme such as Aspergillus oryzae 30% (w/v) (Dhananjay and Mulimani, 2008), tomato (Lycopersicon esculentum) 50% (w/v) (Özer et al, 2010), Baker's yeast (S. cerevisiae) 50% (w/v) , Aspergillus sojae 80% (w/v) (Lincoln and More, 2018), Saccharum officinarum L. 100% (w/v) (Hussain et al, 2009). t-Butanol is the most convenient co-solvent, owing to its size and furcate structure, and also does not cause denaturation (Dennison and Lovrein, 1997;Dhananjay and Mulimani, 2008).…”

“…For instance, invertase from Aspergillus sojae JU12. was purified by size exclusion chromatography with 5.41 fold and 10.87% recovery (Lincoln and More, 2018). Optimization of crude extract to t-butanol ratio for invertase partitioning.…”

“…Thus, we can conclude that M.charantia invertase is an acidic enzyme. The optimum pH values of invertases purified from different sources varies between 3.5 to 8.0 in the literature Lincoln and More, 2018;Tang et al, 1996;Persike et al, 2002 andBelcarz et al, 2002).…”