Abstract
A novel intracellular β-galactosidases produced by Lactobacillus plantarum HF571129, isolated from an Indian traditional fermented milk product curd was purified and characterized. The β-galactosidases is a hetrodimer with a molecular weight of 60 kDa (larger subunit) and 42 kDa (smaller subunit), as estimated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme was purified 7.23 fold by ultrasonication, ultrafiltration and gel filtration chromatography with an overall recovery of 30.41 %. The optimum temperature for hydrolysis of its preferred substrates, o-nitrophenyl- β-D-galactopyranoside (ONPG) and lactose, are 50 °C (both), and optimum pH for these reactions is 6.5 and 7.5, respectively. The β-galactosidases showed higher affinity for ONPG (Km, 6.644 mM) as compared to lactose (Km, 23.28 mM). Galactose, the end product of lactose hydrolysis was found to be inhibited (47 %). The enzyme activity was drastically altered by the metal ion chelators EDTA, representing that this enzyme is a metalloenzyme. The enzyme was activated to a larger extent by Mg2+ (73 % at 1 mM), while inhibited at higher concentrations of Na+ (54 % at 100 mM), K+ (16 % at 100 mM) and urea (16 % at 100 mM). The thermal stability study indicated an inactivation energy of Ed = 171.37 kJ mol−1. Thermodynamic parameters such as ∆H, ∆S and ∆G, were determined as a function of temperature. About 88 % of lactose was hydrolyzed at room temperature within 1 h. The study suggested that this enzyme showed its obvious superiority in the industrial lactose conversion process.
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Abbreviations
- ∆H:
-
Enthalpy
- ∆G:
-
Gibbs free energy
- ∆S:
-
Entropy
- R:
-
Universal gas constant
- Kd :
-
Deactivation rate constants
- Vmax:
-
Maximum reaction velocity
- Km:
-
Michaelis–Menten constants
- Kcat:
-
Turn over number
- Kcat/Km:
-
Second order rate constant
- T:
-
Absolute temperature
- κ:
-
Boltzmann’s constant
- \( t\raisebox{1ex}{$1$}\!\left/ \!\raisebox{-1ex}{$2$}\right. \) :
-
Half-life time
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Acknowledgments
The authors would like to thank VIT University, Vellore, India for supporting and performing the study. ES is obliged to the management of VIT for partial completion of doctoral works.
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The authors declare that they have no conflict of interest.
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Selvarajan, E., Mohanasrinivasan, V. Kinetic studies on exploring lactose hydrolysis potential of β galactosidase extracted from Lactobacillus plantarum HF571129. J Food Sci Technol 52, 6206–6217 (2015). https://doi.org/10.1007/s13197-015-1729-z
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DOI: https://doi.org/10.1007/s13197-015-1729-z