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Purification and Enzymatic Properties of β-galactosidase Produced from Lactobacillus acidophilus isolated from Dairy Waste-Water

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DOI: 10.23977/enzyme.2022.010102 | Downloads: 13 | Views: 502

Author(s)

Oparaji E.H. 1, Okwuenu P. C 1, Onosakponome I. 1, Eze S.O.O 1, Chilaka F.C 1

Affiliation(s)

1 Department of Biochemistry, University of Nigeria, Nsukka

Corresponding Author

Oparaji E.H

ABSTRACT

β-Galactosidase producing Lactobacillus was isolated from the dairy industrial waste water in Rumuekini River State using standard microbiology (MRS), Biochemical and molecular techniques. Crude extract of β-Galactosidase was produced after successful screening of the isolates using mineral broth containing o-NPG through submerged fermentation system with optimized physiologic conditions. Four steps of purification was carried out: Ammonium sulphate, dialysis, ion exchange (DEAE-cellulose) and gel filtration (sephadex G-100). Crude extract was precipitated using 60% saturation of ammonium sulphate at pH 5.0 which gave the optimum precipitation of the protein with specific activity of 260.56 U/mg. Precipitation using ammonium sulphate carried out at pH 6.5 and 8.0 gave specific activity of 245 U/mg and 236 U/mg μmol/min of the precipitated protein respectively. The precipitates were further desalted through dialysis for twelve hours and specific activity of 425.17 U/mg was recorded afterwards. NaCl concentration gradient of 0.2-0.4M was found suitable in eluting the bound proteins from DEAE-cellulose resin with specific activity of 522.11 U/mg recorded from the pooled fractions. Further purification was done using sephadex G-100 and specific activity of 604.20 U/mg was recorded from the active pooled fractions.  The purification table shows a 3.5 purification folds of β-galactosidase was gotten after ion exchange (DEAE-cellulose) and gel filtration (sephadex G-100) with enzyme percentage yield of 2.00%. The specific activity of β-galactosidase increased from 175.78 to 604.20 U/mg. Characterization of β-galactosidase gave optima pH and temperature of the enzyme at 5.0 and 70οC respectively. Kinetic constants: KM and VMAX values obtained at various concentrations of p-NPG where 0.262 mM and VMAX of 270.27μmol/min respectively. Ca2+ and Co2+showed greater effect to β-galactosidase activity in a concentration dependent manner (0.03-0.05 M) when compared to Mn2+ and Fe2+ . The results from this study have shown that β-galactosidase producing Lactobacillus acidophilus has wide range of activity over physiologic conditions as regards to industrial and clinical standard operational procedures. 

KEYWORDS

Lactobacillus acidophilus, β-galactosidase, purification, specific activity

CITE THIS PAPER

Oparaji E.H, Eze C. G, Okwuenu P. C, Onosakponome I,  Eze S.O.O and Chilaka F.C, Purification and Enzymatic Properties of β-galactosidase Produced from Lactobacillus acidophilus isolated from Dairy Waste-Water. Journal of Enzyme Engineering (2022) Vol. 1: 16-38. DOI: http://dx.doi.org/10.23977/enzyme.2022.010102.

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