Characterization of β-Galactosidase from Lactose Utilizing Yeast Isolated from the Dairy Sample
Bivek Dahal1#,
Sujan Karki1#, Nabaraj Adhikari2, Upendra Thapa Shrestha2,
3
1Universal Science
College, Pokhara University, Baneshwor, Kathmandu
2Central Department of
Microbiology, Tribhuvan University, Kirtipur, Kathmandu
3Research Laboratory for
Biotechnology and Biochemistry (RLABB), Kathmandu
# Both the authors have equal contributions.
Corresponding
author: Upendra Thapa Shrestha, Central Department of Microbiology,
Tribhuvan University, Kirtipur, Kathmandu. Email:
upendra.thapashrestha@cdmi.tu.edu.np
ABSTRACT
Objectives: The objective of the
study was to isolate lactose positive yeasts from dairy samples collected
from local markets of Kathmandu, to extract crude β-galactosidase from the
lactose positive yeast and to characterize the enzyme for optimum time
duration, pH, temperature, Michaelis-Menten constant (Km) and maximum activity
(Vmax).
Methods: Four lactose positive
yeast strains were isolated from dairy samples collected from local market of
Kathmandu by pour plate method. Single strain having maximum lactose positive
activity was selected for the study. The mass culture of the lactose positive
yeast strain was lysed by 2% Chloroform and the yeast cell lysate containing
β-galactosidase (i.e. crude enzyme extract) was characterized by using ONPG
(Ortho-Nitrophenyl-β-D-galactopyranoside) as substrate. ONPG is a colorless
substrate for the enzyme assay which is hydrolyzed by the enzyme into yellow colored
product ONP (Ortho-Nitrophenol). The concentration of product formed was
monitored spectrophotometrically at 420 nm to determine the enzyme activity and
to characterize the enzyme.
Results: The enzyme had wide
range of working temperature from 0-50ºC, with optimal temperature of 37ºC.
However, greater than 50% hydrolyzing ability was maintained in the range of
14-40ºC. Optimum time of reaction was 70 min. The enzyme had maximum activity
in the near neutral pH of 6.8. Michaelis-Menten constant of the enzyme was
found to be 2.23 mM of ONPG and Vmax was 58.82 nmol/min/ml. Enzyme activity was
27.88 nmol/min/ml, Specific enzyme activity was 59.97 nmol/min/mg and total enzyme
activity was 3346.33 nmol/min.
Conclusion: The activity over a
wide range of temperature 0-50ºC with low Km value shows that the enzyme has a
commercial application in clearance of lactose pollution in waste water in
different environmental conditions.
Keywords: Yeast, β-galactosidase, enzyme activity, ONPG, Km
