Characterization of β-galactosidase from lactose utilizing yeast isolated from murcha

Characterization of β-galactosidase from lactose utilizing yeast isolated from murcha

Shrestha KO¹, Adhikari S¹, Tiwari KB^1,2 and Agrawal VP^1,2

¹Universal Science College, Maitidevi, Kathmandu, Nepal

²Research Laboratory for Biotechnology and Biochemistry, Maitidevi, Kathmandu, Nepal

Abstract

Lactose utilizing yeasts have a potential application in the lactose pollution management in brewery and dairy industries as the organisms can grow at environmental conditions well. Eleven strains of lactose positive yeast were isolated from murcha samples collected from local markets in Kathmandu. Because of highest lactose positive activity among the isolated strains, L4 was selected for production, partially purification and characterization of β-galactosidase. Mass culture of the strain was treated with 2% (v/v) Chloroform to disrupt the cells. The enzyme was purified with Acetone precipitation method to 7.7 fold activity with 77.6% yield; specific activity was found to be 0.054 nmol/min/mg of protein. Optimum pH and temperature were determined to be 6.6 and 37°C respectively with the reaction time period 180 min. Michaelis-Menten constant (Km) value of the enzyme was found to be 8.798 mM of O-nitrophenyl-β-D-galactopyranoside.

Introduction

Murcha is a mixed starter inoculum, used in production of local alcoholic beverages in India, Tibet, Nepal, Bhutan etc. (Tsuyoshi et al., 2005). Murcha is a round cake, which is mildly acidic and has a pH around 5.2 containing 13% w/w moisture and 0.7% w/w ash (dry weight basis). The Murcha cakes contain mixed microbial population viz. molds, yeasts and bacteria (Tamang and Sarkar, 1995).

Yeasts are the world's premier industrial microorganisms, which have wide exploitation in the production of foods, beverages and pharmaceuticals. Alcoholic beverages are one of the major products in the world’s market. Yeasts can contaminate different dairy products because they have relatively low water activity (a_w, 0.88) (Frazier and Westhoff, 1995), can easily grow at room temperature and can utilize (assimilate and/or ferment) a variety of carbohydrates (Nahvi and Moeini, 2004), eg. pentoses, hexoses, disaccharides and, rarely polysaccharides (Barnett et al., 1990). The capacities of the organisms, thus, can be exploited to manage the biodegradeable wastes of the food, dairy and beverage industries. Most of the yeasts, except Saccharomyces spp., can grow on cellulosic materials, however; only few genera are able to degrade starch. Among disaccharides, lactose is one of the most refractory carbon substrate to most of the yeasts. Among hundreds of genera, only few are lactose positive, viz. many Cryptococcus spp. and Trichosporon spp.; some Debaryomyces spp., Kluyveromyces spp. and Myxozyma spp.; and occasionally Bullera spp., Candida spp., Rhodotorula spp. and Tremella spp. (Barnett et al., 1990). Lactose is one of the major components in whey in cheese industry that is non-friendly in the environment (Nahvi and Moeini, 2004). As they can grow at 25-30⁰C, yeasts can be exploited to manage lactose pollution (Sarova and Nikolova, 2002).

Yeast has been considered the predominant microbial enzyme source for food applications. Lactose utilizing yeast are good source of β-galactosidase. With rigorous search, Tiwari et al (2008) elucidated the presence of abundant lactose positive yeasts in murcha samples collected from various parts of Nepal. Hence, the study was designed to collect various yeasts capable to utilize lactose efficiently, and production, purification and characterization of β-galactosidase.

Materials and Methods

Isolation: Murcha samples were collected from local markets in Kathmandu, Nepal. The pure culture of yeast strains were isolated by serial dilution methods in Potato Dextrose Agar (PDA) (20% potato extract, 2% dextrose, and 1.5% agar, pH 4.5) incubating for 48 hr at 27⁰C. The purified isolates were stored on PDA slant at 4⁰C. The pure culture of the isolates was Gram stained for microscopic morphology.

Sugar assimilation test: The sugars used were glucose, fructose, sucrose, maltose, mannitol, galactose, lactose, and arabinose using a basal medium (4.5gm yeast extract, 7.5gm peptone and 20gm sugar in 1-lit distilled water) with phenol red (1mg/ml) as indicator. The pH was adjusted to 7.0-8.0. The medium was dispensed into tubes and sterilized by autoclaving. The pure culture of the isolates was incubated at 27⁰C till 72 hr. in the tubes and the result was indicated by change of color from red to yellow (Shrestha and Sharma, 1995). Control tubes were used in each set to monitor contamination.

Yeast Propagation: Sterile basal media containing ammonium sulphate, yeast extract and potassium dihydrogen phosphate was prepared with pH 4.5. Filter sterilized lactose (0.05M) was added to the 100 ml broth and yeast was inoculated and was incubated in waterbath shaker at 30°C for 48 hour.

Enzyme Extraction: The broth was centrifuged at 7000 rpm for 10 min. and the pellet was resuspended in 80ml phosphate buffer of pH 6.8. Chloroform (2%, v/v) was added and incubated overnight at 28°C. Incubated cells were centrifuged at 10000 rpm for 20 min. Supernatant was collected and one volume of acetone was added. The mixture was shaken in vertex shaker and centrifuged at 10000 rpm for 20 min. Pellets were collected and dissolved in 16ml of phosphate buffer with pH 6.8.

Enzyme Activity: β-galactosidase assay was done with O-nitrophenol β-D-galactopiranoside (ONPG) as substrate. Two test tubes were filled with 3ml of 0.01M of ONPG prepared in supplemented phosphate buffer of pH 6.8. Enzyme extract (1ml) was added to the test solution and reaction progress was determined spectrophotometrically against the blank tube at 420nm for every 30 min. for 3 hours. The reaction was quenched by adding 1ml of 0.5M sodium carbonate.

Determination of optimum time duration for reaction: Seven test tubes were filled with 3ml of 0.01M ONPG prepared in supplemented phosphate buffer of pH 6.8. Enzyme extract (1ml) was added in each tube and incubated at 37°C. By adding 1ml of 0.5M sodium carbonate, the reaction was quenched at every 30 min interval for respective tube and reaction progress was measured at 420nm against the blank.

Determination of optimum pH: Seven different test tubes were taken and 3ml of 0.01M ONPG prepared in buffer of pH 5.5, 6.0, 6.2, 6.4, 6.6, 6.8 and 7.0 was added to each respective test tube. Enzyme extract (1ml) was added to each test tube and incubated at 37°C for 180 min. The reaction was quenched by adding 1ml of 0.5M sodium carbonate and reaction progress was measured at 420nm against the blank.

Determination of optimum temperature: Seven test tubes were taken and 3ml of 0.01M ONPG prepared in phosphate buffer of pH 6.6 was added to each test tube. Enzyme extract 1ml was added to each test tube and incubated separately at 0°C, 15°C, 28°C, 37°C, 40°C, 45°C and 50°C respectively for 180 min. The reaction was quenched by adding 1ml of 0.5M sodium carbonate and reaction progress was measured at 420nm against the blank.

Determination of K_M value: ONPG solution (0.01M) was prepared in phosphate buffer of pH 6.6, and 0.6ml, 1.2ml, 1.8ml, 2.4ml, 3.0ml of the substrate was added in a series of five test tubes respectively. The final volume was set to 3ml by adding phosphate buffer of pH 6.6. Enzyme extract 1ml was added to each test tube and incubated at 37°C for 180 min. The reaction was quenched by adding 1ml of 0.5M sodium carbonate and reaction progress was measured at 420nm against the blank.

Results:

L4 strain those posses highest lactose positive activity was selected for production, partially purification and characterization of β-galactosidase. The enzyme was purified with Acetone precipitation method to 7.7 fold activity with 77.6% yield; specific activity was found to be 0.054 nmol/min/mg of protein (Table 1). Optimum pH and temperature were determined to be 6.6 (Fig. 1) and 37°C (Fig. 2) respectively with the reaction time period 180 min (Fig. 3). Michaelis-Menten constant (Km) value of the enzyme was found to be 8.798 mM of O-nitrophenyl-β-D-galactopyranoside (Fig. 4).

Discussion:

Yeasts are one of the important organisms having a wide industrial application. Most of the genera are unable to assimilate lactose as carbon source (Barnett et al., 1990). A higher abundance of the yeasts capable to produce β-galactosidase and thus assimilate lactose explored the importance of Nepalese yeast cakes (Murcha). Nahvi and Moeini (2004) reported three (3/30, 10.0%) β-galactosidase positive strains in Iran. In this study out of 148 isolates, only 11 were lactose positive.

The β-galactosidase from the yeast isolated from marcha on this study was characterized by their pH optimum at neutral or on the weekly acidic side. In general, β-galactosidase from yeast and bacteria have pH optimum near the neutral region, whereas the enzyme from molds act well at more acidic level as reported by Wierzbicki and Kosikowski (1973). The β-galactosidase of yeast was relatively heat labile, but they hydrolyze substrate well at lower temperature. The optimal hydrolysis was attended at 37°C and nearly 60% of hydrolyzing ability was maintained at 14°C. An enzyme workable at low temperature as possible is preferred for treatment of foods like milk. The Km value with ONPG as a substrate is slightly higher than reported in K. lactis by Kim and Lim (1981).

β-galactosidase commercially can be extracted from yeast. Its primary commercial use is to break down lactose in milk to make it suitable for people with lactose intolerance. β-galactosidase is also used in the manufacture of ice cream. Because glucose and galactose are sweeter than lactose, β-galactosidase produces a more pleasant taste. Lactose positive yeast strains can be used for removal of whey pollutants, Single cell protein (SCP) and ethanol production and treatment of lactose in dairy industry (Nahvi and Moeini, 2004).

Whey is the aqueous fraction of milk generated a by-product of cheese manufacturing which is produced in large amounts. The main solute in cheese whey is lactose present at a concentration of about 4.5-5% (Rohm et al., 1992; Zadow, 1992). Because of its high organic content, dumping directly to the environment causes serious contamination problems. As a solution, bioconversion of whey into SCP or ethanol has been performed in several countries (Gonzales, 1996; Irvine and Hill, 1985; Mawson, 1994). SCP could be produced from whey with employing of yeasts from different species including Kluyveromyces spp., Candida spp. and Trichosporon spp. as they are naturally able to metabolize lactose (Castillo, 1990). Although species of yeasts may differ considerably in their physiology, those of industrial importance have enough physiological characteristics in common to permit generalizations, provided that it is kept in mind that there will be exceptions to every statement made (Frazier and Westhoff, 1995). The findings explored that these yeasts may be new strains and lead to a search of yeast strains having high β-galactosidase activity (Nahvi and Moeini, 2004) as more efficient bioactive agents.

References

Barnett, J.A., D. Yarrow and R.W. Payne. 1990. The yeasts: Classification and identification. 2^nd Ed., Cambridge University Press. pp. 50-77.

Castillo, F. 1990. Lactose metabolism by yeast. In Verachtert, H. and R. DeMot (eds.) Yeast: Biotechnology and Biocatalysis. Marcel Dekker, New York; pp.: 297-320.

Frazier, W.C. and D.C. Westhoff. 1995. Food Microbiology. 4^th Ed., Tata McGraw-Hill Publishing Company Ltd. p10.

Gonzales, S.M.I. 1996. The biotechnological utilization of cheese whey. A review. Biores. Technol. 57: 1-11.

Irvine, D.M. and R.M. Hill. 1985. Cheese technology. In: Moo-young M (Ed.) Comprehensive Biotechnology. Pergman, Oxford; pp: 523-526.

Kim S.H. and Lim K.P. 1981. Differences in the hydrolysis of lactose and other substrates by β-D-Galactosidase from Kluyveromyces lactis.

Lodder, J. 1984.General classification of yeasts. In J Lodder (ed.), The yeasts. 3^rd ed., North-Holland Publishing Co., Amsterdam.

Mawson, A.J. 1994. Bioconversions for whey utilization and waste abatement. Biores. Technol. 47: 195-203.

Nahvi, I. and H. Moeini. 2004. Isolation and identification of yeast strains with high beta-galactosidase activity from diary products. Biotechnol. 3: 35-40.

Rohm, H., F. Eliskases-Lechner and M. Brauer. 1992. Diversity of yeasts in selected dairy products. J. Applied Bacteriol. 72: 370-376.

Sarova, I. and M. Nikolova. 2002. Isolation and taxonomic study of yeast strain from Bulgarian diary products. J. Culture Collection. 3: 59-65.

Shrestha, B. and A.P. Sharma. 1995. Manual on practical pharmaceutical microbiology. 1^st Ed., pp. 83-84.

Tamang, J.P. and P.K. Sarkar. 1995. Microflora of Murcha: an amylolytic fermentation starter. Microbiol. 81: 115-122.

Tiwari KB, Shrestha ML and Agrawal VPA. 2009. Abundance of lactose utilizing yeasts from Nepalese Murcha (Yeast Cakes). Nepalese Journal of Microbiology. In press

Tsuyoshi, N., R. Fudou, S. Yamanaka, M. Kozaki, N. Tamanag, S. Thapa and J. P. Tamang. 2005. Identification of yeast strains isolated from murcha in Sikkim, a microbial starter for amylolytic fermentation. Int. J. food Microbiol. 99: 135-146.

Wierzbicki, L. E., and F. V. Kosikowski. 1973. Food syrups from acid whey treated with/3-galactosidase of Aspergillus niger. J. Dairy Sci. 56:1182.

Zadow, J.G. 1992. Whey and lactose processing. Elservier Applied Science, London and New York.