Reduction of Lactose in Milk by Purified Lactase Produced by Kluyveromyces lactis

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1 30 Journal of Food Protection, Vol, 52, No, I, Pages (January 1988) Copyright International Association of Milk, Food and Environmental Sanitarians Reduction of Lactose in Milk by Purified Lactase Produced by Kluyveromyces lactis L. HUSSEIN 1 *, S. ELSAYED 1 AND S. FODA 2 Nutrition and Microbial Chemistry Laboratories, National Research Center, Glza, Dokki, Egypt (Received for publication May 28, 1987) ABSTRACT Lactase preparations were purified from cell-free extracts of Kluyveromyces lactis to homogeneity, as determined by disc SDS polyacrylamide gel electrophoresis. A combination of techniques including ammonium sulfate or acetone precipitation and hydroxy apatite chromatography was used for the purification of the enzyme. The enzyme has a ph optimum of with Km s' of 1.25 and 28mM for the substrates O-nitrophenylgalactopyranoside and lactose respectively. Activation energies for denaturation of enzyme and conversion of substrate to product were determined to be 15.1 and 10.0 Kcalmol, respectively. When reconstituted skim milk containing 20% total solids was treated with the lactase preparation (60,000 lactase units1 of milk) at I8 C, 82% of the milk lactose was hydrolyzed within 24 h. Such treatment lends itself for application in developing countries and where the incidence of lactose intolerance is high. Though milk is a highly nutritious food with a potential for providing high quality protein to disadvantaged populations, its consumption by lactose intolerant individuals is normally associated with clinical symptoms as well as interference and losses in the utilization of its nutrients (2,20). In Egypt, recent work demonstrated that 76% of the young adults are lactose intolerant (10). Therefore, the reduction of lactose in milk is desired for nutritional reasons. From a technological point of view, hydrolysis of lactose produces the more soluble monosaccharides, glucose and galactose, and is associated with a considerable increase in sweetness, equivalent to 75% of the sweetening power of sucrose (8,21). Utilization of cheese whey is limited by a very high lactose content (about 70% on a dry weight basis) and, as a result, whey disposal has become a major problem for the dairy industry. Thus, whey imposes heavy loads of waste on the facilities for sewage treatment. Culturing whey with Kluyveromyces fragilis prior to disposal has been suggested, since the yeast is reported to utilize all of the lactose during its growth in whey (7). In an earlier paper (6) we reported the production of lactase from a locally 'Nutrition Laboratory 2 Microbial Chemistry Laboratory selected strain of Kluyveromyces lactis propagated on Domiatti cheese whey with or without 5% NaCl. The present work presents data on the purification of lactase from cell-free extracts of K. lactis, some of its characteristics and its catalytic properties with lactose of milk as substrate. MATERIALS AND METHODS Reagents: O-nitrophenyl-B-D-galactopyranoside was from Fluka: Hydroxyapatite was from Serva, Heidelberg; acrylamide and bisacrylamide were from Canalco, U.S.A. Milk samples: Skim milk powder was reconstituted in water to final concentrations of 6.2 and 20% (wv) equivalent to 2.1 and 6.8% lactose, respectively. Whey permeate was obtained fresh from the Dairy Laboratories, National Research Center. It was kept frozen at -40"C until subsequent use. Purification procedures: Preparation of cell-free extract: Kluyveromyces lactis was grown at 30 C for 65 h on whey permeate containing 0.07% NH 4 OH and 0.25% ammonium phosphate at ph 4.8 in shake flasks (2.5 1) to provide optimum enzyme yield as previously described (6). The cells were harvested by centrifugation in the cold at 6,000 rpm for 20 min. The cells were suspended in phosphate buffer, ph 7.0 and autolyzed in 2% toluene for 48 h. The autolysate was freed from excess toluene by gassing with N 1 and the cell debris were then sedimented by centrifugation in the cold at 10,000 rpm for 30 min. Acetone precipitation: One volume of cold acetone (4"C) was slowly added to one volume ( ml) cell-free extract also at 4"C. The precipitate was collected by centrifugation for 10 min at 10,000 rpm. The acetone step was repeated on the supernatant followed by collection of the precipitate. The precipitates were combined and suspended in 0.05 M potassium phosphate buffer, ph 7.0, containing 0.5 mm MgCl,, 0.1 mm MnCl, and 0.5 mm dithiothreitol (Buffer A). Precipitation with ammonium sulfate: Solid ammonium sulfate was added to a second volume of the cell-free extract to make 50% saturation (26.5%). The sedimented protein was separated by centrifugation. Solid ammonium sulfate was added to the supernatant to increase its final concentration to 31.5%. This addition was followed by centrifugation. The precipitates containing the lactase activity were pooled, resuspended in buffer A and dialyzed overnight against several changes of 0.01 M phosphate buffer A. JOURNAL OF FOOD PROTECTION, VOL. 52, JANUARY 1989

2 REDUCTION OF LACTOSE BY PURIFIED LACTASE 3 1 Chromatography on hydroxyapatite column: The dialyzed enzyme (972 enzyme units) was applied to the top of a 2 X 16 cm column of hydroxyapatite equilibrated with 0.01 M phosphate buffer A. The elution was carried out with a stepwise phosphate buffer gradient of M, ph 7.0. The fractions from the hydroxyapatite column which contained lactase activity were pooled, the protein was precipitated with 31% final ammonium sulfate concentration in solution, the pellet dissolved in a minimum volume of 0.05 M phosphate buffer A and dialyzed in the cold against 0.01 M phosphate buffer A with several changes. Disc polyacrylamide gel electrophoresis: Sodium dodecyl sulfate (SDS) electrophoresis was performed in 5.9% polyacrylamide gels containing 0.2% SDS as described by Fairbanks et al. (5). Enzyme sample containing u.g protein was mixed with equal volume of 10% SDS, a few crystals of sucrose, one drop of bromophenol blue (tracking dye) and was layered on top of the gel under the surface of the running buffer. The SDS electrophoresis was performed for 4 h (6 matube) at room temperature. The protein was detected by staining with Coomassie Blue overnight and destaining with 7% acetic acid, according to the instructions indicated by Fairbanks et al. (5). Protein determination: Protein was determined by the Folin-Ciocalteau method (14) with bovine serum albumin used as the standard. Characterization of beta galactosidase: determinations: the enzymatic activity was assayed using O-nitrophenyl- 6-D-galactopyranoside (ONPG) and lactose according to the methods described by Mahoney and Whitaker (15), scaled down to volumes stated by Foda et al. (6). For determination of K m and V max, the ONPG concentration was varied from 1 to 10 mm and that of lactose from 10 to 207 mm. Values of K and V were determined from plots according to Hanes (9) and Lineweaver and Burk (13). The plots were analyzed for best fit by least squares analyses. Effect of ph on enzyme activity was investigated by utilizing ONPG or lactose as substrate. The substrate was dissolved in phosphate buffer, which was adjusted to the appropriate ph ( ) and 500 u.1 portions were pipetted in a reaction vessel. The enzymatic assay was started by addition of u.1 of the enzyme preparation and it was terminated as described in the standard assay (6,15). Activities were calculated from the change in absorbance at 436 and 420 nm for the determination of ph optimum with respect to lactose and ONPG, respectively. The percentage activity was calculated at each ph relative to the ph which displayed the greatest activity. Effect of temperature on rate of conversion of substrate to product was determined in the temperature range 25-70"C in 0.05 M potassium phosphate buffer A. Proteolytic activity: This was measured on azocasein by a modification of the method of Kunitz (12). The enzyme solution m max (0.5 ml) was added to 0.5 ml of 1% azocasein solution in a 1.6 ml assay mixture using tris-hcl buffer, ph 8.0 and in the presence of M calcium chloride. The assay mixture was incubated at 37 C for 15 min. The reaction was terminated by adding 1.5 ml of 5% trichloracetic acid solution. After mixing and centrifuging, the A 40 _ nm of the supernatant was taken as a measure of proteolytic activity when compared to a control in which TCA was added before the enzyme. Treatment of the milk with the lactase preparation: The reconstituted milk was distributed in equal volumes in conical flasks and flasks were autoclaved at 115 C for 5 min. After cooling to room temperature, the enzyme preparation was added in increasing amounts ranging between ,000 lactase units per liter of milk. The enzymatic hydrolysis was allowed to proceed for 18 or 24 h at 10 C or 18 C. Milk samples were taken and tested for its glucose content by the enzymatic GOD-PERID method (according to instructions given by the manufacturer, Boehringer, Mannheim). The relative activity of lactase was expressed as percent of lactose hydrolyzed in reconstituted skim milk under the experimental conditions. RESULTS AND DISCUSSION Purification of the enzyme: The data in Table 1 represent typical results obtained for the purification of lactase from the cell-free extracts of Kluyveromyces lactis. Acetone purification resulted in recovery of 34% of the initial enzyme activity (not shown in the table); on the other hand, 39% of the original enzyme activity was retained after ammonium sulfate precipitation with an 8-fold purification. An elution profile from the hydroxyapatite column chromatography is illustrated in Figure 1. Fractions containing lactase activity were eluted as sharp peaks between fractions 4 and 15 with 21.6% recovery of the original enzyme activity applied. The overall recovery amounted to 9% compared to initial activity in the cell-free extract. The extinction coefficient of the purified enzyme at 280 nm was 1.23 cm 2 - mg protein '; this figure is slightly lower than the one of 1.58 obtained for purified K. fragilis (16). The purified enzyme preparation obtained after hydroxyapatite chromatography was completely devoid of any proteolytic activity with A 4()5nm = 0. Corresponding absorbancy of 2.0 was obtained after incubating the crude enzyme with azocasein as substrate, reflecting the presence of proteolytic activity in the crude preparation. The protein patterns obtained by SDS-polyacrylamide gel electrophoresis are shown in Figure 2. The protein pattern of the pure TABLE 1. Summary of the purification of lactase from K. lactis Step Procedure Volume (ml) 1. Cell-free extr 2. (NH 4 ),S0 4 precipitation 3. Dialysis 4. Hydroxyapatite b Lactase (units ml) Total (lactase units) 28,500 11,400 11,600 2, "Measured by a Folin-Ciocalteau method (Lowry et al., 12). b A 2-ml portion of the dialyzed enzyme was applied to the hydroxyapatite column. 'The yield which would have been obtained, if all the sample had been applied. Protein" (mgml) (lactase unitsmg) Specific Fold Purification Yield C JOURNAL OF FOOD PROTFCTION. VOL. 52. JANUARY 1989

3 32 HUSSEIN, ELSAYED AND FODA enzyme obtained by hydroxyapatite chromatography was quite distinct and gave no evidence for more than one band. The protein pattern of the crude enzyme showed diffused edges of the stained protein bands and a fast moving band. Electrophoretically pure B-galactosidases has been prepared from Streptococcus lactis (18), from Saccharo- myces fragilis (22), from K. fragilis (16) and from Lactobacillus helveticus (3) by the more classical chromatographic techniques or by the use of affinity chromatography using a thiogalactosidase inhibitor to purify the enzyme. Overall enzyme yields of 5 (3), 22 (22) and 60% (16) have been reported by the different groups of workers. McFeters et al. (18) reported the lability of the galactosidase of S. lactis after gel filtration on Sephadex G-200 column with 9% enzyme recovery. The authors successfully improved the enzyme recovery to 41% by stabilizing the crude enzyme in 0.85 M ammonium sulfate for 30 h prior to gel filtration on a Sephadex G-200 column equilibrated with buffer containing 0.85 M ammonium sulfate FRACTION NUMBER Figure 1. Elution profile of lactase from the hydroxyapatite column (2 x 16 cm). The colum was equilibrated with.01 M buffer (A), ph 7 and 8 C. The enzyme was eluted with a stepwise phosphate buffer gradient ( M). Characteristics of the pure lactase preparation. Effects of substrate concentration and chemical nature on activity. The rates of lactose and ONPG hydrolysis by K. lactis lactase are shown in Figures 3,4. The Michaelis constants calculated from the Hanes plot were 1.5 and 28 mm for ONPG and lactose, respectively. When a Lineweaver-Burk plot was made of the same data, a straight line was generated with intercepts corresponding to a K m of 1.25 and 29 mm for ONPG and lactose, respectively. 350 HANES PLOT 300 Km= 28 mmoll :S K m \X 50^ v max " ^0 60 fto 120 HO S Lactose concentration mmoll LINEWEAVER-BURK PLOT K 5 Km= 29 mmoll \ t) i Figure 2. Sodium dodecyl sulfate disc polyacrylamide gel electrophoresis of crude (a) and purified enzyme from hydroxyapatite (b) column chromatography. Enzyme sample contained 30 \lg protein. The run was carried out at 6 matube for 4 h. Protein stain. Coomassie blue. Migration is from cathode to anode. -O02 t ^ ] mmoll Lactose Figure 3. Effect of ONPG concentration on rate of hydrolysis by K. lactis R-galactosidase. The data are plotted by the methods of Hanes (upper) and Lineweaver-Burk (lower). JOURNAL OF FOOD PROTF.CTION. VOL. 52, JANUARY 1989

4 REDUCTION OF LACTOSE BY PURIFIED LACTASE 33 In this respect, the K m for the Saccharomyces lactis enzyme on ONPG was reported to be 1.18 mm (1). Uwajima et al. (22) found the K m s for Saccharomyces fragiis for lactose and ONPG to be 21 and 4 mm, respectively, at ph 7. The K.fragilis enzyme had K values of 13.9 and 2.72 'V yi HANES PLOT K m =1.5mmolL ONPG 2 U 6 8 S ONPG mmoll «, 0-8 mmol" Figure 4. Effect of lactose concentration on rate of hydrolysis by purified K. lactis lactase. The data are plotted by the methods of Hanes and Lineweaver-Burk. mm on lactose and ONPG, respectively at ph 6.6 (15). De Macias et al. (3) reported a much lower value of mm for the K rn on ONPG of Lactobacillus helveticus ea lactosidase. Effect of ph on activity. Figure 5 illustrates the ph-profile for the activity of the enzyme preparation. Optimum ph values of 6.5 and 7.0 were obtained when lactose and ONPG, respectively were the tested substrates. This coincides with several reports (1,3,4,15,22) in which lactase from different sources was studied. Effect of temperature on Kluyveromyces lactis lactase. The enzyme was shown to be stable up to 34 C. This temperature optimum is within the reported range of temperature optima of C (4,15). Figure 6 shows an Arrhenius plot of the effect of temperature on the activity and stability of the enzyme employing two substrates, namely ONPG and lactose. Activation energy for the conversion of substrate to product was 9.87 and kcalmol for lactose and ONPG, respectively. Respective activation energies for denaturation were and kcal as calculated from the left side slope (based on seven points obtained by measuring the enzyme activities between C. Values of 9.08 and 58 kcal-mol' were reported for the activation energy of catalysis and of denaturation, respectively, for the Kluyveromyces fragilis (15). However, Marin and Marshall (17) reported a higher figure of 25 kcal-mol" 1 for the activation energy for catalysis of a E 200 o c 150 LU 50 en o ONPG Lactose -o P H Figure 5. ph profile of the pure lactase preparation using the synthetic substrate ONPG ( ) or the natural substrate lactose (-0-) Figure 6. Arrhenius plot for ONPG and lactose. _i i_ JOURNAL OF FOOD PROTECTION, VOL. 52, JANUARY 1989

5 34 HUSSEIN, ELSAYED AND FODA cell filtrate from Pseudomonas fluorescens with ONPG as a substrate. Generally, values of E a for transformation of reactants to products in enzyme catalyzed reactions are in the range of 6-15 kcal-mol"y2?). Marin and Marshall (17) attributed the high E a value to the different factors which affect the velocity of a reaction, including presence of activators or inhibitors. Hydrolysis of milk lactose by purified lactase preparations. Treating the reconstituted skim milk (20% total solids containing 6.8 g lactose) with purified lactase containing 60,000 lactase unitsl of milk for 18 and 24 h at 18 C resulted in the hydrolysis of 73 and 82% of the milk lactose, respectively (Table 2). When enzymatic treatment of the milk was carried out at 10 C, the respective hydrolysis rates were 64 and 76%. At lower enzyme concentrations equivalent to lactase unitsl of milk, the percentage hydrolysis of lactose did not exceed 10%. Kosikowski and Wierzbicki (11) used a commercial Saccharomyces lactis lactase (Gist-Brocade, Holland) for the lactose hydrolysis of raw and pasteurized milk. At an enzyme concentration of 25 mgl of milk and after an incubation period of 48 h at 4 C, the percentage hydrolysis of lactose was 75 and 80% in raw and pasteurized milk, respectively. Nakanishi et al. (19), using immobilized enzyme reported high hydrolytic activity of Bacillus circulans 13-galactosidase for the hydrolysis of milk lactose. Also the authors indicated that the formation of oligosaccharides during the process was considerable, reflecting a shift in the equilibrium reaction towards the left. Yeast lactase is rich in its carbohydrate content, therefore it is difficult to immobilize. In Egypt, the cheese industry annually disposes around one million tons of whey, 80% of which contains about 5% sodium chloride. Results obtained from previous (6) and present work demonstrate that the propagation of the yeast Kluyveromyces lactis on sweet and salt whey gave promising yields. The lysis of the cells by toluene autolysis liberated lactase in the cell-free extract, which amounted to lactase units per liter of salt and sweet whey, respectively. Hence, there is a potential for producing lactase TABLE 2. Hydrolysis of milk lactose by purified Kluyveromyces lactis lactase obtained after hydroxy apatite column chromatography. Enzyme preparation added Temperature of hydrolysis 10 C 18"C 10'C 18 C Duration of hydrolysis (h) Lactase unitsliter* US 24 Lactose Hydrolysed (%) Reconstituted skim milk containing 20% total solids = 6.8% lactose. in amounts equivalent to 3 X 10" lactase units annually, enough for the hydrolysis of 10,000 tons of milk lactose. The cheap equipment and small amounts of capital and external energy required for the development of the whey lactose-fermenting yeast and the extraction of lactase would offer some interesting challenges. RKFERENCES 1. Bierman, L. and M. D. Glantz, Isolation and characterization of 6-galactosidase from Saccharomyces lactis. Biochem. Biophys. Acta 167: Calloway, D. and W. Chenoweth, Utilization of nutrients in milk and wheat-based diets by men with adequate and reduced abilities to absorb lactose. I. Energy and nitrogen. Am. J. Clin. Nutr. 26: Dc-Macias, M. E., M. DcNadra, A. Saad, A. Holgado, G. Oliver, Isolation and properties of B-galactosidase of a strain of Lactobacillus helveticus isolated from natural whey starter. J. Appl. Biochem. 5: Edgar, D. G Beta galactosidase activity of free and immobilized cells of Kluyveromyces fragilis. M. Sc. Thesis, Univ. Manitoba, Winnipeg. 5. Fairbanks, G T. L. Steck and D. F. Wallach Electrophoretic analysis of the major polypeptide of the human erythrocyte membrane. Biochemistry 10: Foda, M. S., S. Mohammed and Laila Hussein, Production of lactase from Kluyveromyces lactis propagated in media with different sodium chloride concentrations. Zbl. Mikrobiol. (in press). 7. Gilliland, S Measuring chemical oxygen demand of cottage cheese whey cultured with Kluyveromyces fragilis. J. Dairy Sci. 62: Gregory, K. Dec New dairy processes using lactase. Food Processing Industry Hanes, C. S Reversible formation of starch from glucose-1- phosphate catalyzed by potato phosphorylase. Proc. Roy. Soc. B129: Hussein, L., S. Flatz, W. Kiihnau and G. Flatz Distribution of human adult lactase phenotypes in Egypt. Human Hcred. 32: Kosikowski, F. V. and L. E. Wierzbicki Lactose hydrolysis of raw and pasteurized milks by Saccharomyces lactis lactase. J. Dairy Sci. 56: Kunitz, M Crystalline soybean trypsin inhibitor 2. General properties. J. Gen. Physiol. 30: Lineweaver, H. and D. Burk The determination of enzyme dissociation constant. J. Am. Chem. Soc. 56: Lowry, O. H., N. J. Rosebrough, A. L. Farr and R. J. Randall Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: Mahoney, R. R. and J. R. Whitaker Stability and enzymatic properties of beta galactosidase from Kluyveromyces fragilis. J. Food Biochem. 1: Mahoney, R. R. and J. R. Whitaker Purification and physicochemical properties of beta galactosidase from Kluyveromyces fragilis. J. Food Sc. 43: Marin, A. and R. T. Marshall Characterization of glycosidases produced by Pseudomonas fluorescein. J. Food Prot. 46: McFcters, G. A., W. E. Sandine and P. R. Elliker Purification and properties of Streptococcus lactis B-galactosidase. J. Bacterid. 93: Nakanishi, K., R. Matsuno. K. Torij and K. Yamamoto Properties of immobilized 6-D-galactostdasc from Bacillus circulans. Enzyme and Microbial. Technol. 5: Paige, D. M., T. Bayless, S. Huang and R. Wexler Lactose hydrolyzed milk. Am. J. Clin. Nutr. 28: Rentier. E Ansatzpunkte zum Abbau von Milchiibcrschiissen. Deutsche Molkerei Zeitung 109: Uwajima, T., H. Yagi and O. Terada Purification, crystallization and some properties of betagalactosidase from Saccharomyces fragilis. Agric. & Biol. Chem. 36: Whitaker. J. R Principles of cnzymology for the food science. Marcel Dekker, New York. JOURNAL OF FOOD PROTECTION. VOL. 52, JANUARY 1989