J. Natn. Sci. Foundat ion Sri L a n k a 2001 29(3&4): 97-106 KINETIC STUDIES OF EXTRACELLULAR a - GALACTOSIDASE FROM CITROBACTER FREUNDII M.A. LOKUGE and C. DEEPAL MATHEW Department of Biochemistry & Molecular Biology, Facul ty ofMedicine, University of Colombo, Colombo (Receiued: 29 December 1999 ; accepted: 24 J a n u a r y 2002 ) Abstract: Extracellular a-galactosidase producing bacteria were isolated from soil. Bacteria that showed a-galactosidase activity were identified as Escherichia coli, Klebsiella pneumonrae and Citrobacter freundii by morphological and biochemical tests. Citrobacter freundii showed the highest enzyme production of 19 milliunitsl ml af ter 36 h r s of cultivation i n pH 8 phosphate buffer containing peptone. a-Galactosidase from Citrobacter freundii was purified by ammonium sulphate- fractionation and DEAE ion exchange chromatography. One a-galactosidase activity peak was observed indicating the presence of a single enzyme form. A 164 fold purification was obtained with a yield of 8%. Polyacrylamide gel electrophoresis of the enzyme showed 2 protein bands. The kinetic properties of the enzyme were studied using p-nitrophenyl a-D-galactopyranoside. The Michaelis constant and maximum reaction velocity obtained were 2.85 x M and 14/mmol/min/mg of protein respectively. Studies on the effect of pH on enzyme activity showed a broad pH optimum from 6.0 to 8.0 with maximum activity a t pH 7.5 a t 2g°C. The enzyme was stable between pH 5.5 to 8.0. The optimum enzyme activity was observed a t 40% a t pH 7.5. The enzyme was stable upto 40 OC. The enzyme preparation did not contain any invertase activity. Key Words : Citrobacter freundii, Extracellular, ct - Galadosidasq Kinetics. INTRODUCTION a-Galactosidase (a-D-galactoside galactohydrolase) hydrolyses a-1,4 galactosidic linkages of galactose containing polysaccharides. It has been reported to occur widely in microorganisms, plants and animals. This enzyme is used in industry and in medicine. In Japan and in USA, the raffinose content in molasses of the beetsugar industry is reduced using a-galactosidase by the addition of Mortierella vinacea mycelial pe1lets.l Partially purified a-galactosidase from Aspergillus satoi2 and Cladosporium cladosporides3 have been shown to be capable of removing flatulence causing oligosaccharides from soy milk. In addition to these, this enzyme has been used in the pulp and paper indust@ and in the manufacture of gelling agentsa5 ' Corresponding author SB M. A. Lokuge and Deepal Mathew Microorganisms are the most promising sources for large scale enzyme production. They can be easily grown and it is usually not difficult to scale up the production process. With microbes, it is possible to increase the production by changes in the growth conditions. In this paper, we report the partial purification of extracellular a-galactosidase from Citrobacter freundii and kinetic studies carried out with the same enzyme preparation. METHODS AND MATERIALS Materials: Analytical grade Serva Fein Biochemicals and Sigma Chemicals were used. DEAE Sephadex A-25 was obtained Fom Pharmacia Fine Chemicals. Absorbance meas~rements were carried out using a Shimadzu W 120-02 spectrophotometer. Bench centrifuge (MSE) and high speed centrifuge (Beckman Model 52-21) were used for centrifugation. Enzyme Assay: To 1 ml of culture supernatant, lml of 0.15M McIlvaine buffer (pH7.5) was added and mixed well. lml of this mixture was incubated with 0.5ml of 1mM p-nitrophenyl a-D-galactosidase (PNGP) solution for 30 minutes a t 29 OC. The reaction was terminated by the addition of 5ml of O.1N Na,CO,. Absorbance was measured at 405 nm.6 A unit of enzyme activity is defined as the amount that hydrolyses lpmol of substrate per minute under specified conditions. Protein Estimation: The protein determinations were done by the method of Lowry et a17 voing crystalline bovine serum albumin as standard. Isolation of bacteria producing extracellular a-galactosidase: Soil was incubated in a culture medium containing raffinose, peptone, yeast extract and salt solution. After incubation the bacteria present in the medium were isolated in pure form. These pure bacterial cultures were grown in the same culture medium and the supernatant was tested for a-galactosidase activity. Three bacterial species that showed high enzyme production were identified as Escherichia coli, Klebsiella pneumoniae and Citrobacter freundii by morphological and biochemical tests.8 Citrobacter freundii gave the highest enzyme production of 14mu/ml after 18h of cultivation when grown in peptone culture medium with an initial pH of 8. Extracellular a-galactosidase production of Citrobacter freundii could be increased upto 19mu /ml when cultivated ifi pH.8 buffer culture medium for ,36h.8 a- Galactosidase from Citrobactor freundii 99 Purification of a-galactosidase Extraction: The a-galactosidase enzyme present in the culture medium was obtained by centrifugatioll of the culture broth at 5000g at 4OC for 20 minutes. The supernatant was retained. Ammonium Sulphate Fractionation: The supernatant was brought upto 75% (NH,),SO, saturation using solid (NH,),SO,. The 75% saturated solution was centrifuged at 25000g for 20 minutes at 4 OC. The precipitate was dissolved in a minimum volume of 0.001M McIlvaine buffer (pH71 and dialysed with the same buffer. DEAE Sephadex A-25 Ion Exchange Chromatography: A column of DEAE A-25 (1.6cm x 40cm) was prepared as described by Andrews? The dialysed (NH4),S04 fraction was applied and eluted with a 2:l McIlvaine buffer containing NaC1. The mixing chambers contain 100ml of 0.001M McIlvaine buffer (pH 7) (starting buffer) and 50ml of 0.1M McIlvaine buffer (pH 7) containing 10% NaCl. Fractions (10ml) were collected in a refrigerated fraction collector at a flow rate of lmllmin. (Figure 1) The fractions containing enzyme activity were pooled, dialysed and retained. Polyacrylamide gel electrophoresis: (PAGE): PAGE was carried out by the method described by Weber & Osborne1° using the Shandon apparatus. Polyacrylarnide gels were prepared and loaded with 40pl of the enzyme preparation. 0.01M Phosphate buffer pH 8.0 was used as the reservoir buffer. Gels were si;ained for prc.teins using Coomassie blue. The enzyme preparation was tested for invertaseactivity using sucro~e.~ Kinetic studies were carried out using PNGP as the substrate. RESULTS Purification Purification of a-galactosidase from Citrobacter freundii is summarized in Table 1. The multistep purification gave an overall yield of 8% and the a-galactosidase was purified 164 fold. (Table 1). A high decrease in activity is observed in DEAE chromatography (Figure 1) indicating that the enzyme could be inactivated in solutions with a low protein concentration. Test for purity: PAGE of the enzyme preparation gave two protein bands and one a-galactosidase activity'band. 100 M. A. Lokuge and Deepal Mathew Table 1: Purification of cx - Galactosidase from Citrobacter freundii spent culture medium. Purification stage Total Total Total Specific Recovery Purification Volume Activity Protein Activity (%) (fold) (ml) (mu) (mg) (mUlmg Protein) 5000g supernatant- SI 2100 11603 2241 5 100 1 Ion Exchange 10 968 1.1 848 8 164 Chromatography Figure 1: Fractionation of the partially purified alpha- galactosidase preparation (0-75% ammonium sulphate fraction) from Citrobacter freundii on DEAE sephadex A-25. The enzyme preparation did not display invertase activity Effect of subtract concentration on enzyme activity: a-Galactosidase was incubated at 29 OC in (0.15M) McIlvaine buffer (pH 7.5) containing PNGP for 30mins. The Krn and Vmax values of a-galactosidase for the substract detemined from the linear part of the Lineweaver-Burk plot were 2.85 x loa3 M and 14 pmoYmg of protein (Figure 2). No significant difference in Kin and Vmax values were observed when Krn and Vmax values were calculated by the Hofstee plot. a- Galactosidase from Citrobactor fi-eundii Figure 2: Lineweaver Burk double reciprocal plot for a-galactosidase from Citrobacter fi-eundii Figure 3: Effect of. pH on the activity of alpha-galactosidase from Citrobacter freundii. Effect of pH on enzyme activity: a-Galactosidase in (0.15M) McIlvaine buffer incubated for 30 minutes at 29 OC showed high activity between pH 5.5 to 8.0 with a maximum activity at pH 7.5 (Figure 3). Stability of the enzyme at differentpH values : The enzyme was incubated in 0.01M McIlvaine buffer at pH 5.0, 5.5, 6.0, 6.5, 7.0, 7.5,8.0, 8.5 at 29 W and assayed for activity at 1 hour intervals using 0.1M McIlvaine buffer (pH7.5 ) at 29 OC. 102 M. A. Lokuge and Deepal Mathew The enzyme is stable over a broad pH range of 6.0 - 8.0. The enzyme is relatively less stable at low pH values than at high pH values (Figure 4). Figure 4: Effect of pH on the stability of alpha- galactosidase from Citrobacter freundii. Figure 5: Effect of temperature on the stability of alpha- galactosidase from Citrobacter freundii. a- Galactosidase from Citrobactor freundii Figure 6: Effect of temperature on the stability of alpha- galactosidase from Citrobacter freundii.(incubated for 2h.) Effect of temperature on enzyme activity: The enzyme when incubated at temperatures from 10 OC to 50 OC in 0.15M McIlvaine buffer (pH 7.5) for 30 minutes has an optimum activity at 40 OC. The activity of the enzyme increases with temperature from 10 OC to 40 OC, thereafter it decreases with further increase in temperature. (Figure 5). Stability of the enzyme at different temperatures: ~ h e h incubated for 2hours at temperatures varying from 10 OC to 50 OC in 0.15M McIlvaine buffer (pH 7.5) and assayed for enzynie activity at 29 OC it was observed that the thermal stability of the enzyme gradually decreases upto 40 OC. It retained about 60% activity at 40 OC (Figure 6). The enzyme lost almost 90% activitywhen incubated at 50°C for 30mins. DISCUSSION a-Galactosidase has been isolated from several bacterial species. KZebsiella sp.No.PG-211,E.coli sub sp.Communior IAM 127212 and Baci l lus s tearotherm~phi lus .~~ Michaelis constant (Km) for .extracellular a-galactosidase of C. freundii was less than the Km value obtained for intracellular a-galactosidase of Klebsiella sp No. PG.211 for the same substrate indicating a higher affinity for the substrate. a-Galactosidase from plant14 and animal tissues15 have shown optimum pH in the acidic range 2.5-6.0. In the present investigation, a-galactosidase from C. freundii appears to have a broad pH range from 5.5 - 8.0 with a maximum activity at pH 7.5. A neutral a-galactosidase activity has been reported in Klebsiella sp. No. PG 211, E. coli, sub sp, communior IAM 127212 & B. stearothermophil~s.~~ ' 104 M. A. Lokuge and Deepal Mathew a-Galactosidase from Aspergillus niger16 is stable at pH 3.5-5.8 a-galactosidase from Dichoderma reesei RUTC-30" is stable at pH 4.5-6.5. Thus a-galactosidase from C. freundii is stable over a broader pH range. The optimum temperature of 40 OC, observed in the present study is higher than the optimum temperature reported from a-galactosidase of E.colil?, Hebsiella sp. No. PG.2.11 The thermal stability of this enzyme is higher than a-galactosidase from E-coli sp. Communior IAM 1272l" KZebsiella sp.ll and is relatively less than a-galactosidase from B.stear~thermophilus.~~ a-Galactosidase from C. freundii is extracellular and inducible in nature. In industry, extracellular enzymes are preferred. Most of the a-galactosidase is optimally active at acidic pH values. In the sugar beet industry, low pH tends to cause inversion of sucrose or precipitation of proteins. Thus, it is important to have an enzyme with the optimum activity at pH 7.5. a-Galactosidase from C. freundii is stable at room temperature and the enzyme solution does not contain invertase activity. A combination of a-galactosidase and invertase activity results in an undesirable hydrolysis of raffinose to galactose. Acknowledgement The authors thank Prof. Jennifer Perera, Dept. of Microbiology, Faculty of Medicine, University of Colombo, for help in identifying the bacterial species. References 1 Suzuli H., OzawaY., Ohta H. &Yoshida H. (1969). Studies on the decomposition of raffinose by a-galactosidase of mold: a-galactosidase formation and hydrolysis of raffinose by the enzyme preparation. Agricultural and Biological Chemistry 33: 501-513. 2 Sugimoto H. & Van B.J.P. (1970). Removal of oligosaccharides from soymilk by an enzyme from Aspergillus satoi. Journal of Food Science 35: 655. 3 Cruz R. & Park Y.K. (1982). Production of fungal a-galactosidase and its application to the hydrolysis of oligosaccharides in soybean milk. Journal of Food Science 47: 1973-5. 4 Talbot G. & Sygusch T. (1990). Purification and characterization of thermosta- ble p-mannase and a-galactosidase from Bacillus stearothermophilus. Applied and Environmental Microbiology 56: 3505-3510. a- Galactosidase from Citrobactor freundii 105 5 Mier H. & Reid J.S.G. (1983). Reserve polysaccharides other than starch in higher plants. Encyclopaedia of Plant Physiology 418-471. 6 Dey P.M. (1969). Inhibition, transgalactosylation and mechanism of action of Sweet Almond a-galactosidase. Biochemica et Biophysica Acta. 191: 644-652. 7 Lowry 0. H, Rosebrough N. J, Farr A.L. & Randall R.J. (1951). Protein measurements with the Folin phenol reagent. Journal of~iological Chemistry 193: 265-275. 8 Lokuge M.A. & Mathew C.D. (2000). Isolation from soil of bacteria producing extracellular a-galactosidase. Journal of the National Science Foundation 28(4): 243-252. 9 hdrews P. (1964). Estimation of the molecular weight ofproteins by sephadex gel-filtration. Biochemical Journal 92: 222-223. 10 Weber K. & Osborn M. (1969). Journal of Biological Chemistry 244: 4406- 4412. 11 Shah V. & Parekh L.I. (1987). Purification and properties of a-galactosidase from Klebsiella sp. No. PG.2. Indian Journal of Biochemistry and Biophysics 27: 103-7. 12 Kawamura S., Kasai T. & Janusi S. (1976). Purification and properties of a-galactosidase from Escherichia colf sub sp. Communior IAM 1272. Agricultural and Biological Chemist? 40: 641-8. 13 Delente J., Johnson J.H. Kuo M.J., O'Conner R.J. &- Weeks L.E. (1974). Production of a new thermostable natural a-galactosidase from a strain of Bacillus stearothermophilz~s. Biochemistry and Bioengineering 16:1227-1243. 14 William J., Villarroya H. & Petek F. (1977). Purification and properties of a-galactosidase galactohydrolase from seeds of fiifolium repens (1977). Biochemical Journal 161: 509-515. 15 Beutler E. & Kuhi W. (1972). Biochemical and electrophoretic studies of a-galactosidase' from normal man, in patients with Fabry's disease and in Equidae. American Journal of Human Genetics 24:.237-249. 16 Bahl 0, N.P. & Agrawal K.M.L. (1969). Glycosidases of Aspergillus niger Journal of Biological Chemistry 244: 2970-8. 106 M. A. Lokuge and Deepal Mathew 17 Zeilinger S., Kristufek D, Arisan A.T., Hodits R. & Kubicek C.P. (1993). Conditions of formation, purification and characterization of an a-galactosidase of Trichoderma reesei RUTC-30.Applied and Environmental Microbiology 59: 1347-1353. JNSF 29_384_97.pdf JNSF 29_384_97 (2).pdf JNSF 29_384_97 (3).pdf JNSF 29_384_97 (4).pdf JNSF 29_384_97 (5).pdf JNSF 29_384_97 (6).pdf JNSF 29_384_97 (7).pdf JNSF 29_384_97 (8).pdf JNSF 29_384_97 (9).pdf JNSF 29_384_97 (10).pdf