PropertiesOfLactase (1)

PropertiesOfLactase (1) - 5 \. ‘1'? Properties of Lactate...

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Unformatted text preview: 5 \. ‘1'? Properties of Lactate Produced by Candida pseudo tropics/is FRANCISCO J. CASTILLO Ind BELISARIO MORENO Laboratorio de Farmentaeion Centm dc Microbiolocia y Bialogla Calular Instituto Venezolano du Investigaciones Cientifieat [I.V.I.C.) ABSTRACT Studies were of factors affecting activ‘ ity of the lactase produced by the yeast Candida pseudotrops'cah'r NCYC 744. The enzyme was inactivated by ethylenedi~ aminetetraacetic acid tetrasodium salt or dialysis against distilled water, and activity was recovered partially upon ad- dition of magnesium“ or manganese” salts at optimal concentrations of 10 mM and 1 mM. respectively. Heavy metals and p-chioromercuribenzoate Strongly in- activated the enzyme. indicating sulphy- dtyl groups and their requirement for lactose activity . O-nitro phenyl-B—D- galactopyranoside hydrolysis was in- hibited competitively by lactose (inhibi- tion constant 13 mM). methyl-BI)- gaIactoside (27 mM). galactosr: (32 mM), galacturonic acid (160 mM). ribose (175 mM). and galactitol (180 mM); but neither glucose nor melibiose affected enzyme activity. MoIecular weight of the iactase was estimated at 200,000. The information will be of significance in the design of pmcesses for lactose hydrolysis with this enzyme. INTRODUCTION Interest in lactase (B-D-galactosidase. EC 3.2.1.23) originates from its potential for the treatment of milk and milk products and by- products to reduce lactose content. Production of lactase by the lactose-utiiiaing yeast Candida pseudatmpicnlis NCYC 744 grown in deproteinized whey has been studied. and apparent optimal conditions for growth of cells in batch and continuous culture were established (5. 9). Synthesis of the enzyme is inducible by galactose and lactose and subject to catabolite repression (9. 19). The Michaelis Received December 6. 1982. 1983 J Dairy Sci 66:1616—1621 Adpo. 1827. Caracas 1010A. Venezuela constants of the lactase have been estimated 3.1 to 3.4 mM for o-nitrophenyl-B—D-gaiacro— pyranoside (ONPG) and 32.6 to 33.7 mM for lactose. Maximum activity occurs at pH 6.2 and between 45 and 475°C (19). The objective of this work was to gain more information on properties of the enzyme to optimize conditions for its utilization. Effects of dialysis, salts, and sugars on enzyme activity are reported. MATERIALS AND METHODS Microorganism: Ind Maintenance Medium Candida pseudntropicalis NCYC 744 was ob- tained from the National Collection of Yeast Cultures, Surrey. England. It was maintained at room temperature on slants consisting of (Will vol) 2% lactose (Merck). 3% malt extract. 3% yew extract, .596 peptone. and 2% agar (all from Difco) and subcultured monthly. Culture Medium Sweet. dry whey was purchased from Indus‘ trials Lacteas Torondoy. Caja. Seca. Zulia. Venezuela. It contained (wtlvol): 73% lactOSC. 10.8% protein, and .7596 phosphorus. For yeast propagation. 2% deproteinized WHEY solutions were prepared by acidification. heat treatment. and filtration as in (4). and SDPPlC' mented with (wtlvol) .196 yeast extract and 1% (NHa);SOQ. The pH finally was adjusted to 5 (5)- YM Propulsion , Yeast was propagated in BOO-ml cotton- plugged Erlenmeyer flasks containing 45 m1°f medium and 5 ml of 18-h old inoculum. Flasks were agitated (120 strokcslmin) in a TCCiPm‘ eating shaker bath at 30°C for 18 h. Cells WC": harvested by centrifugatian at 5.000 x g for m min. 1616 “fir—.j _‘_. CANDIDA PSEUDOTROPICALIS LACTASE Enzyme Extraction Yeast cells were washed with 100 mM potas sium phosphate buffer supplemented with .5 mM M350. and .1 mM MnClz (phosphate buf~ fer), pH 7. resuspended in the same buffer, and treated with 2% (vol/vol) chloroform at 37°C for 16 h. Suspensions were centrifuged at 7,000 X g for 5 min at 4°C, and supernates were treated with one volume of acetone at 4°C. The precipitates formed were resuspended in phos- phate buffer, pH 6.2. and used as partially puri- fied enzyme solutions (S). 5’2va Assays Routine assays used 10 mM o~nitrophenyl- fl-D-galacmpyranoside (ONPG, Sigma) in phos phate buffer, pH 6.2. One unit of enzyme activity was defined as the amount of enzyme required to liberate 1 ,umol of o-nitrophcnol (GNP) in 1 min. Effects of salts, sugars. and other compounds on enzyme activity were studied with ONPG at three concentrations (2.5. 5 . and 10 mM) and varying concentrations 0f compounds tested as potential inhibitors. TYPES of inhibition and inhibition constants (Ki) were determined as described by Dixon (7). Mulemlar Weight The molecular weight of lactase was esti- mated by gel filtration on Ultrogel ACA 34 (“(3) (Figure 1), Partially purified enzyme was a~i’l’lied to a column (2.5 X 40 cm) of Ultrogel AcA 34 equilibrated with phosphate buffer, pH 6.2, containing .02% sodium azide, and the enzyme was eluted with the same buffer. Fraci lions of 2.4 ml were collected and assayed for [HMS-C activity as described. Marker proteins used were: hen ovalbumin (45,000), bovine serum albumin (67,000), rabbit musele aldolase (158-000). and bovine liver catalasc (232.000) (3” f'I’Om Pharmacia). Logarithms of molecular weights were plotted against distribution con- su-“ts (14-). R ESULTS The enzyme was inactivated completely by iCldirion of 1 mM ethylenediamineretraacetate (_EDTA). Also. dialysis of 5 ml of enzyme solu- “ml against 2 liters of distilled water at 4°C led to almost total loss of activity within 24 h I617 3 u m' N: ‘ m‘ Nubia!»- wag»: Figure 1. Molecular weight determination of lac- tue by chromatography on Ultrogel ACA 34. erker proteins (0). C. puudorropicalis lactose (O). (a. D-antlmldml Iii:de Arvme I'I-l c I .= i a 5 I»... [Noun] Figure 2. Loss of lactose activity by diafysis against distilled water. Five milliliters of enzyme were diaiyzed against 2 liters of distilled Watch l_ —r 1 6 IV ,I .e " -:" _ 3‘ ‘ i. .5 a i“’ , . i = ~ __ so wav-lP-wlfivh'n' Figure 3. Activation of locus: activity by Mn“ and MgH salts The enzyme was dialyzed against dis- tilled water for 24 h and the salts added at the concen- trations shown. The relative enzyme activity shown corresponds to the ratio: activity in the presence! absence of added salt. MnCl; (0). MnSO‘ (o), M‘gCl1 (A). M350. w— }ournal of Dairy Science Vol. 66. No. 8. 1983 1618 CASTILLO AND MORENO Residual Enzyme Aclivil'v [Vol 10" to" to" m“ lo" 10' Salt Concentration [Moioriiyl Figure 4. inactivation of iactase activity by different cations. H30' (0). AgNO, (0). CuSO. (A). ZnCl, (9. Cacl, (a). can, (-1. (Figure 2), whereas undialyzed controls last no activity. Thes: results suggested involvement of divalent cation(s) in the enzyme activity. To study this possibility, salts of Ca“, Co”, Fe“, Mn++, and Mg‘H were tested by adding them at concentrations ranging from 10‘s and 3 x 10" M to dialyzed enzyme solutions and measuring their ability to reactivate the en- zyme. Activity was recovered partially (Figure 3) with additions of Mg” and MnH salts (chlorides and sulfates). and optimal concentra— tions were 10 mM and 1 mM. respectively. Salts of Ca”. Co“. and FeM did not activate the enzyme. Over 90% inactivation was caused by Hg” (chl2) and Ag“ (AgNOg) at concentrations above 10'a M; Cu++ (0.180.) at MIT4 M or higher; and Cd“, Zn”, and CaH (as chlo- rides) at 10'1 M concentrations (Figure 4; Table 1). The enzyme was unaffected by FeM at IO‘3 M (data not shown) but was inhibited by p-chloromercuribenzoate (PCMB) (15“ ca. 5 x 10'7M)(Figure 5). Glucose. methyl-fl-D-glucoside, fucose, or Journal of De‘ny Science VOL 66. No. 8. 1983 melibiose at 100 mM concentrations did nor inhibit the enzyme nor did isopropylfl-l?‘ thiogalactoside at 10 mM concentration. AS In TABLE 1. The [:03 of different compounds on hydrolysis by laczase from Candida pseudorroprcabs chc 744)] M Compound added I” ___—________._ (M) HgCl, 2.5 x 10-' AgNO, 3 x to" CoSO‘ 10" CdClz s ur’ not, a x 10" cm, 3 x 10” ——-—-———-—I—-——-——"-.__ I1:,“:Concentration of compound that inhibited El" enzyme nctivity by 50%. bObrained from data in Figure 3. Each comP‘fund added to mixtures of enzyme and 10 mM Gnl‘w' phenyl—fl-D—galnctopy'ranos'dc. _s _._.,:_.. _. __ firx q ‘11-? ‘ CANDID/i PSEUDDTROPICALIS LACTASE IO U1 ENZYME ACTIVITY (nMol. GNP/min) | O I 2 3 PC M B CONCENTRATION (pMi Figure 5. Inhibition of laetnse by p-ehloromercur'r benzoate (PCMB). Table 2, several sugars inhibited ONPG hydroly- sis by lactase (Figure 6). Galactosamine had the highest inhibitory activity followed by lactose. methyl-fi-D-galactoside. galactose. galacturonic acid. ribose, and galactitol. All neutral sugars inhibited competitively. However, it could not be determined whether the effect of galactos- amine was competitive or noncompetitive, because we were unable to obtain reproducible results from effects of different concentrations of the amino sugar. DISCUSSION That divalent cations restituted enzyme activity after dialysis indicates lactase require- ments for cationic cofactor(s). Of several cations tested, only Mn” and to less exrent Mg“ activated the dialyzed enzyme, Similar stimulatory effect was reported for lactases of Yeast: Kluyueromyces fragiiis (21. 23). K. “"155 (2, 6), Saecberamyees anamensis (1), Tomlapn's versatiiis, Ti rpbaerica, and Candida Puudotropicnlis (12) and for the lactase of 1619 Streptococcus tbermapbilus (20). Although Mn++ and Mg’H stimulated the activity, they also inhibited when added at concentrations above optimal (I and 10 mM, respectiVely); similar observations have been reported for Mg‘H on K. lactis lactase and for Co” and K” on K. fragilis lacrases (16, 21, 24). Wendorff (24) showed that additions of 10'“ M MnCl1 increased the enzymatic lactose hydrolysis in milk and whey. According to the literature (Table 3), average concentrations of Mn++ in milk and whey (acid and sweet) are at least two orders of magnitude below the optimal conccna {ration (1 mM) required for recovery of enzyme activity (Figure 3). However, concentrations of MgM are near optimal 10 mM, even though only 73% of the Mg++ in milk is soluble (22). The enzyme inactivation caused by the alky- lating sulfhydryl-group reagent (PCMB) and the heavy metals indicates sulfhydryl groups in the enzyme and their importance for expression of hydrolytic activity, as has been pointed out for other yeast lactases (9, 12.17.21, 23). Milk solids other than lactose inhibited hydrolysis of lactose by S, fiagilis lactase in milk and whey (24). As in Table 3, concentra- tions of Cd++ and 2n++ in milk and whey are between two and three orders of magnitude below their 150 (Table 1). However. concentra- tions of Hg++ and Cu” are near their esti- mated 159. and those of Ag" and Ca“ TABLE 2. Inhibitory effect of sugars on O—nitrn- phenyl-fi-D—galaetopyranoside hydrolysis by lutase from Candida pseudarropirnl'is NCYC 744.3 Sugar added Klb __#_____——— (mM) Calm-1,6 6— 10 Lactose 13 Mgal 27 Galaetose 32 Calacturonie acid 160 Ribose ‘ 175 Galactitui 180 _____—___._———-————- IObtained from data in Figure 4 and calculated for Guamr bKi: equilibrium constant of the reversible com- bination of the enzyme with a competitive inhibitor. cGalNH,: galactosamine; Mgal: methylafl-D—galse- rose. journal of Dairy Science Vol. 66. No. B. 1985 ORENO 1620 CASTILLO AND M 1:"! >200 460 ‘li‘D 10 40 0 10 K) I?!) -IDD Jan 420 10 ‘59 0 10 DO '20 '100 'lbfl 420 '60 10 D ‘0 80 '3: Guernol Ribau Gulucw'w Au Concme (mM) Cummirohm ImM) (menial-en [li .1 i IJV l “Mo-l ONPI’mIn IJ'V 40‘70 0 10 IO 60 80 ID!) NO 10 '20 010 IO 90 IO IUD I?!) 'll) '20 0 20 ‘0 b0 30 "’05?" (Solution Mnlwl- fi-Gubclawfi lones- Cmcmuum [mm Emmi-mm [mm Comma-anon thI Figun: 6. Comgetieive inhibition of 0~nitrophenyl-a-D-gala:topyranasidc (ONPG) hydrolysis by din-"em sugus. (A) saiacdm]. (B) ribosc, (C) galzcruronic acid. (D) gallatOse. (E) methyl-u-D-galactOSidC. (F) lactose. Conccntrntions of DNPG: 2.5 mM (0). 5 mM (A), 10 mM (D). (although oniy 39% is in soluble form in milk whey solutions. (22)) are higher than their rcspcctivc 150. These The enzyme was inhibited by galaptose and last four cicmcnrs. especially Ag” and Ca“. some galactosidcs but not by glucosm this may may have important negative effects in the use be of significance in a process for lactose M" of iactasc for trcatment of some canccntratcd droliysis. This response was similar to that Ctf TABLE 3. Average cations contentsof milkI and whcy.b ___—__—_—____________—— Concentration (M) __. Whey ____________d_._ Cation Milk Sweet Add ________—________.____—-—— Hg” ... _6 x 10" .9 X 10-. Ar 4.35 x 10” . .. 4 Cu“ 2.04 x 10-‘ 2.73 mo“ 5.17 x 10” Cd“ 2.3 x 10-1 a x 10" 7.7 x 10 5 Zn" 5.95 x 10-! 1,9 x 10-: 7,5 x 10:i ca" (mm 3 x In") (soluble 1.11 x 10-2) L3 x 10-: 3.7 X l0 9 Mn” 4.0 x :o-5 17 x m-s 2.7 x 10‘3 MS" 4.93 x 10" 453 x 10*! 5.71 Y- 10- ________________—________.__—- 'Cnlcullted from data in us. 22). bCIkuhted from am in (s. 15). Journal at Dliry Science Vol. 66. No. 8. I983 x 1“- .‘_‘._._ .___ ___ __I _i CA NDIDA PSEUDO TROPICALIS LACTASE K. fregilis (16). K. mention“ (10). Aspergillus foetidus (3). A. crime (18). and Bacillus (2-125 (11) but opposite to the glucose sensitivity of the lactases from K. fragiiis (11) and Saeeb. memensis (l). The inability of fucose to in- hibit the enzyme would indicate the impor- tance of the —H20H group in the C6 of the sugars for interaction with active centers of the enzyme. Also, the a bond between galactose and glucose made lactose more inhibitory of ONPG hydrolysis than melibiose (or linkage be- tween galactose and glucose). All this indicates the high stereospecificity of the lactase. The inv creased inhibitory effect of galactosamine in relation to galactose also has been reported for K. fragilis (16) and is possibly related to the positive charge on C, due to the presence of the amino group. Nieves and Castillo (unpublished results) have obtained supporting evidence of this with two K, fragilis lactases. Subsrirution of the —NH; group renders N-acetyl-galactosv amine ineffective as inhibitor of the enzymes. The estimated molecular weight (200,000) was higher than for K. iacris enzyme (6) similar to those of K. fregii'is (17. 21). two 0th" strains of C. pseudotropicalis, T. versatilis, and 7'. spbuerica (12), and lower than K. murxiamrs (10). This large molecular weight may indicate more than one subunit, as proposed for the lactases of K'. fragilis (17) and K. lactis (6): however, for strain 744 we have no evidence for this. and the number of subunits remains to be determined. The information will be of value in the design or Proce55es aimed at lactose hydrolysis by C. pseudarropimiis lactaSe. HEFERENCES l Emelicr. M.. A. Chakraharty. and 5. K. Majumdar. 1982. Immobilization of yem cells containing 0- galactosidase. Biotechnol. Bioeng. 24:18”. 2 Riemann, 1.... and M. D. Giantz. 1968. Isolation and Chll’ltteriznion of B-galaetosidase from Succbommym Inuit. Bioehim. Biophys. Act: 167: 373. in(It'lzlum. G. 13., and M. 2. Slernberg. 1972. Prop- erties of: fungal lactase. J. Food Sci. 37:619. 4 cmmo. F. L. and S. B. de Sanchez. 1978. Studies on “‘9 Emweh of Kiuyueromycer fragilrs in whey for the production of yeast protein. Aeta Cient. Venez. 29:113. 5 dc B-Ics. 5.. and F. J. Castillo. 1979. Production at lactase by Candida pseudotrapicalis grown in Whey. Appl. Environ. Microbiol. 37:1201. ‘5 Dickm. R. c. L R. Dickson. and 3. s. Markin. 1621 1979. Purification and properties of an inducible li—gulactosidase isolated from the yeast Kiuyuero- my“: inctis. J. Bacteriol. 137:51. 7 Dixon. M. 1953. The determination of enzyme in. hibition constants. Biochem. J. 55:170. 8 Glam, 1... and T. l. Hedriek. 1977. Nutritional com- position of sweet- and acid-type dry whey. ll. Vitamin. mineral. and caloric contents J. Dairy Sci. 60:190. 9 Gomez, 2’... and F. J. Castillo. 1983. Production of biomass and a-galaetosidase by Candida pseudo- :rapicalir grown in continuous culture on whey. Biotechnol. Bioeng. 25:1341. 10 Goncalves, J. A.. and F. J. Castillo. 1982. Partial purification and characterization of li—D-galaetosi— dase from Kluyveromyees mamiamts. J. Dairy Sci. 65:2088. 11 lkura. Y.. and K. Horikoshi. 1979. p-galaetosidase in alkalophilie Bacillus Agrie. Biol. Chem. 4:: 1359. 12 ltoh. T., M. Suzuki, and S. Adachi. 1982. Froduc- tion and characterization of fl—galactosidase from lactose-fennenting yclsts. Agric. Biol. Chem. 46: 899. 13 Lunpen, L. M, 1975. Modern dairy products. Chem. Pub]. Co., Inc.. New York, NY. l4 Laurent, T. C., and J. K. Killander. 1964-. A theory of gel filtration and its experimental verification. ,1. Chromatogr. 14:317. 15 Mavrapoulou, l. P., and F. V. Kosikowski. 1973. Composition, solubility, and stability of whey powders. J. Dairy Sci. 5621128. 16 Mahoney, R. It. and J. R. Whitaker. 1977. Stabil- ity and enzymatic properties of a-gaiaetosidase from Kluyveromyces fragilis. J. Food Biochem. J: 327. 1'1 Mahoney, R. 8.. and J. R. Whitaker. 1978. Purifi- cation and physicoehemieal properties of fl-galac- tosidase from Kluyvemmyces fragilis. J. Food Sci. 43:584. 18 Park, Y, K.. M.S.S. De SantiI and G. M. Pasture. 1979. Production and charaCterization of fl—galaev rosidase from Aspergillus myzae. J. Food Sci. 44: 100. 19 Pedrique. M.. and F. J. Castillo. 1982. Regulation of fi-D-galactosidase synthesis in Candida pseudo- !ropicalis. Appl. Environ. Microbiol. 43:303. 20 Ramma Rao, M. V.. and S. M. Dutta. 1981. Puri- fication and properties of beta-galaetosidsse from Slre’plococcrls tbermopbiius J. Food Sci. 46:14-19. 2i anjirna. T., H. Yagi. and O. Terada. 1972. Puri- fication. crystallization and some properties of a—galactosidase from Suecbaramyees fragilis. Agric, Biol. Chem. 36:570. 22 Webb. B. H.. A. Pb Johnson, and J. A. Alford. 1974, Fundamentals of dairy chemistry. Avi Publ. Co., Inc.. Westport. CT. 23 Wendorff. W. L, and C. H. Amundmn. 1971. Characterization of beta-galactosidase from Saccbaromyres fragilis. J. Milk Food Technol. 34: 300. 24 Wenderfl’, W. i... C. H. Amundson, and N. F. Olson. 1971. Use of yeast betavgallcton'dase in milk and milk products J. Milk Food Technol. 34:294. Journal of Dairy Science Vol. 66. No. 8. 1933 ...
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PropertiesOfLactase (1) - 5 \. ‘1'? Properties of Lactate...

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