Lipase of Mucor pusillus"

Transcription

1 APPLIED MICROBIOLOGY, Apr. 1969, p Vol. 17, No. 4 Copyright 1969 American Society for Microbiology Printed in U.S.A. Lipase of Mucor pusillus" G. A. SOMKUTI,2 F. J. BABEL, AND A. C. SOMKUTI3 Department of Animal Sciences, Purdue University, Lafayette, Indiana 4797 Received for publication 8 January 1969 Lipase of Mucor pusillus NRRL 2543 was recovered with ammonium sulfate precipitation, gel filtration on Sephadex G-75, and anion-exchange chromatography on diethylaminoethyl-sephadex A-5. Maximal glycerol ester hydrolase (lipase) activity was observed at ph 5. to 5.5 and 5 C when trioctanoin and olive oil were used as substrates. The enyme also showed esterase activity; it hydrolyed, with the exception of methyl butyrate, all methyl esters tested. A minimum chain length of six carbons appeared to be a requirement for esterase activity, which was maximal at about ph 5.5 with methyl dodecanoate (C12) as the substrate. Neither the glycerol ester hydrolase (lipase) nor the esterase activity of the enyme appeared to be affected by thiol group inhibitors, chelating agents, and reducing compounds. On the other hand, hydrolysis of triolein and methyl dodecanoate was arrested to the same extent in the presence of diisopropyl fluorophosphate, which suggested the involvement of serine in the active center of the enyme. The enyme remained stable during a 3- day storage at -1 C. Mucor pusillus synthesies an extracellular protease (6, 15) which, owing to its powerful milk-clotting activity, has been recommended for the production of cheese (K. Arima and S. Iwasaki, U.S. Patent 3,212,95). The synthesis of the acid protease appears to be accompanied by lipase production. Previously obtained data in our laboratory showed that M. pusillus NRRL 2543 secretes a lipase into the environment, when grown in a liquid wheat bran medium (14). The effect of this presumably extracellular enyme on natural oils, fats, and synthetic triglycerides also has been studied. Excessive amounts of active lipase in a milkclotting enyme preparation endangers the development of proper flavor in cheese. The development of possible means for the elimination of lipase from the protease preparation would require more detailed knowledge of the lipase. Therefore, in the second phase of our studies we examined some of the properties of M. pusillus lipase and compared them with that of other fungal lipases. It was also hoped that the investigation would provide further information with regard to microbial lipases in general, the classification of which is difficult owing to their wide range of substrate specificity. This problem has been recently discussed in detail by Lawrence (7 ). MATERUILS AND METHODS Organism. The culture of M. pusillus NRRL 2543 was maintained on potato-malt-agar (Difco) slants at 37 C and transferred weekly. Enyme production and recovery. A 4% wheat bran suspension in distilled water was sterilied by autoclaving at 121 C for 3 min. After cooling, 2,-ml Erlenmeyer flasks, each with 35 ml of medium, were inoculated with a spore suspension of M. pusillus, prepared as described previously (13). Flasks were agitated on a New Brunswick (model 3-25) gyratory shaker for 5 days at 35 C. The broth (ph 6.6 to 6.8) was filtered through a 3.8-cm layer of Celite analytical filter aid. The filtrate was cooled to 4 C and the recovery of lipase was carried out by ammonium sulfate precipitation. To 2, ml of cell-free filtrate at 4 C, ammonium sulfate was added to 8% saturation. After standing overnight at 4 C, the precipitate was collected by centrifugation in an International (model B-2) centrifuge at 25, X g. The supernatant fluid was decanted and the pellet was dispersed in.5 M acetate buffer (ph 5.45) and stirred for 3 min. Insoluble matter was removed by centrifugation. The supernatant fluid was used for the gel filtration and the ion-exchange chromatography of the lipase. Glycerol ester hydrolase (lipase) and esterase assays. The substrates used in the glycerol ester hydrolase (lipase) or esterase assays were prepared in an identical manner; 1 g of substrate,.4 g of sodium taurocholate, 1 ml of.1 M CaC12, and 5 ml of.1 M acetate buffer (ph 5.45) were emulsified in a Waring Blendor ml of the enyme was mixed with 'Published with the approval of the Director of the Purdue for 1 min. Then, 1 University Agricultural Experiment Station as Journal Series 5 ml of the substrate emulsion in a 5-ml Erlenmeyer Paper No flask, and the assay mixture was incubated at 35 C for 2 Present address: Department of Biological Sciences, Duquesne University, Pittsburgh, Pa min on a reciprocal shaker (16 strokes per min). 3 Present address: Department of Microbiology, School of At the end of incubation, the reaction was stopped by Medicine, University of Pittsburgh, Pittsburgh, Pa adding 1 ml of 1 N H2SO4. The mixture was trans- 66 Downloaded from on November 29, 218 by guest

2 VOL. 17, 1969 LIPASE OF MUCOR PUSILLUS 67 ferred into a 1-ml separatory funnel and combined with 1 ml of absolute ethyl alcohol that had been used to rinse out the reaction vessel. Fatty acids were extracted (twice) with 25 ml of petroleum ether. The solvent phases were combined and titrated with.1 N alcoholic KOH to the phenolphthalein end point. The control was an assay mixture containing boiled enyme. The net uptake of alkali was taken as the measure of enyme activity (micromoles of fatty acids liberated by 1 ml of enyme solution per 12 min). Gel filtration. All column chromatographic runs were carried out in a 6 C cold room. A portion of the protein solution was percolated through a Sephadex G-75 column (2.4 by 55 cm) equilibrated with.1 M acetate buffer (ph 5.45). Elution was conducted with the same buffer; 5-ml fractions were collected automatically. The protein content of each fraction was estimated by determining the optical density at 28 nm in a Beckman DU spectrophotometer. The distribution profile of glycerol ester hydrolase (lipase) and esterase activity was checked by mixing 1 ml of each fraction with 1 ml of trioctanoin or 1 ml of methyl dodecanoate substrate emulsion in.1 M acetate buffer (ph 5.45). Tubes were incubated for 6 min without agitation in a 35 C water bath. Then, 1 ml of absolute ethyl alcohol and 1 drop of phenolphthalein were added to each tube, and titration was carried out with.1 N alcoholic KOH directly in the test tubes with vigorous stirring, employing a Vortex Junior mixer. Anion-exchange chromatography. A column (2. by 2 cm) of diethylaminoethyl (DEAE)-Sephadex A-5 was equilibrated with.2 M phosphate buffer (ph 6.2) containing.1 M NaCl. Active protein fractions from several gel filtration runs were pooled and applied to the column. The following stepwise elution schedule was used:.2 M phosphate-.1 M NaCl,.2 M phosphate-.5 M NaCl,.2 M phosphate-.1 M NaCl,.2 M phosphate-.2 M NaCl, and.2 M phosphate-.5 M NaCl. Lipase and esterase activity of each 5-mlfractionwas determined as before. The active fractions were pooled, dialyed against distilled water, and applied to a DEAE-Sephadex column previously equilibrated with.2 M phosphate buffer (ph 6.2) containing.1 M NaCl. Elution of protein was stepwise with.2 M phosphate buffers having NaCl concentrations of.1 and.2 M. The active fractions were pooled and dialyed against distilled water. The enyme solution was dispensed into test tubes (3 ml per tube) and kept froen until needed. Effect of ph. The effect of hydrogen ion concentration on enyme activity was determined between ph 3. and 8., in McIlvaine buffers, with olive oil, trioctanoin, and methyl dodecanoate as substrates. In these assays, CaCl2 was omitted from the reaction mixture since calcium would have reacted with the components of the buffer. One milliliter of enyme solution (.4 mg of protein) was incubated with 5 ml of substrate emulsion for 12 min. Effect of temperature. Enyme protein (.4 mg in 1 ml of acetate buffer, ph 5.5) was incubated with triolein emulsion (5 ml) at temperatures of 25 to 6 C for 12 min. Hydrolysis of fatty acid methyl esters. Reaction mixtures contained.6 mg of protein and 1 g of emulsified substrate in a total volume of 6 ml. Incubation was at 35 C for 5 hr. The amount of free fatty acid was determined as before. Since it is known that the recovery of fatty acids with <C8 is low by solvent extraction (1), the activity of the enyme against methyl butyrate was also checked by direct titration (14). Effect of reducing agents, thiol poisons, and chelating agents. One milliliter of enyme solution (.8 mg of protein) was incubated for 6 min at 35 C with 1 ml of the respective chemical agent to give a 1-3 or 1-4 M final concentration. Then, 1 ml of the enymeinhibitor mixture was combined with 5 ml of substrate emulsion (trioctanoin or methyl dodecanoate), and incubation was continued for 12 min. Effect of diisopropyl fluorophosphate (DIFP). The enyme was preincubated with DIFP (1-3 and 1-4 M) for 6 min. Residual lipase and esterase were tested against triolein and methyl dodecanoate as substrates. Incubation time was 4 hr at 35 C. Chemicals. Synthetic triglycerides and fatty acid methyl esters were products of Nutritional Biochemicals Corp., Cleveland, Ohio. DIFP and N-ethyl maleimide were obtained from Mann Research Laboratories, Inc., New York, N.Y. Monoiodoacetic acid and 2-mercaptoethanol were products of Matheson Coleman and Bell, East Rutherford, N.J., and o-phenanthroline and p-chloromercuribenoate (pcmb) were products of J. T. Baker Chemical Co., North Phillipsburg, N.J., and of Calbiochem, Los Angeles, Calif., respectively. Ethylenediaminetetraacetic acid (EDTA) and olive oil were obtained from Fisher Scientific Co., Pittsburgh, Pa. Cysteine was purchased from K & K Laboratories, Inc., Jamaica, N.Y. Sephadex G-75 (medium) and DEAE-Sephadex A-5 (medium) were products of Pharmacia Fine Chemicals, Inc., New Market, N.J. Buffers. Buffer solutions were prepared according to Dawson and Elliott (1). Protein determination. Protein was measured by the method of Lowry et al. (9), with crystalline bovine albumin as the standard. RESULTS AND DISCUSSION It was shown previously that M. pusillus lipase hydrolyed natural oils and fats as well as synthetic triglycerides (14) The definition (7) of lipase as a "glycerol ester hydrolase" (EC ) is applicable to enymes isolated from animal systems in which the so-called simple esterases can be distinguished on the basis of their ability to hydrolye substrates like methyl butyrate; triglycerides are hydrolyed very sluggishly or not at all. Such a definition in regard to microbial lipases is of questionable merit, for there is evidence that certain microbial lipases attack both triglycerides and fatty acid esters (4, 5, 8). Because of this property, it was of interest to study some of the properties of the lipase of M. pusillus and to examine the enyme for esterase activity. By means of ammonium sulfate precipitation, approximately 5% of the original lipase activity present in the crude broth was recovered. In addition to hydrolying triglycerides, lipase of M. pusillus displayed esterase activity against Downloaded from on November 29, 218 by guest

3 SOMKUTI, BABEL, AND SOMKUTI 68 APPL. MICROBIOL..6 TABLE 1. Hydrolysis offatty acid methyl esters by M. pusillus lipasea.4,.2.1 FRACTIONS (5.l s 9 FIG. 1. Gel filtration of lipase of M. pusillus on Sephadex G-75. Sample load: 54 mg. Flow rate:.25 ml min-' cm2, at 6 C. (-) Protein; () lipase activity (against trioctanoin); (A) esterase activity (against methyl dodecanoate)..8 _.4 Z.2 a..1 Q.1M >,.5M l ><.2 OM.. O.SI > NaCI NaCI NaCI NaCI NaI O.IM Nod FRACTIONS (5 ml),.1 M NaCI vi.2 M NdCl L 7i 4 o3 3F, O~c FIG. 2. Anion-exchange chromatography of lipase on DEAE-Sephadex A-5. Sample load: 125 mg. (-) Protein; () lipase activity (against trioctanoin); (A) esterase activity (against methyl dodecanoate). s.8.4 wji. 2.1 FRACTIONS (Smi) 2 5 E 4' O FIG. 3. Rechromatography of M. pusillus lipaseesterase on DEAE-Sephadex A-5. Sample load: 26 mg.(-)protein; () lipase activity (against trioctanoin); (A) esterase activity (against methyl dodecanoate). a 3- Z 6 2 o E' Substrate Amt of fatty acid liberated Methyl butyrate... Methyl hexanoate... 1 Methyl octanoate Methyl decanoate Methyl dodecanoate Methyl tetradecanoate Methyl oleate Triolein (control) a Reaction mixtures (.6 mg of protein and 1 g of substrate in a 6-ml total volume) were incubated at 35 C for 5 hr. The results are expressed as nanomoles of fatty acid liberated per micromole of substrate ph FIG. 4. Effect of ph on the lipase and esterase activity of M. pusillus enyme. Substrates: () olive oil; () trioctanoin; (A) methyl dodecanoate. methyl esters of fatty acids. Gel filtration experiments on the crude enyme showed that glycerol ester hydrolase and esterase activities of the enyme coincided, when measured by the hydrolysis of trioctanoin and methyl dodecanoate, respectively (Fig. 1). Further treatment of the active protein, obtained by gel filtration, by means of --.-A S 6 7 Downloaded from on November 29, 218 by guest

4 VOL. 17, 1969 LIPASE OF MUCOR PUSILLUS 69 i ICJ!aJ ci Co -J 12 1 so 4 6)I- I.-!i 444 In J cc 2 I- _ for the hydrolysis of trioctanoin, olive oil, and methyl dodecanoate were ph 5. to 5.5 (Fig. 4). These values were similar to those reported for other fungal lipases (3, 4, 12). In contrast to the protease secreted by this thermophilic organism (15), the lipase was somewhat less resistant to heat, and maximal hydrolysis of triolein was noted at about 5 C (Fig. 5). Reducing compounds (cysteine, 2-mercaptoethanol), chelating agents (EDTA, o-phenanthroline), thiol group poisons (monoiodoacetate, pcmb, N-ethyl maleimide), and exhaustive dialysis showed no effect on either the glycerol ester hydrolase or esterase activity of the enyme, indicating that the lipase was not a metalloenyme and did not require either a free -SH group(s) or an intact S-S bridge(s) for activity. This observation supported the suggestion that -SH groups are not required for activity by microbial lipases (11). Furthermore, the presence of a dialyable cofactor evidently was not required by the lipase. TEMPERAATURE, C The glycerol ester hydrolase and esterase aconai. FIG. 5. Effect of temperature tivities of M. pusillus were equally inhibited by Substrate: triolein. on lpase activity. DIFP, which suggested that serine may be involved in the active center of the enyme (Table 2). DIFP caused a 9% inhibition of the hyanion-exchange chromatograp)hy on DEAE- drolysis of either substrate by the enyme, when Sephadex again concentrated liipase and esterase the inhibitor was present at 1-3 M concentration. activities in a single symmetric,al peak (Fig. 2), The lipase preparation was also stored at -1 suggesting the possible presencee of only one en- C in a freeer for 3 days. It appeared that the enon DEAE-Sepha- yme remained stable during this duration of yme. Upon rechromatography dex, active fractions showed both lipase and storage. esterase activities (Fig. 3). Estcerase activity was The coincidence of glycerol ester hydrolase dependent on the chain length of the fatty acid and esterase of M. pusillus, purified by gel filtra- maximal against tion and anion-exchange chromatography, the moiety of the substrate and wass methyl dodecanoate (Table 1). This finding was proximity of the ph optima, the identical re- on the glyc- sponse to DIFP, and the negative response to all different from data obtained earlier erol ester hydrolase activity of the enyme (14), other chemical agents suggested that both types which was shown to be maximal when the fatty of activity were displayed by the same enyme. acid moiety in the triglyceride Ihad eight carbons (trioctanoin). With the excelption of methyl TABLE 2. Inhibition of lipase and esterase butyrate, the enyme hydrolyed all of the tested activity of M. pusillus enyme by fatty acid methyl esters. Deteirmination of free diisopropyl fluorophosphate fatty acid by direct titration ailso indicated the (DIFP)a failure of the enyme to hydrol1 ye methyl butyr- chain length of Substrate DIFP Relative ate. It appeared that a minimuim concn activity six carbons was required by ti he enyme to hy- requirement was M g7c drolye simple esters. A similarr reported for the lipase of Asper, gillus niger which, Triolein 1 with the exception of methyl tbutyrate, also hyon to triglycerides drolyed simple esters in additi( (5). However, the enyme of A. niger showed Methyl dodecanoate maximal esterase activity wheni methyl esters of fatty acids with 8 and 16 carbsons were used as substrates. On the other hanid, the lipase of a Reaction mixtures (.4 mg of protein and 1 g Rhiopus delemar has been repc)rted to hydrolye of substrate in a 6-ml total volume) were incubated methyl butyrate as well (4). at 35 C for 4 hr. The ph optima of the enymee from M. pusillus b Before addition of substrate. Downloaded from on November 29, 218 by guest

5 61 SOMKUTI, BABEL, AND SOMKUTI APPL. MICROBIOL. The data also underlined the difficulties encountered in attempting to classify microbial lipases in terms accepted for glycerol ester hydrolases and esterases of animal systems. ACKNOWLEDGMENTS This investigation was supported by Public Health Service grant EF from the National Institute of Environmental Engineering and Food Protection. We thank Stephen S. Somkuti for preparing the illustrations. LITERATURE CITED 1. Dawson, R. M. C., and W. H. Elliott Buffers and physiological media, p In R. M. C. Dawson, D. C. Elliott, W. H. Elliott, and K. M. Jones (ed.), Data for biochemical research. Oxford University Press, Oxford. 2. Dirks, B. M., P. D. Boyer, and W. F. Geddes Some properties of fungal lipases and their significance in stored grain. Cereal Chem. 32: Fukumoto, J., M. Iwai, and Y. Tsujisaka Studies on lipase. L Purification and crystalliation of a lipase secreted by Aspergillus niger. J. Gen. Appl. Microbiol. (Tokyo) 9: Fukumoto, J., M. Iwai, and Y. Tsujisaka Studies on lipase. IV. Purification and properties of a lipase secreted by Rhiopus delemar. J. Gen. Appl. Microbiol. (Tokyo) 1: Iwai, M., Y. Tsujisaka, and J. Fukumoto Studies on lipase. II. Hydrolytic and esterifying actions of crystalline lipase ofaspergillus niger. J. Gen. Appl. Microbiol. (Tokyo) 1: Iwasaki, S., G. Tamura, and K. Arima Milk clotting enyme from microorganisms. I. The enyme production and the properties of the crude enyme. Agr. Biol. Chem. (Tokyo) 31: Lawrence, R. C Microbial lipases and related esterases. I. Detection, distribution and production of microbial lipases. Dairy Sci. Abstr. 29: Lawrence, R. C., T. F. Fryer, and B. Reiter The production and characteriation of lipases from a Micrococcus and a Pseudomonad. J. Gen. Microbiol. 48: Lowry,. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: Nashif, S. A., and F. E. Nelson The lipase of Pseudomonas fragi. I. Characteriation of the enyme. J. Dairy Sci. 36: Patel, V., H. S. Goldberg, and D. Blenden Characteriation of leptospiral lipase. J. Bacteriol. 88: Shipe, W. F A study of the relative specificity of lipases produced by Penicillium roqueforti and Aspergillus niger. Arch. Biochem. Biophys. 3: Somkuti, G. A., and F. J. Babel Conditions influencing the synthesis of acid protease by Mucor pusillus Lindt. Appl. Microbiol. 15: Somkuti, G. A., and F. J. Babel Lipase activity of Mucor pusillus. Appl. Microbiol. 16: Somkuti, G. A., and F. J. Babel Purification and properties of Mucor pusillus acid protease. J. Bacteriol. 95: Downloaded from on November 29, 218 by guest