APPUED MICROBIOLOGY, JUIY 1970, P. 16-22Copyright © 1970 American Society for Microbiology

Vol. 20, No. 1Printed in U.S.A.

Purification and Properties of a Glycerol EsterHydrolase (Lipase) from Propionibacterium

shermaniiANDERS OTERHOLM,1 Z. JOHN ORDAL, AND LLOYD D. WITTER

Departments ofFood Science and Microbiology, University ofIllinois at Urbana-Chlampaign, Urbana, Illinois 61801

Received for publication 12 March 1970

An intracellular glycerol ester hydrolase (lipase) from Propionibacteriwn sher-manii was recovered from cell-free extracts and purified by ammonium sulfate pre-cipitation, gel filtration, and ion-exchange chromatography on diethylaminoethyl-cellulose. Maximum enzyme activity was observed at pH 7.2 and 47 C when anemulsion of tributyrin was used as substrate. The enzyme was stable betweenpH 5.5and 8. Heating the enzyme solution at 45 C for 10 min resulted in a 75% decreasein activity. Maximum rate of hydrolysis of triglycerides was observed on tripropio-nin, followed in order by tributyrin, tricaproin, and tricaprylin. The lipase wasstrongly inhibited by mercury and arsenicals, but specific sulfhydryl reagents hadlittle or no inhibiting effect on the enzyme activity. The enzyme also showed someesterase activity, but the hydrolysis of substrates in solution was small as compared tothe hydrolysis of substrates in emulsion.

The formation of monocarboxylic acids fromcarbohydrates by the propionic acid bacteriahas been extensively studied, and the pathway isknown to contain a number of enzymes, manyof which have been purified and characterized (2).The formation of monocarboxylic acids fromtriglycerides by propionic acid bacteria, however,has not been investigated. In another report(Oterholm, Ordal, and Witter, J. Dairy Sci., inpress), we presented evidence that Propionibac-terium shermanii possesses a glycerol ester hy-drolase (EC 3.1.1.3) which forms monocarboxylicacids from triglycerides as the substrate. Thepresent work is concerned with the purificationand characterization of this enzyme in an effortto obtain further information of microbial lipasesin general and of the glycerol ester hydrolyzingproperties of the propionic acid bacteria inparticular.

MATERIALS AND METHODS

Organism and cultural conditions. The strain of P.shermanii used in this work was obtained from Chr.Hansen's Laboratory, Inc., Milwaukee, Wis. Theorganism was routinely grown in 5-liter batches asstationary culture at 30 C for 38 to 40 hr in a mediumcontaining 5 g of yeast extract (Difco), 20 g of Tryp-ticase, and 5 g of glucose per liter (20). Stock culturesand cultures for daily use were prepared as previously

I Present address: Norske Meieriers Salgssentral, Oslo 1,Norway.

described (20), with the above broth as the basalmedium.

Preparation of cell-free extract. After harvesting bycentrifugation, the cells were washed three times in0.01 M ammonium chloride buffer and resuspended in200 ml of the same buffer. Cell-free extract was thenprepared as described by Oterholm, Ordal, and Witter(20). The protein content of the cell-free extract wasdetermined by the method of Lowry et al. (16) withcrystalline serum albumin as the standard.

Lipase assay. The glycerol ester hydrolase (lipase)activity of the cell-free extract was determined bymeasuring the initial rate of hydrolysis by continuoustitration of the liberated acids with 0.1 N C02-freeKOH by using an automatic recording pH-stat (E.H. Sargent & Co., Chicago, Ill.). Controls containingboiled enzyme were similarly titrated. An emulsion oftributyrin in 10% gum arabic (5) was routinely usedas the substrate. The assays were conducted at 35 Cand at pH 7.2. One unit of enzyme activity is definedas the amount of enzyme which catalyzes the forma-tion of one nano-equivalent of acid per minute. Thesimple triglycerides, esters of fatty acids and aromaticesters, used as substrates were all reagent grade com-mercial preparations. Emulsions of the various sub-strates were prepared by ultrasonic treatment by theprocedure described for the assay of lipase from thelactic acid bacteria (20).

Purification procedure. Pulverized ammoniumsulfate (Mallinckrodt Chemical Works, St. Louis,Mo.) was slowly added to the crude cell-free extractwith constant stirring. During this step, a pH of 6.8was maintained. Material which precipitated between

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LIPASE FROM PROPIONIBACTERIUM SHERMANII

0.30 and 0.50 saturation after standing overnight at4 C contained most of the glycerol ester hydrolaseactivity. The precipitate was collected by centrifuga-tion and resuspended in distilled water. The salts re-

maining in the enzyme protein solution after am-monium sulfate fractionation were removed by gelfiltration by using Sephadex G-25 as described byFlodin (8). The packed column (2.5 by 3.5 cm) wasequilibrated with 0.01 M tris(hydroxymethyl)amino-methane (Tris)-hydrochloride buffer (pH 7.2) beforedesalting.

Further purification of the enzyme was obtained byion-exchange chromatography. Type 40 diethylamino-ethyl (DEAE)-cellulose (Schleicher and Schuell Co.,Keene, N.H.) was prepared by the procedure ofPeterson and Sober (21). The DEAE-cellulose column(1.5 by 2.5 cm) was equilibrated with 0.01 M Tris-hydrochloride buffer (pH 7.2) by slowly percolatinglarge volumes of the buffer through the packed ma-terial. A sample of the desalted-enzyme preparationwas applied to the column, and the protein waseluted by a linear gradient of NaCl obtained by using270 ml of 0.6 M NaCl in 0.01 M Tris-hydrochloridebuffer (pH 7.2) in the reservoir and 270 ml of the samebuffer in the mixing chamber. The column was con-nected to an automatic fraction collector, and frac-tions of 5 ml were collected and analyzed for proteinand glycerol ester hydrolase activity. The flow ratewas controlled at 0.5 ml/min by a Beckman SolutionMetering Pump (model 746). The active fractionswere pooled and dialyzed against 50 volumes of 0.01 MTris-hydrochloride buffer (pH 7.2) for 30 hr with onechange of buffer. The enzyme preparation was con-centrated by using coarse beads of Sephadex G-25. Aweighed and precooled amount of Sephadex was addedto the protein solution under stirring and allowed toswell for 25 to 30 min. The swollen gel grains werethen removed by centrifugation with modified centri-fuge tubes (Occomy Associates, Chicago, Ill.). Thisconcentrated enzyme preparation was stored at 0 Cand used in all of the subsequent work on the prop-erties of the enzyme.

Effect of pH. The effect of hydrogen ion concentra-tion on enzyme activity was determined by measuringthe initial velocity of the reaction at various pH valuessimply by changing the setting of the pH-stat. Theeffect of pH on enzyme stability was studied by ex-posing the enzyme to different pH values for 5 min at35 C and then measuring the remaining activity afterreadjusting the solution to pH 7.2 (6).

Effect of temperature. The effect of temperature onboth enzyme activity and enzyme stability was de-termined. The influence of temperature on lipase ac-tivity was studied over the range of 20 to 50 C bymeasuring the reaction rate at pH 7.2. The varioustemperatures were obtained by changing the settingof the pH-stat. Data were collected at 5-degree incre-ments, and the activation energy was calculated fromthe linear portion of an Arrhenius plot of log k versuslIT (where k is the initial velocity and T the absolutetemperature). The effect of temperature on thestability of the enzyme was studied over a range of 35to 47.5 C at pH 7.2 as follows. A 0.15-ml amount ofthe enzyme solution was added to 9.85 ml of distilled

water which had been previously equilibrated at theappropriate test temperature. The diluted enzyme solu-tion was kept at the temperature being tested for 10min. At the end of this incubation period, the solutionwas brought to the assay temperature of 35 C by addi-tion of 2 ml of an ice-cold tributyrin emulsion, and theactivity was determined by continuous titration of theliberated acids.

Michaelis constant. The relation between substrateconcentration and enzyme activity was obtained bydetermining the reaction rate on various concentra-tions of tributyrin emulsion at pH 7.2 and 35 C. TheMichaelis constant, Km, for the enzyme was deter-mined by the double-reciprocal plot method of Line-weaver and Burk (15).

RESUTLTS AND DISCUSSIONThe scheme of treatment used for the purifica-

tion of glycerol ester hydrolase from P. shermanjiis given in Table 1. The specific activity of thepurified enzyme was 141-fold over that of thestarting cell-free extract, and the overall yieldwas about 44%.The lipase protein was eluted from the DEAE-

cellulose column at a concentration gradientranging from 0.32 to 0.37 M NaCl and was con-tained in the very last portion of the eluted pro-teins. An attempt to purify further the enzyme bygel filtration on Sephadex G-75 was not success-ful, because the lipase activity was found in themajor protein peak which came off the columnwith the void volume. This indicated, however,that the lipase protein was a relatively large mole-cule since the molecular weight exclusion limit ofSephadex G-75 is approximately 50,000.The rate of hydrolysis of the tributyrin emul-

sion obeys zero-order kinetics as shown in Fig. 1.

TABLE 1. Purification of lipase from Propioni-bacterium shermaniia

Total TotalSpe-Treatment cfiTotalTal cifi Yieldprotein activity activ-ityb

mg unilsc %Cell-free extract .... 2,167 420,000 193 100(NH4)2SO4..........798 331,000 415 78.8Sephadex G25.. 684 299,200, 437 71.2DEAE-cellulose ..... 6.85 187,500127,300 44.6

a Reaction mixture contained 58 mmoles oftributyrin sonically dispersed in 10% aqueousgum arabic, water, and enzyme in a total volumeof 6 ml. Rate of hydrolysis was determined atpH 7.2 and at 35 C by continuous titration of theliberated acids with a pH-stat.

b Expressed as units per milligram of protein.c One unit of enzyme activity is defined as the

amount of enzyme which catalyzes the formationof one nano-equivalent of acid per minute.

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OTERHOLM, ORDAL, AND WITTER

5000 50o r

4001F1-U,.2

- 300

-J

00< 200.La

I

100

0

2 3 4 5 6 7 8 9

Time (Minutes)

FIG. 1. Time course of hydrolysis of tributyrin bylipase. The reactioni mixture conltained 58 mmoles oftributyrin sonically dispersed in 10% gum arabic,water, and liDase in a total volune of 6 ml. Hvdrolvsiswas determintedmethod.

600O

480 -

360 F

240[

1201

0 P

FIG. 2. Effectactivity. Assay (

legenid to Fig. 1.

This linear rel

min, after whicrate of hydrol5findings of De

F3 4 5 6 7 8 9 10

pHFIG. 3. Effect of pH on lipase activity. Except for

the pH changes, the assay con2ditions were those de-scribed in the legend to Fig. 1.

at pH 7.2 anid 35 C by the pH-stat down relatively early and that assay systemsbased on long incubation times may lead to in-accurate results.A linear relationship was also evident when

enzyme activity was measured as a function ofprotein concentration (Fig. 2). The same direct

* proportionality was also found when crude ex-tract was used as the source of enzyme.The effect of the hydrogen ion concentration on

* the activity of the enzyme (Fig. 3) shows that thelipase had a rather broad pH optimum withmaximum activity in the region of pH 7.2. This

o was in agreement with the general findings thatmost bacterial lipases have their optimum ac-tivity at a neutral or slightly alkaline pH (1, 13,

/ 17, 23, 24). The fairly rapid decline in the reac-tion velocity on either side ofpH 6 and 8 may bedue to a decreased saturation of the enzyme withsubstrate because of a decreased stability of theenzyme, or a combination of the two. These effectscan be distinguished experimentally by exposingthe enzyme to a range of pH values and then

2 3 4 5 testing the activity after readjusting the pH to astandard value. The results of such an experi-

elotive Concentration of Lipase ment are shown in Fig. 4. Apparently, the fall onof enizyme concentration ont lipase the alkaline side of pH 8 is primarily due to de-

conditions were those described in the struction of the enzyme, whereas the fall on theacid side of pH 6 is partly due to a decreasingaffinity of the lipase for its substrate and partly

Lationship continued for about 20 due to an irreversible destruction of the enzymeh a slow but steady decrease in the protein.ysis was noted. This supported the The majority of microbial lipases have been2snuelle (4) that hydrolysis slows reported to be most active within a temperature

'5

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LIPASE FROM PROPIONIBACTERIUM SHERMANII

loo r

,. 75

4

a 50

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800.r

I-

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,200

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pH

FIG. 4. Effect ofpH on lipase stability. The enzymepreparation was held at the indicated pH for 5 min.The sample was then adjusted to pH 7.2 and assayed.The conditions were those described in the legend toFig. 1.

range of 30 to 40 C (9, 10, 19, 23). The lipase ofP. shermanii was an exception to this as it had anapparent optimum activity at 47 C (Fig. 5). Attemperatures as high as about 45 C, the maineffect of heat was an increase in the reaction rate.Above 45 C, the thermal inactivation of the en-zyme increased, and a rapid decrease in the ac-

tivity of the enzyme was experienced.The rather marked difference between the

optimal temperature of the lipase of P. shermaniiand most other lipases was probably due to differ-ences in the assay conditions used. Many workershave based their temperature-activity studies on

the relatively long incubation periods necessitatedby most lipase assay systems, rather than on theinitial velocity as reported here. Since thermaldestruction of an enzyme is progressive, the shapeof the curve and the apparent optimum tempera-ture depend on time and would cause the optimumtemperature to fall as the time interval was in-creased.The activation energy for the lipase-catalyzed

reaction was obtained from the linear portion ofan Arrhenius plot of the log of the reaction rateagainst the reciprocal of the absolute temperatureand was found to be 8,100 cal/mole. The cor-responding temperature coefficient, Qlo, was 1.6.Activation energies of 7,000 and 8,500 cal/molegiven by Sizer and Josephson (26) and Schwartz(24), respectively, for the action of pancreaticlipase on tributyrin are not much different fromthe value obtained for the lipase of P. shermaniion the same substrate. A somewhat higher value,11,800 cal/mole, was reported by Shah and Wilson

Temperature ( C )

FIG. 5. Effect of temperature on lipase activity. Thereaction mixture conitained 58 mmoles of tributyrinsonically dispersed in 10% gum arabic, water, andlipase in a total volume of 6 ml. Hydrolysis was de-termined at pH 7.2 at the indicated temperature by thepH-stat method.

(25) for Staphylococcus aureus on triolin as thesubstrate, whereas Kvamme, Clagett, andTreumann (14) found the energy of activation ofwheat germ lipase to be 8,000 cal/mole. Theactivation energies which hitherto have been re-ported for bacterial lipases appear to be of thesame order of magnitude as that of P. shermanii.The procedure used for studying the thermal

inactivation of the lipase (see above) should elimi-nate the inactivation resulting from the timenecessary to heat or cool the enzyme solution tothe given temperatures. The inactivation of P.shermanii lipase by exposure to various tempera-tures is given in Table 2. After 10 min at 35 Cin the absence of substrate, there was some lossin activity. This loss increased to greater than 80%after 10 min at 47.5 C. Apparently, the lipase ofP. shermanii does not belong to the group ofmarkedly thermostable bacterial lipases found inPseudomonas fragi (17), Achromobacter lipolyti-cum (18), Pseudomonas mucidolens (22), andothers. The lipase of P. shermanii, however, wasmuch more stable in the presence of its substratethan it was in aqueous solution. The enzyme lostonly 12% of its activity in 10 min at 47.5 in thepresence of an emulsion of tributyrin as comparedto a loss of 82% in aqueous solution at the sametemperature. Exactly the opposite effect has beenreported for a "tributyrinase" from wheat germ(23), whereas the thermostability of the lipase ofP. fragi appears to be increased in the presenceof fat or protein in the medium (17).For the action of pancreatic lipase, Desnuelle

(4) has shown that the interfacial area is more

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TABLE 2. Thermal stability of the lipasea

Temp Activity units Relative activityb

C %35.0 278 85.837.5 267 82.440.0 189 58.342.5 145 44.745.0 83 25.647.5 58 17.9

aAn 8.95-ml amount of distilled water wasequilibrated at the respective temperatures fol-lowed by the addition of 0.15 ml of the enzymesolution. After 10 min of incubation at the appro-priate temperatures, the enzyme solution wasbrought to the assay temperature, 35 C, by theaddition of 2 ml of an ice-cold tributyrin emulsion,and the remaining activity was determined by con-tinuous titration of the liberated acids at pH 7.2.

b No decrease in the reaction rate was observedwhen the enzyme was incubated for 10 min at 35 Cand at pH 7.2 in the presence of substrate, and theactivity under these conditions was taken as 100%.

500

4001

300

200

100

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/ ° 301-

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TABLE 3. Ratio ofesterase activity to lipase activityduring purificationi

Specific activity Ratio ofesterase

Purification procedure - activity toOn tri- On lipasebutyrina triacetinb activity

Crude extract. 193 9.26 0.048(NH4)2SO4 fraction-

ation .......... 415 15.8 0.038Desalting by Sepha-

dex G-25......... 437 19.7 0.045DEAE-cellulosechromatography.. 27,300 1,310 0.048

a Assay conditions were those described infootnote a of Table 1.

I Esterase assay was performed in 0.2 M aqueoussolution of triacetin. Rate of hydrolysis wasdetermined at pH 7.2 and at 35 C by continuoustitration of the liberated acids with a pH-stat.

TABLE 4. Hydrolytic activity of the lipaise onlsubstrate in solution and in emulsioni

Substratea Physical stateb Activityc

Triacetin Solution (0.25 S) oTriacetin Solution (0.50 S) 4.8Triacetin Solution (1.00 S) 9.9Triacetin Emulsion (1.50 S) 22.0Triacetin Emulsion (3.0 S) 21.7Methylbutyrate Solution (0.1 M) 6.7a-Naphthylacetate Solution (10-s M) 0Tween 60 Solution (1%) 0Tween 80 Solution (1%) 0

OL I I I I I a Emulsion of triacetin was prepared by ultra-o 10 20 30 40 50 60 70 sonic treatment according to the procedure de-

/S x lo2 scribed for the assay of lipase.I Values of 0.25 S, 0.50 S, etc, are equivalent to

25% saturation, 50% saturation etc.I0 15 20 25 c Reaction rate on tributyrin emulsion under

Tributyrin Concentration (mrM)

FIG. 6. Effect of tributyrin concentration on thereaction rate of lipase. Except for the changes in theconcentration of tributyrin, the assay conditions werethose described under Fig. 1. Km (2.0 X 1-3 M) wascalculatedfrom these data.

important than the substrate concentration per se.Therefore, when evaluating the effect of substrateconcentration on enzyme activity, all differentconcentrations were made from the same 10%tributyrin emulsion. When the substrate concen-tration in the reaction mixture is increased, theinterfacial area should increase proportionally.No attempt was made, however, to define theMichaelis constant in terms of interfacial area.The relation between substrate concentration

the conditions described in footnote a of Table 1

was taken as 100%.

and lipase activity is presented in Fig. 6. Fromthe inserted double reciprocal plot of reactionrate versus tributyrin concentration, the Kmvalue was calculated to be 2 X 10-3 M. This valueis somewhat larger than the Km of 6 X 10-4 Mfound for pancreatic lipase (27).As indicated in another report (Oterholm,

Ordal, and Witter, J. Dairy Sci., in press), thecrude extract of P. shermanii exhibited smallamounts of esterase activity when assayed on atypical esterase substrate such as triacetin insolution. The various preparations obtained dur-ing purification were also found to contain a weakesterase activity. However, the ratio of esterase

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LIPASE FROM PROPIONIBACTERIUMSHERMANI2

TABLE 5. Comparison of the relative rates ofhydrolysis of triglycerides by the lipase

Substratea C atoms Activity (%)b

Triacetin ........... 2 24.2 (24.1)cTripropionin. 3 100.0 (100.0)Tributyrin.......... 4 83.3 (76.8)Tricaproin ......... 6 68.3 (15.0)Tricaprylin......... 8 16.7Tricaprin........... 10 NilTrilaurin ........... 12 Nil

a Assay conditions were those described infootnote a of Table 1.

b Activity on tripropionin was taken as 100%.¢ Data on pancreatic lipase (29).

TABLE 6. Effect ofenzyme inhibitors on the activityof the lipasea

Compound Concn Activity

None 100NaN3................. 2.86 X 10-i 96NaF................. 8.30 X 10-3 79Na2HAsO4............. 1.67 X 10-' 0HgCl2................. 1.67 X 10-4 0CaC12................. 8.3 X 10-3 52lodoacetate.................4.44 X 10-4 100p-Hydroxymercuri-

benzoate ............. 4.44 X 10-4 85Ethylenediaminetetra-

acetate............... 5.0 X 10-2 55Sodium taurocholate.... 2.7 X 10-2 68

a Assay conditions were those described infootnote a of Table 1 after addition of the enzymeto the reaction mixture containing the inhibitor.

activity to lipase activity was almost constantthroughout purification, indicating that the ester-ase activity was due to an activity of the lipaseitself (Table 3).To evaluate further the dual esterase and lipase

activity of the glycerol ester hydrolase, the enzymeactivity toward substrates in solution as well asin emulsion was determined. The rates of hy-drolysis given in Table 4 are all relative to therate of the enzyme activity on an emulsion oftributyrin. The results show that no activity wasfound at the lowest concentration of triacetin,whereas 0.5 saturation gave the same slight ac-tivity found in the cell-free extract and partiallypurified preparations. Moreover, a relativelysharp increase in the reaction rate was evidentwhen triacetin in solution was changed to tri-acetin in emulsion. The activity on methylbutyratewas the same as that observed during purification,whereas no activity was found on such typical

esterase substrates as a-naphthyl acetate, Tween60, and Tween 80. These results were evidencethat the enzyme from P. shermanii preferentiallyhydrolyzed substrates in emulsion and did notact, or acted very slowly, on substrates in solu-tion. It was therefore concluded that the glycerol-ester-hydrolyzing enzyme from P. shermaniiwas a lipase and that the small esterase activitytowards substrates in solution was due to anactivity of the lipase itself. Previously presentedkinetic studies were further evidence for this con-clusion. Similar dual substrate properties havebeen reported for many other microbial lipases,including the crystalline lipase of Aspergillusniger (12) and the purified lipase of Rhizopusdelemar (9) and P. fragi (17).

Wills (29) was one of the first investigators toobserve that mammalian lipases preferentiallyhydrolyze triglycerides of short chain fatty acids,notably tripropionin and tributyrin. With theapparent exception of the lipase from P. fragi(17), microbial lipases also preferentially hy-drolyze triglycerides of short-chain fatty acids.The substrate specificity of the lipase of P. sher-manii (Table 5) corresponded to other microbiallipases (7, 11, 23, 25) by showing a maximum rateof hydrolysis toward tripropionin and a decreas-ing rate with increasing fatty acid chain length.The effect of selected inhibitors on the activity

of the lipase of P. shermanii is given in Table 6.In common with many hydrolytic enzymes (3,28), this lipase was strongly inhibited by mercuricions and arsenate. Inhibition by heavy metals andarsenate suggested the possible participation ofsulfhydryl groups in the active sites of enzymeactivity. This suggestion was negated, however,by the lack of or very little inhibition caused bythe more specific sulfhydryl inhibitors of p-hydroxymercuribenzoate and iodoacetate. Sincethe rate at which lipases hydrolyze triglyceridesis dependent upon the interfacial area between theemulsified substrate and soluble enzyme, agentswhich reduce this area may act as inhibitors eventhough they do not combine with the enzyme it-self. Inhibition of lipolytic activity by the sur-face-active agents ethylenediaminetetraacetateand sodium taurocholate may act in this man-ner. The sensitivity of this lipase to calcium ionsdiffers from some other microbial lipases whichhave been shown to be activated by equivalentconcentrations (12, 27, 30), but generally thepattern of inhibition of P. shermanii lipase issimilar to that of other microbial lipases.

ACKNOWLEDGMENTS

This investigation was supported by Public Health ServiceTraining Grant FD 00-004.

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LITERATURE CITED

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3. Chandan, R. C., and K. M. Shahani. 1964. Milk lipases. Areview. J. Dairy Sci. 47:471-480.

4. Desnuelle, P. 1961. Pancreatic lipase, p. 129-161. In F. F.Novel (ed.), Advances in enzymology, vol. 23. lntersciencePublishers, Inc., New York.

5. Desnuelle, P., M. J. Constatene, and J. Baldy. 1955. Techniquepotentio-metrique pour la mesure de l'activit6 de la lipasepancreatique. Bull. Soc. Chem. Biol. 37:285-290.

6. Dixon, M., and E. C. Webb. 1964. Enzymes. AcademicPress Inc., New York.

7. Drummond, M. C., and M. Tager. 1959. Enzymatic activityof staphylocoagulase. I. Characterization of an esteraseassociated with purified preparations. J. Bacteriol. 78:407-412.

8. Flodin, P. 1961. Methodological aspects on gel filtrationwith special reference to desalting operations. J. Chroma-togr. 5:103-115.

9. Fukumoto, J., M. Iwai, and Y. Tsujisaka. 1964. Studieson lipase. IV. Purification and properties of a lipase se-

creted by Rhizopus delemar. J. Gen. Appl. Microbiol.10:257-265.

10. Gorbach, G., G. Dedic, and K. Lorenz. 1955. Zur Anreiche-rung und Wirksamkeitsbestimmung von Bakterien-Lipasen.Arch. Mikrobiol. 21:237-247.

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qualitative study of the lipolytic activity of single strainsof seven bacterial species. J. Appl. Bacteriol. 25:72-82.

12. Iwai, M., Y. Tsujisaka, and J. Fukumoto. 1964. Studies on

lipase. I1I. Effect of calcium ion on the action of the crystal-line lipase of Aspergillus niger. J. Gen. Appl. Microbiol.10:87-93.

13. Kahn, F. M., R. C. Chandan, C. W. Dill, and K. M. Shahani.1964. Production and properties of the extracellularlipase of Achromobacter lipolyticum. J. Dairy Sci. 47:675.

14. Kvamme, 0. J., C. 0. Clagett, and W. B. Treumann. 1949.Kinetics of the action of the sodium salt of 2,4-dichloro-phenoxyacetic acid on the germ lipase of wheat. Arch.Biochem. 24:321-328.

15. Lineweaver, H., and D. Burk. 1934. The determination of

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enzyme dissociation constants. J. Amer. Chem. Soc. 56:658--666.

16. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Ran-dall. 1951. Protein measurement with the Folin phenolreagent. J. Biol. Chem. 193:265-275.

17. Lu, J. Y., and B. J. Liska. 1969. Lipase from Pseudonionasfragi. I. Purification of the enzyme. Appl. Microbiol. 18:104-107.

18. Nashif, S. A., and F. E. Nelson. 1953. The extracellularlipases of some gram-negative non-sporeforming rod-shapedbacteria. J. Dairy Sci. 36:698-706.

19. O'Leary, W. M., and J. T. Weld. 1964. Lipolytic activitiesof Staphylococcus aureus. I. Nature of the enzyme producingfree fatty acids from plasma lipids. J. Bacteriol. 88:1356-1363.

20. Oterholm, A., Z. J. Ordal, and L. D. Witter. 1968. Glycerolester hydrolase activity of lactic acid bacteria. AppI.Microbiol. 16:524-527.

21. Peterson, E. A., and H. A. Sober. 1962. Column chroma-tography of proteins: substituted celluloses, p. 3-27.In S. Colowick and N. Kaplan (ed.), Methods in enzy-mology, vol. 5. Academic Press Inc., New York.

22. Pinheiro, A. J. R., B. J. Liska, and C. E. Parmelee. 1965.Heat stability of lipases of selected psychrophilic bacteriain milk and Purdue Swiss type cheese. J. Dairy Sci. 48:983-984.

23. Rottem, S., and S. Razin. 1964. Lipase activity of Myco-plasma. J. Gen. Microbiol. 37:123-134.

24. Schwartz, B. 1943. The effect of temperature on the rateof hydrolysis of triglycerides by pancreatic lipase. J.Gen. Physiol. 27:113-118.

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