Purification and Some Properties of Two Types of Penicillium. Lipase, I and II, and Conversion of Types I and II under

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1 [Agr. Biol. Chem., Vol. 31, No. 11, p `1366, 1967] Purification and Some Properties of Two Types of Penicillium Lipase, I and II, and Conversion of Types I and II under Various Modification Conditions By Susumu OI, Akira SAWADA* and Yukio SATOMURA Faculty of Science, Osaka City University *Osaka Bacteriological Research Institute Ltd. Received June 26, 1967 The lipase produced by a strain of Penicillium crustosum Thom was fractionated into three lipase components, I `III by DEAE cellulose column chromatography, and two of hem, I and II were purified and obtained in crystalline form respectively, which proved homogeneous by electrophoresis and ultracentrifugal analysis. Lipase I was an ordinary lipase with molecular weight about 29,000 hydrolyzing olive oil and tributyrin favourably in almost the same degree, while II, rather, a so-called tributyrinase with M. W. about 32,000 hydrolyzing tributyrin more efficiently than olive oil. The site of the activity on olive oil in these lipase was generally sensitive to sodium desoxycholate, ethylenediamine -tetraacetate (EDTA), and p-chloromercuribenzoate (PCMB), and lipase I was converted to a lipase II by a treatment with these reagents. Also, partial degradation of I by proteinase ('pronase') yielded the enzyme fragment of type II. On the other hand, treatment of the enzymes with hydrogen peroxide or sodium borohydride caused the conversion of type II into I. From the observation of UV difference spectrum during incubation with sodium desoxycholate it was indicated that the situation of tryptophane residue in enzyme molecule may have a significance in the activity of lipase I on olive oil. INTRODUCTION A strict separation between esterases and lipases is difficult, and, in lipase itself, there are also the two types of enzymes. The relative specificity of them is qualitative and variable with the emulsion state of substrate. Also, the effects of bile salts and other emul sifiers on lipolysis are various with enzyme sources. It may be due to a wide variations in the chemical properties of lipase protein. With the purification and specificity of micro bial lipase, a number of studies have already 1) Y. Satomura and S. Oi, J. Agr. Chem. Soc. Japan, 31, 202 (1957). 2) Y. Satomura, S. Oi, A. Sawada and J. Fuku moto, Bull. Agr. Chem. Soc. Japan, 24, 329 (1960). 3) Y. Satomura, M. Ono, S. Oi and J. Fukumoto, This journal, 25, 15 (1961). 4) N. Tomizuka, Y. Ota and K. Yamada, This Journal, 30, 576 (1966). been performed,1 `8) hitherto. However, the characteristic nature of the lipase protein for determining the relative specificity is not yet made clear. In the present work, the chemical and enzymological properties of two types of Penicillium lipase which could be purified and obtained in crystalline form were investigated and compared employing various methods of modification. As a result, it was found that the two types of lipase, one behaves like an ordinary lipase and the other, rather, like an 5) N. Tomizuka, Y. Ota and K. Yamada, This Journal, 30, 1090 (1966).6 ) C. Matsumura, Hakko-kyokai-shi, 9, 423 (1964).7 ) J. Fukumoto, M. Iwai and Y. Tsujisaka, J. G eneral Applied Microbiol., 9, 353 (1963). 8) M. Iwai, Y. Tsujisaka and J. Fukumoto, Symposium on Enzyme Chemistry in Japan, p. 304 (1966).

2 1358 Susumu OI, Akira SAWADA and Yukio SATOMURA esterase, are mutually convertible under an carried out using a Hitachi Tieselius Electrophoresis appropriate condition. MATERIALS AND METHODS Apparatus HT-B. Partial specific volume was measured using a 5ml pycnometer at enzyme con centrations of 0.8 to 0.4%, at 30 Ž. Strain and Culture Condition. A strain of Penicillium mold which was isolated in our laboratory from leaves of a bamboo, and identified as Pen. crustosum Thom was cultured in 50ml of the medium taken in a 500ml flask, which consisting of 2% of wheat bran, 0.5% of soybean meal and 0.3% of CaCO3, adjusted at ph 5.6, with reciprocal shaking at 25 Ž for 50hr. Crude Enzyme Solution. The culture filtrate, of which final ph reached about 7.8, was salted out with 0.9 saturantion of ammonium sulfate and allowed to stand overnight at 4 Ž. The resulting precipitate was collected by centrifugation at 4,000 r. p. m. for 20min., dissolved in a small volume of water, and dialyzed again distilled water. The dialyzed solution was used as crude enzyme solution. Lipase Activity. Reaction mixture containing 5 ml of 0.1M NH2-NH4Cl buffer (ph 9.0), 1ml of enzyme solution, 0.2ml of 1 M. CaCl2 solution, 1ml of substrate (olive oil or tributyrin), and 2.8ml of distilled water in a test tube (1.5 ~ 12.5cm) plugged with a rubber stopper was incubated at 36 Ž for 60 min. with constant shaking (180 times oscillation per min. in 3.0cm amplitude). Then, the mixture was poured into a flask with 30ml of 95% ethanol, and after warming at 55 Ž for 20min., free fatty acid liberated was titrated against 0.05N NaOH using a potentiometer. One unit of lipase activity was defined as that which produces 1 p mole of free fatty acid under the condition. Specific activity was expressed by units per mg protein, and protein quantity was determined spectrophotometrically at 280mƒÊ applying the factor E_??_=13.2 which was estimated from the dry weight measurement of the crystalline preparation. Ultracentrifugal Analyses. These were carried out at Institute for Protein Research, Osaka University with a Hitachi Analytical Ultracentrifuge UCA-1. The sedimentation coefficient was calculated as describ ed by Schachman9) and was corrected to 20 Ž in water according to the method described by Svedberg.10) Electrophoresis. Electrophoretic experiments were 9) H. K. Schachman, "Methods in Enzymology", Academic Press Inc., New York, (1957) p ) T. Svedberg and D. K. Pederson, "The Ultracentrifuge", Claredon Press, Oxford (1940) p Gel Filtration. Sephadex G-200 (140 `400 mesh, Pharmacia, Uppsala, Sweden) was after swelling for a month in a 0.75M NaCl solution containing 0.025M MgCl2 used as the gel-filtration medium of column. With the column, the elution volumn, (Ve) was calibrated for estimating the molecular weight of proteins in the manner as described by Andrews,11) using as standards the proteins of known molecular weights. Molecular weights were derived from the calibration graph which indicated a minimum mole cular weight of at least 106 for proteins excluded from the gel pores, (Ve 67ml). Difference Spectrum. UV difference absorption spectra were measured in a matched quartz cells of 1cm path on Ito-Chotanpa QU-2 Spectrophotometer. EXPERIMENTAL AND RESULTS I) Purification and Crystallization The purification procedure is summarized in Table I. To precipitate the lipase, acrinol solution was added to the crude enzyme solution in a 1% final concentration. The resulting precipitate was collected by centri fugation, and extracted with a small volume of 0.2M NH3-NH4Cl buffer (ph 8.9) containing 0.5M acetate, followed by dialysis against 0.002M NH3-NH4Cl buffer (ph 8.0). To the dialyzed solution was added 4 volumes of acetone. The resulting precipitate was col lected by centrifugation, and after dissolving in a small volume of 0.025M Tris-HC1 buffer (ph 9.1) passed through DEAE cellulose column (2.2 ~ 60cm) which had been equili brated with 0.025M Tris-HCl buffer at ph 9.1, whereby the lipase was adsorbed on the column. The column was eluted with a linear gradient ph change of Tris-HC1 buffer from 9.1 to 7.2 by using the mixing chamber containing 2.01 of 0.025M Tris-HCl buffer (ph9.1) and the reservior containing 2.01 of 0.05M Tris-HCI buffer (ph 7.2). The elution 11) W. K. Downey and P. Andrews, Biochem. J., 94, 642 (1965).

3 Purification and Some Properties of Two Types of Penicillium Lipase 1359 TABLE I. PURIFICATION OF LIPASE I AND II. (Peak 1) and I (peak 2) were respectively eluted at ph 8.0 and 7.6. The lipase III (peak 3) was, at last, eluted with adding NaCl in 1M concentration to 0.05M Tris-HCl buffer (ph 7.2). The lipase I and II were subjected to a rechromatography on DEAE cellulose column under the same condition as the above. Then, the lipase fractions thus obtained were brought to 0.9 saturation with ammonium sulfate, and allowed to stand overnight at 4 Ž, respectively. The resulting FIG. 1. Chromatography on DEAE Cellulose Column. precipitates were collected by centrifugation, dissolved in a small volume of distilled water, and dialyzed against distilled water. These pattern is presented in Fig. 1. The lipase II

4 1360 Susumu 01, Akira SAWADA and Yukio SATOMURA dialyzed solutions were freeze-dried without any appreciable loss of activity. The freezedried preparation were redissolved in a small volume of distilled water containing 0.002M calcium acetate and, after adjusting ph to 7.8 with dilute NaOH solution, the insoluble impurities were removed by centrifugation. These supernatant solutions were respectively mixed with a half volume of acetone and allowed to stand for about 3 days at 4 Ž, whereupon the crystals appeared as shown in Fig. 2. In this step, the activity of lipase I was attained to 181 fold per mg protein, and that of II attained to 113 fold. FIG. 2. Micrographs of Crystalline Lipase I and II. II) Physical Properties i) Electrophoresis. As shown in Fig. 3, both lipase I and II proved homogeneous electro phoretically. Also, the isoelectric point was determined by the electrophoresis using cellulose-acetate, and those of lipase I and II were shown to be approximately 8.0 and 7.8 respectively. ii) Ultracentrifugal analysis. The experi ments were operated at 54,400 r. p. m. and 20.2 Ž in a standard 12mm, 4 sector cell. The sedimentation coefficient S20,w was caluclated from the sedimentation velocity runs shown in Fig. 4, and a value of 2.90 ~10-13 and 3.20 ~10-13 was obtained respectively, for lipase I and II. Also, the single peak shown in the sedimentation pattern again revealed the homogeneity of the lipase preparations. The molecular weight was determined by the Archibald method12), and the sedimentation was conducted at 13,700 r. p. m. and 22.9 Ž, where the photograph was taken at intervals of 8 minutes, and FIG. 3. Electrophoretic Patterns of Lipase I and II. I; 0.66 % of lipase I in 0.02 M veronal buffer (ph 8.75) II; 0.76 % of lipase II in 0.02 M veronal buffer (ph 8.75) The electrophoresis was carried out at 67 volts, 6 mamp. and 18 Ž, adjusting the ionic strength to 0.1 with NaCl. protein concentration was evaluated from a run in synthetic boundary cell. The mole cular weight of lipase I and II was calculated as approximately, 29,300 and 32,200 respec tively. iii) Partial specific volume. Duplicate de- 12) H. K. Schachman, "Methods in Enzymology" ed. by S. P. Colowick and N. O. Kaplan, Vol. IV, Academic Press Inc., New York, 1957, p. 32.

5 Purification and Some Properties of Two Types of Penicillium Lipase 1361 FIG. 4. Sedimentation Patterns of Lipase I and II. I; 0.66% of lipase I in 0.02M veronal buffer (ph 8.75), II; 0.76% of lipase II in the same buffer as I. The ionic strength was adjusted to 0.1 with NaC1. The photographs were taken from right to left at the above minutes after reaching full speed. terminations of partial specific volume of the lipases yielded respectively a value of and for lipase I and II. III) Enzymatic Properties i) Optimum ph and Stability. As seen in Fig. 6, ph optima of lipase I, II and III were all present at nearly 9.0. Thermostability is shown in Fig. 7. These lipase was stable below 45 Ž while, very unstable over that temperature. They almost completely lost their activity above 80 Ž within 15 min.

6 1362 Susumu OI, Akira SAWADA and Yukio SATOMURA In Fig. 8, ph-stability is presented. The activity of these lipase in NH3-NH4Cl buffer was comparatively stable between ph 6.0 and 9.0, while, that of lipase II in phosphate buffer with the same ph range was unstable, and especially between ph 7.0 and 9.0 it rapidly deteliolated within 24hr. ii) Substrate specificity. As shown in Table II, lipase I hydrolyzed olive oil most efficiently, but also tributyrin favourably in the next. On the other hand, lipase II and III hydrolyzed tributyrin much more efficiently than olive TABLE II. SUBSTRATE SPECIFICITY OF LIPASE 1, II AND III. TABLE III. EFFECT OF METAL, EDTA AND SURFACE ACTIVE AGENT ON LIPASE I AND II. oil. Also, the activity of these lipase, I, II and III on tributyrin was almost the same value. The relative specificity of lipase I was the following, i. e. [Olive oil>tributyrin> Tween 80 > Methyl laurate > Tween 40> Ethyl laurate > Triacetin > Span 20, 60>The others], and that of II, i. e. [Tributyrin> Triacetin, Olive oil > Span 60, 20, 80> Ethyl laurate> Methyl laurate> The others]. The specificity of lipase III was analogous to II with some exceptions. iii) Effect of metal, EDTA and surface active agent. As shown in Table III, the activity of lipase I and II on olive oil was increased by such bimetallic ions as Ca++, and Mn++ ion, but inhibited by Cu++ ion, and the inhibitory effect of Cu++ ion on lipase I was marked. EDTA and some surface active agents such as sodium laurylsulfate and TEGO-51 remarkably inhibited the activity of both lipase. Sodium desoxycholate slightly inhibited the activity of lipase I while it rather stimulated that of lipase II in some degree. The inhibitory effect of sodium desoxycholate on the former was, however, reversed by Ca++ ion to a certain extent. iv) Effect of emulsifier. As shown in Table IV, hydrophilic emulsifier such as Tween 20 and polyvinyl alcohol slightly increased the TABLE IV. EFFECT OF EMULSIFIER ON LIPASE I AND II. * C12H25-NH-C2H4-NH-C2H4-NH-CH2COOH * Polyvinylalcohol %.

7 Purification and Some Properties of Two Types of Penicillium Lipase 1363 TABLE V. MODIFICATION OF ACTIVITY OF LIPASE I AND II BY TREATMENT WITH REAGENTS activity of lipase I on tributyrin though, it rather inhibited that on olive oil. The inhibitory effect of Tween 20 in the latter case was especially serious. The activity of lipase II on tributyrin was also inhibited in some degree by these emulsifier. On the other hand, lipophillic emulsifier such as Span 80 did not so influence the activity of lipase I, while, it inhibited the activity of lipase II especially on tributyrin considerably. v) Conversion of Lipase Types, I and II by Modification. When these enzymes were treated with various reagents under an appropriate condition, respectively, as shown in Table V, it was observed that a different TABLE VI. MODIFICATION OF ACTIVITY OF LIPASE I BY INCUBATION WITH Ca-ACETATE ALONE OR PROTEINASE ('PRONASE') PLUS Ca-ACETATE FIG. 9. Gel-Filtration of Original Lipase I, II and III on a Sephadex G-200 Column. Column size: 2.2 ~58cm (Pronase: Product of Kaken-Kagaku Ltd. (a Strepto myces proteinase) 1u. produced 1ƒÊg tyrosine/ml. reaction mixture (0.3% casein)/min.) Flow rate: about 6ml/hr.

8 1364 Susumu OI, Akira SAWADA and Yukio SATOMURA FIG. 10. Gel-Filtration of Modified Lipase I on a Sephadex G-200 Column change occured between the relative activity on olive oil and on tributyrin. The treat ments with sodium desoxycholate, EDTA and PCMB, all, generally blocked the activity on olive oil more severely than on tributyrin. Accordingly, with lipase I, the order of the relative activity on both substrates was con verted and yielded the activity of a type of II. On the other hand, the treatment with hydrogen peroxide or sodium borohydride specifically blocked the activity on tributyrin, and thus, the conversion of the lipase types II into I occurred. Also, when lipase I was either incubated with Ca-acetate alone or pro teinase 'pronase' plus Ca-acetate under an appropriate condition, respectively, as shown in Table VI, the conversion of the relative activity of type I into II occurred. Then, the molecular weights of original, and some of modified lipases were estimated by gel-filtration method, as presented in Fig. 9 and 10, and they were shown together in Table VII. The M. W. of lipase I, II and III was respectively 29,000, 32,900 and 11,000. Although lipase I was partially denatured yielding a protein fraction with M. W. 6,000 during a prolonged dialysis, the remaining fraction was composed of the original enzyme TABLE VII. MOLECULAR WEIGHTS OF ORIGINAL, AND SOME MODIFIED LIPASES ESTIMATED BY GEL-FILTRATION

9 Purification and Some Properties of Two Types of Penicillium Lipase 1365 FIG. 11. Difference Spectra of Lipase I during Incubation with Sodium Desoxycholate. protein with M. W. 29,000. A partial degradation of lipase I by mixture of 'pronase' and Ca-acetate produced the two enzyme fragments, one with a M.W. 18,200 and the other with a M. W. 6,000, of which the later showed an enzyme activity of type II. The incubation of lipase I with sodium desoxy - cholate under a condition as described above produced also the two enzyme fragments, one with a M. W. 36,500 and the other with a M. W. 9,000, of which the former, however, gave an enzyme activity of type II. There may occur a reconstruction of the protein structure. During the incubation it was found that lipase I exhibited the UV differ ence absorption spectrum, as presented in Fig. 11. After an incubation for about 20hr, a blue shift appeared in the absorption region of mp, indicating the appearance of a tryptophan residue in the lipase molecule. If sodium desoxycholate was removed by dia lysis at that time, the difference spectrum disappeared, and lipase I restored the original enzyme activity. With an incubation for 70 hr, a red shift appeared with accompanying the inactivation of enzyme activity. DISCUSSION As shown in the above experiment, the activities on olive oil of two types of Penicillium lipase, I and II, one is an ordinaly lipase and the other, rather, a tributyrinase are both inhibited remarkably by EDTA and some surface active agents such as sodium lauryl -sulfate and TEGO-51, but are both activated by calcium ion. In the activating effect of Ca++ ion on lipolysis, although various mechanisms are involved, it seems of most significance in the above case to stabilize the active configuration of the enzyme. However, when lipase I is incubated with Ca-salt for a long time, converted to a lipase II changing the relative specificity on olive oil and tributyrin, as shown in Table VI. It indicates a change in the protein structure of lipase I per se. Also, treatment of the enzyme with sodium desoxycholate, EDTA and PCMB all bring about a change in the relative specificity of type I into II, as described above. From the observation of UV difference spectrum during incubation with sodium desoxycholate, it is shown that the situation of tryptophan residue buried in a hydrophobic region of the lipase protein may have a significance in the heterogeneous lipase reaction on olive oil. Although-SH groups are also important for the lipase activity on olive oil, they are not considered to be a component of the active center of the enzyme. The active site of the lipase on tributyrin is rather contained in a hydrophillic region, and comparatively stable to the above reagents. While, treatment of the enzyme with hydrogen peroxide or sodium borohydride causes the conversion of enzyme, type II into I, as shown above. For such rearrangement of the active site of lipase, com plicated mechanisms are possibly involved in the different way, respectively. In the active region of lipase I, a lipophillic emulsion state of the substrate must be required for lipolysis, since hydrophillic emulsifier such as Tween 20 inhibits the lipolysis especially on olive oil as shown in Table IV It is of interest that from lipase I a type of lipase II with the small molecular weight is produced by a proteolysis, and there is also in nature a milk lipase with the similar properties and a small mol. wt. as II.

10 1366 Susumu 01, Akira SAWADA and Yukio SATOMURA Acknowledgement. The authors wish to express their sincere gratitude to Dr. S. Abe of Tokyo Research Inst., Kyowa Fermentation Industry Ltd. for identification of species of the mold, and to Dr. K. Kakiuchi of Institute for Protein Research, Osaka University, Ultracentrifugal analysis. They are grateful to Mrs. C. Oi for her assistant part of the experiments.

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