/. Biochem., 80, 367-370 (1976)
Effective Method for Activity Assay of Lipase from Chromobacterium viscosuml Yosifumi HORIUTI, Hajime KOGA, and Sinobu GOCHO2 Research Laboratory, Toyo Jozo Co., Ltd., Mifuku, Ohito-cho, Tagata-gun, Shizuoka 410-23 Received for publication, February 19, 1976
A method was devised for activity assay of the lipase [triacylglycerol acyl-hydrolase, EC 3.1.1. 3] excreted from Chromobacterium viscosum into the culture medium; olive oil emulsified with the aid of Adekatol 45-S-8 (a non-ionic detergent, the ethoxylate of linear seoalcohols having chain lengths of 10—16 carbon atoms) was used as the substrate. This method was specifically effective for Chromobacterium lipase activity assay, and was approximately twice as sensitive as the conventional method, in which polyvinyl alcohol is used for the emulsification of the substrate.
Various partial or extensive purifications and characterizations of lipases from many microorganisms such as fungi, yeasts, and bacteria have been reported in the last 10 years (1—33). The present paper deals with an effective method for the assay of the lipase [EC 3.1.1.3] excreted from Chromobacterium viscosum into the culture medium. MATERIALS AND METHODS Measurement of Lipase Activities—Lipase activity was measured by two methods, the Adekatol emulsion method and the polyvinyl 1
Reported at the annual meeting of the Japanese Conference on the Biochemistry of LJpids held at Matsumoto-shi (1974). 1 Present address: Research Laboratory, Hasegawa Koryo Co., Ltd., Kariyado, Nakahara-ku, Kawasakishi, Kanagawa 211. Abbreviations: pi, isoelectric point; PVA, polyvinyl alcohol. Vol. 80, No. 2, 1976
alcohol (PVA) emulsion method. These methods were based on the method of Tomizuka et al. (26) with some modifications as described below. The Adekatol emulsion method was used unless otherwise indicated. 1. Adekatol emulsion method: Twenty grams of olive oil, 20 ml of Adekatol 45-S-8 (see below) and 60 ml of deionized water were mixed, and the mixture was blended at 25° for 5 min in a homogenizer (HD-2 Universal Homogenizer, Ninon Seiki Co., Tokyo) at a rate of 10,000 rpm. The resulting Adekatol emulsion was stable for at least 24 hr at room temperature. Two ml of 0.1 M KH,PO 4 Na2HPO4 buffer (pH 7) and 5 ml of Adekatol emulsion were mixed in a test tube (2.4x20 cm) and preincubated for 10 min at 37°. The reaction was started by adding 1 ml of enzyme solution, continued at 37° for 20 min, and stopped by adding 20 ml of an acetone-ethanol (1 : 1) mixture. The solution thus obtained was supplemented with three drops of 1% alcoholic phenolphthalein solution, and titrated 367
368
with 50 mM NaOH under a continuous stream of nitrogen gas ("sample"). The control experiment was carried out in the same manner, except that the enzyme solution was added after the addition of the acetone-ethanol mixture. One unit of lipase was defined as the amount of the enzyme which could liberate 1 /imole of fatty acids per min at 37°. Adekatol SO-120 (a non-ionic detergent, the ethoxylate of linear sec-alcohols) was found to be as effective as Adekatol 45-S-8. 2. PVA emulsion method: A polyvinyl alcohol (PVA) mixture comprising 18.5 g of Poval #117 and 1.5 g of Poval #205 was mixed with 1 liter of deionized water and heated at 75-85° until the PVA was completely dissolved : this took about 1 hr. The resulting solution was then filtered through filter paper (PVA solution). Seventy-five milliliters of the filtered solution and 23 g of olive oil were mixed, and the mixture was blended at 0—5° for 10 min in a homogenizer at a rate of 10,000 rpm. The resulting PVA emulsion was stable for 12 hr at 5°, and was used after standing for 1 hr at 5°. The standard reaction mixture and the experimental procedures for activity assay were the same as for the Adekatol emulsion method, except that PVA emulsion was used instead of Adekatol emulsion. Culture of Cells—The culture medium contained 3 g of Pharmamedia, 1 g of lard, 10 mg of MgS0«-7H,0 and 0.2 g of K2HPO4 per 100 ml, and the pH was adjusted to 7.0. Portions of the medium (100 ml each) were placed in 500-ml shaking flasks, sterilized, and inoculated with Chromobacterium viscosum. The flasks were then incubated at 26° with continuous shaking on a rotary shaker. During incubation, aliquots of the medium were withdrawn at regular intervals in order to determine the lipase activity and pH. The pH of the medium decreased slightly during the initial 2 days, followed by a significant increase (Fig. 1). On the other hand, the lipase activity of the medium gradually increased and reached a maximum after 4 days. Reagents and Equipment—Olive oil and lard were purchased from Sanko Seiyaku Kogyo Co., Tokyo, and Nippon Oils & Fats Co., Tokyo, respectively. Pharmamedia (derived from cot-
Y. HORIUTI, H. KOGA, and S. GOCHO 1
II
'
'
Activity
\
pH
0
4 2 3 Cultivation tini t ( day»)
5
Fig. 1. Lipase activity in the culture medium as related to the growth of Chromobacterium viscosum. The experimental conditions are described in the text.
tonseed) was a product of Traders Oil Mill Co., Texas. Adekatol 45-S-8, Adekatol SO-120, and polyvinyl alcohol (PVA) in the form of Poval #117 and #205 were products of Asahidenka Kogyo Co., Tokyo, and Kurare Co., Kurashiki, respectively. Lipases from Aspergillus niger, Geotrichum candidum, and Rhizopus delemar were purchased from Seikagaku Kogyo Co., Tokyo. The lipase of Candida cylindracea nov. sp. (Lipase MY) was from Meito Sangyo Co., Nagoya, and the lipase of porcine pancreas (B grade) was from Calbiochem, La Jolla, California. The lipase of Rhizopus arrhizus was purchased from Boehringer Mannheim GmbH, Germany. The absorbance at 280 nm (AIM nm) was measured at room temperature (25°) with a Hitachi spectrophotometer, model 101 (Hitachi Co., Tokyo). Values of pH were measured with a Hitachi-Horiba pH meter, model F-5, with a Horiba 6028-10T combined electrode (Horiba Co., Kyoto). RESULTS AND DISCUSSION
It was found that the non-ionic detergent, Adekatol 45-S-8 (the ethoxylate of linear secalcohols having chain lengths of 10—16 carbon atoms), was an excellent emulsifier of the olive I. Biochtm.
FUNDAMENTAL STUDIES ON Chromobaderium viscosum LIPASE
oil used as the substrate for activity assay of the lipase present in the culture medium of Chromobactenum viscosum. Emulsion containing 20 or 30% of Adekatol yielded a higher apparent lipase activity than that containing 10% of the detergent (Table I). When the TABLE I. Effect of Adekatol concentration used for emulsification of olive oil on activity of Chromobactenum lipase. Various volumes of Adekatol 45-S-8 as indicated were mixed well with 20 g aliquots of olive oil in a motor-driven mixer, and deionized water was added to make the total volume 100 ml. The resulting mixture was homogenized at 10,000 rpm for 5 min. The emulsions thus obtained were allowed to stand at 25° for 2 or 18 hr, and were then used for the assay of lipase activity. The enzyme source used was the supernatant obtained by centrifugation at 3,000 X Q for 20 min from the culture medium of Chromobaderium viscosum. Other experimental conditions are described in the text. Concentration of Adekatol 45-S-8
Lipase activity (titration volume of 50 mM NaOH) (ml)
(%, v/v)
After 2 hr
After 18 hr
10 20 30
2.5 3.1
2.4 3.0 3.0
3.3
10 20 30 Reaction tlmt (min)
Fig. 2. Time course of hydrolysis by Chromobacterium lipase of olive oil present in Adekatol emulsion. The enzyme source was the same as in Table I. Other experimental conditions were as described in the text, except that the reaction time was changed as indicated. Vol. 80, No. 2, 1976
369
emulsion was allowed to stand for 2 hr and 18 hr at 25°, almost identical lipase activities were observed with both emulsions. However, 20% Adekatol was more advantageous than 30% of the detergent, because the former was less viscous. When emulsion containing 20% Adekatol was allowed to stand at 5 or 25° for 24 hr, the lipase activities were hardly affected. In the following experiments, emulsion containing 20% Adekatol was used for the activity assay of Chromobacterium lipase, unless otherwise stated. With Adekatol emulsion, the rate of hydrolysis of olive oil by Chromobacterium lipase was almost linear for the initial 20 min, then gradually decreased under the experimental conditions used (Fig. 2). The initial rate increased with increasing amounts of the lipase sample with either the Adekatol emulsion or PVA emulsion; with both emulsions, the increase in- the rate was practically linear when the amount of the lipase sample was low (Fig. 3). It is noteworthy that the lipase activity with the Adekatol emulsion was approximately twice that with the PVA emulsion.
0
10 20 30 Amount of llpait sampU(jjl)
Fig. 3. Relation between the amount of Chromobacterium lipase sample and the rate of hydrolysis of olive oil in Adekatol emulsion and PVA emulsion. The enzyme source was the same as in Table I. Other experimental conditions were as described in the text.
Y. HORIUTI, H. KOGA, and S. GOCHO
370
TABLE II. Comparison of activities of various Upases in PVA emulsion and Adekatol emulsion. The experimental conditions were as described in the text, except that lipase preparations from various sources were used as indicated. The Chromobaderium lipase source used was the same as in Table I. Lipase activity (units) Source of lipase
PVA emulsion method
Adekatol emulsion method
Aspergillus niger Geotrichum candidum
2.9
0
3.0
0
Rhizopus delemar Rhizopus arrhizus Candida cylindracea nov. sp. Porcine pancreas Chromobaderium viscosum
3.0
0
4.5
0
4.7
0
4.7
0
4.0
8.6
The activities of several Upases from various microorganisms and porcine pancreas were measured with Adekatol and PVA emulsions (Table II). All the lipases tested showed activity with the PVA emulsion, whereas lipases other than Chromobacterium lipase were not active with the Adekatol emulsion. This suggests that only the Chromobaderium lipase was resistant to inactivation by Adekatol. Using the assay method described above, Chromobacterium lipase has been highly purified by procedures involving affinity chromatography on palmitoyl cellulose. The purification procedures and properties of the lipase will be described elsewhere. REFERENCES 1. Fukumoto, J., Iwai, M., & Tsujisaka, Y. (1963) J.' Gen. Appl. Microbiol. 9, 353-361 2. Laboureur, P. & Labrousse, M. (1966) Bull. Soc. Chim. Biol. 48, 747-770 3. Semeriva, M., Benzonana, G., & Desnuelle, P. (1967) Biochim. Biophys. Ada 144; 703-705 4. Semeriva, M., Benzonana, G., & Desnuelle, P. (1969) Biochim. Biophys. Ada 191, 598-610 .5. Fukumoto, J., Iwai, M., & Tsuzisaka, Y. (1964) . / . Gen. Appl. Microbiol. 10, 257-265 6. Nagaoka, K. & Yamada, Y. (1969) Agr. Biol. Chem. 33, 986-993
7. Nagaoka, K. & Yamada, Y. (1973) Agr. Biol. Chem. 37, 2791-2796 8. Saiki, T., Takagi, Y., Suzuki, T., Narasaki, T., Tamura, G., & Arima, K. (1969) Agr. Biol. Chem. 33, 414-423 9. Ishihara, H., Okuyama, H., Ikezawa, H., & Tejima, S. (1975) Biochim. Biophys. Ada 388, 413-422 10. Oi, S., Sawada, A., & Sotomura, Y. (1967) Agr. Biol. Chem. 31, 1357-1366 11. Tsujisaka, Y., Iwai, M., & Tominaga, Y. (1973) Agr. Biol. Chem. 37, 1457-1464 12. Liu, W.-H., Beppu, T., & Arima, K. (1973) Agr. Biol. Chem. 37, 157-163 13. Lawrence, R.C., Fryer, T.F., & Reiter, B. (1967) / . Gen. Microbiol. 48, 401-418 14. Rottem, S. & Razin, S. (1964) / . Gen. Microbiol. 37, 123-134 15. Finkelstein, A.E., Strawich, E.S., & Sonnino, S. (1970) Biochim. Biophys. Ada 206, 380-391 16. Troller, J.A. & Bozeman, M.A. (1970) Appl. Microbiol. 20, 480-484 17. O'leary, W.M. & Weld, J.T. (1964) / . Baderiol. 88, 1356-1363 18. Vadehra, D.V. & Harmon, L.G. (1967) Appl. Microbiol. 15, 292-295 19. Renshaw, E.C. & San Clemente, C.L. (1967) Develop. Ind. Microbiol. 8, 214-216 20. Oterholm, A., Ordal, Z.J., & Witter, L.D. (1970) Appl. Microbiol. 20, 16-22 21. Chorvath, B. & Fried, M. (1970) / . Baderiol. 102, 879-880 22. Patel, V., Goldberg, H.S., & Blenden, D. (1964) / . Baderiol. 88, 877-884 23. Henderson, C. (1971) / . Gen. Microbiol. 65, 81-89 24. Hassing, G.S. (1971) Biochim. Biophys. Ada 242, 381-394 25. Fulton, J.E., Noble, N.L. Bradley, S., & Atwad, W.M. (1974) Biochemistry 13, 2320-2377 26. Toraizuka, N., Ota, Y., & Yamada, K. (1966) Agr. Biol. Chem. 30, 576-584 27. Motai, H., Ichishima, E., & Yoshida, F. (1966) Nature 210, 308-309 28. Ota, Y, Nakamiya, T., & Yamada, K. (1970) Agr. Biol. Chem. 34, 1368-1374 29. Breuil, C. & Kushner, D.J. (1975) Can. J. Microbiol. 21, 434-441 30. Yamaguchi, T., Muroya, N., Isobe, M., & Sugiura, M. (1973) Agr. Biol. Chem. 37, 999-1005 31. Sugiura, M., Isobe, M., Muroya, N., & Yamaguchi, T. (1974) Agr. Biol. Chem. 38, 947-952 32. Sugiura, M. & Isobe, M. (1974) Biochim. Biophys. Ada 341, 195-2C0 33. Sugiura, M. & Isobe, M. (1975) Chem. Pharm. Bull. 23, 1226-1230 / . Biochetn.
Effective Method for Activity Assay of Lipase from Chromobacterium viscosuml Yosifumi HORIUTI, Hajime KOGA, and Sinobu GOCHO2 Research Laboratory, Toyo Jozo Co., Ltd., Mifuku, Ohito-cho, Tagata-gun, Shizuoka 410-23 Received for publication, February 19, 1976
A method was devised for activity assay of the lipase [triacylglycerol acyl-hydrolase, EC 3.1.1. 3] excreted from Chromobacterium viscosum into the culture medium; olive oil emulsified with the aid of Adekatol 45-S-8 (a non-ionic detergent, the ethoxylate of linear seoalcohols having chain lengths of 10—16 carbon atoms) was used as the substrate. This method was specifically effective for Chromobacterium lipase activity assay, and was approximately twice as sensitive as the conventional method, in which polyvinyl alcohol is used for the emulsification of the substrate.
Various partial or extensive purifications and characterizations of lipases from many microorganisms such as fungi, yeasts, and bacteria have been reported in the last 10 years (1—33). The present paper deals with an effective method for the assay of the lipase [EC 3.1.1.3] excreted from Chromobacterium viscosum into the culture medium. MATERIALS AND METHODS Measurement of Lipase Activities—Lipase activity was measured by two methods, the Adekatol emulsion method and the polyvinyl 1
Reported at the annual meeting of the Japanese Conference on the Biochemistry of LJpids held at Matsumoto-shi (1974). 1 Present address: Research Laboratory, Hasegawa Koryo Co., Ltd., Kariyado, Nakahara-ku, Kawasakishi, Kanagawa 211. Abbreviations: pi, isoelectric point; PVA, polyvinyl alcohol. Vol. 80, No. 2, 1976
alcohol (PVA) emulsion method. These methods were based on the method of Tomizuka et al. (26) with some modifications as described below. The Adekatol emulsion method was used unless otherwise indicated. 1. Adekatol emulsion method: Twenty grams of olive oil, 20 ml of Adekatol 45-S-8 (see below) and 60 ml of deionized water were mixed, and the mixture was blended at 25° for 5 min in a homogenizer (HD-2 Universal Homogenizer, Ninon Seiki Co., Tokyo) at a rate of 10,000 rpm. The resulting Adekatol emulsion was stable for at least 24 hr at room temperature. Two ml of 0.1 M KH,PO 4 Na2HPO4 buffer (pH 7) and 5 ml of Adekatol emulsion were mixed in a test tube (2.4x20 cm) and preincubated for 10 min at 37°. The reaction was started by adding 1 ml of enzyme solution, continued at 37° for 20 min, and stopped by adding 20 ml of an acetone-ethanol (1 : 1) mixture. The solution thus obtained was supplemented with three drops of 1% alcoholic phenolphthalein solution, and titrated 367
368
with 50 mM NaOH under a continuous stream of nitrogen gas ("sample"). The control experiment was carried out in the same manner, except that the enzyme solution was added after the addition of the acetone-ethanol mixture. One unit of lipase was defined as the amount of the enzyme which could liberate 1 /imole of fatty acids per min at 37°. Adekatol SO-120 (a non-ionic detergent, the ethoxylate of linear sec-alcohols) was found to be as effective as Adekatol 45-S-8. 2. PVA emulsion method: A polyvinyl alcohol (PVA) mixture comprising 18.5 g of Poval #117 and 1.5 g of Poval #205 was mixed with 1 liter of deionized water and heated at 75-85° until the PVA was completely dissolved : this took about 1 hr. The resulting solution was then filtered through filter paper (PVA solution). Seventy-five milliliters of the filtered solution and 23 g of olive oil were mixed, and the mixture was blended at 0—5° for 10 min in a homogenizer at a rate of 10,000 rpm. The resulting PVA emulsion was stable for 12 hr at 5°, and was used after standing for 1 hr at 5°. The standard reaction mixture and the experimental procedures for activity assay were the same as for the Adekatol emulsion method, except that PVA emulsion was used instead of Adekatol emulsion. Culture of Cells—The culture medium contained 3 g of Pharmamedia, 1 g of lard, 10 mg of MgS0«-7H,0 and 0.2 g of K2HPO4 per 100 ml, and the pH was adjusted to 7.0. Portions of the medium (100 ml each) were placed in 500-ml shaking flasks, sterilized, and inoculated with Chromobacterium viscosum. The flasks were then incubated at 26° with continuous shaking on a rotary shaker. During incubation, aliquots of the medium were withdrawn at regular intervals in order to determine the lipase activity and pH. The pH of the medium decreased slightly during the initial 2 days, followed by a significant increase (Fig. 1). On the other hand, the lipase activity of the medium gradually increased and reached a maximum after 4 days. Reagents and Equipment—Olive oil and lard were purchased from Sanko Seiyaku Kogyo Co., Tokyo, and Nippon Oils & Fats Co., Tokyo, respectively. Pharmamedia (derived from cot-
Y. HORIUTI, H. KOGA, and S. GOCHO 1
II
'
'
Activity
\
pH
0
4 2 3 Cultivation tini t ( day»)
5
Fig. 1. Lipase activity in the culture medium as related to the growth of Chromobacterium viscosum. The experimental conditions are described in the text.
tonseed) was a product of Traders Oil Mill Co., Texas. Adekatol 45-S-8, Adekatol SO-120, and polyvinyl alcohol (PVA) in the form of Poval #117 and #205 were products of Asahidenka Kogyo Co., Tokyo, and Kurare Co., Kurashiki, respectively. Lipases from Aspergillus niger, Geotrichum candidum, and Rhizopus delemar were purchased from Seikagaku Kogyo Co., Tokyo. The lipase of Candida cylindracea nov. sp. (Lipase MY) was from Meito Sangyo Co., Nagoya, and the lipase of porcine pancreas (B grade) was from Calbiochem, La Jolla, California. The lipase of Rhizopus arrhizus was purchased from Boehringer Mannheim GmbH, Germany. The absorbance at 280 nm (AIM nm) was measured at room temperature (25°) with a Hitachi spectrophotometer, model 101 (Hitachi Co., Tokyo). Values of pH were measured with a Hitachi-Horiba pH meter, model F-5, with a Horiba 6028-10T combined electrode (Horiba Co., Kyoto). RESULTS AND DISCUSSION
It was found that the non-ionic detergent, Adekatol 45-S-8 (the ethoxylate of linear secalcohols having chain lengths of 10—16 carbon atoms), was an excellent emulsifier of the olive I. Biochtm.
FUNDAMENTAL STUDIES ON Chromobaderium viscosum LIPASE
oil used as the substrate for activity assay of the lipase present in the culture medium of Chromobactenum viscosum. Emulsion containing 20 or 30% of Adekatol yielded a higher apparent lipase activity than that containing 10% of the detergent (Table I). When the TABLE I. Effect of Adekatol concentration used for emulsification of olive oil on activity of Chromobactenum lipase. Various volumes of Adekatol 45-S-8 as indicated were mixed well with 20 g aliquots of olive oil in a motor-driven mixer, and deionized water was added to make the total volume 100 ml. The resulting mixture was homogenized at 10,000 rpm for 5 min. The emulsions thus obtained were allowed to stand at 25° for 2 or 18 hr, and were then used for the assay of lipase activity. The enzyme source used was the supernatant obtained by centrifugation at 3,000 X Q for 20 min from the culture medium of Chromobaderium viscosum. Other experimental conditions are described in the text. Concentration of Adekatol 45-S-8
Lipase activity (titration volume of 50 mM NaOH) (ml)
(%, v/v)
After 2 hr
After 18 hr
10 20 30
2.5 3.1
2.4 3.0 3.0
3.3
10 20 30 Reaction tlmt (min)
Fig. 2. Time course of hydrolysis by Chromobacterium lipase of olive oil present in Adekatol emulsion. The enzyme source was the same as in Table I. Other experimental conditions were as described in the text, except that the reaction time was changed as indicated. Vol. 80, No. 2, 1976
369
emulsion was allowed to stand for 2 hr and 18 hr at 25°, almost identical lipase activities were observed with both emulsions. However, 20% Adekatol was more advantageous than 30% of the detergent, because the former was less viscous. When emulsion containing 20% Adekatol was allowed to stand at 5 or 25° for 24 hr, the lipase activities were hardly affected. In the following experiments, emulsion containing 20% Adekatol was used for the activity assay of Chromobacterium lipase, unless otherwise stated. With Adekatol emulsion, the rate of hydrolysis of olive oil by Chromobacterium lipase was almost linear for the initial 20 min, then gradually decreased under the experimental conditions used (Fig. 2). The initial rate increased with increasing amounts of the lipase sample with either the Adekatol emulsion or PVA emulsion; with both emulsions, the increase in- the rate was practically linear when the amount of the lipase sample was low (Fig. 3). It is noteworthy that the lipase activity with the Adekatol emulsion was approximately twice that with the PVA emulsion.
0
10 20 30 Amount of llpait sampU(jjl)
Fig. 3. Relation between the amount of Chromobacterium lipase sample and the rate of hydrolysis of olive oil in Adekatol emulsion and PVA emulsion. The enzyme source was the same as in Table I. Other experimental conditions were as described in the text.
Y. HORIUTI, H. KOGA, and S. GOCHO
370
TABLE II. Comparison of activities of various Upases in PVA emulsion and Adekatol emulsion. The experimental conditions were as described in the text, except that lipase preparations from various sources were used as indicated. The Chromobaderium lipase source used was the same as in Table I. Lipase activity (units) Source of lipase
PVA emulsion method
Adekatol emulsion method
Aspergillus niger Geotrichum candidum
2.9
0
3.0
0
Rhizopus delemar Rhizopus arrhizus Candida cylindracea nov. sp. Porcine pancreas Chromobaderium viscosum
3.0
0
4.5
0
4.7
0
4.7
0
4.0
8.6
The activities of several Upases from various microorganisms and porcine pancreas were measured with Adekatol and PVA emulsions (Table II). All the lipases tested showed activity with the PVA emulsion, whereas lipases other than Chromobacterium lipase were not active with the Adekatol emulsion. This suggests that only the Chromobaderium lipase was resistant to inactivation by Adekatol. Using the assay method described above, Chromobacterium lipase has been highly purified by procedures involving affinity chromatography on palmitoyl cellulose. The purification procedures and properties of the lipase will be described elsewhere. REFERENCES 1. Fukumoto, J., Iwai, M., & Tsujisaka, Y. (1963) J.' Gen. Appl. Microbiol. 9, 353-361 2. Laboureur, P. & Labrousse, M. (1966) Bull. Soc. Chim. Biol. 48, 747-770 3. Semeriva, M., Benzonana, G., & Desnuelle, P. (1967) Biochim. Biophys. Ada 144; 703-705 4. Semeriva, M., Benzonana, G., & Desnuelle, P. (1969) Biochim. Biophys. Ada 191, 598-610 .5. Fukumoto, J., Iwai, M., & Tsuzisaka, Y. (1964) . / . Gen. Appl. Microbiol. 10, 257-265 6. Nagaoka, K. & Yamada, Y. (1969) Agr. Biol. Chem. 33, 986-993
7. Nagaoka, K. & Yamada, Y. (1973) Agr. Biol. Chem. 37, 2791-2796 8. Saiki, T., Takagi, Y., Suzuki, T., Narasaki, T., Tamura, G., & Arima, K. (1969) Agr. Biol. Chem. 33, 414-423 9. Ishihara, H., Okuyama, H., Ikezawa, H., & Tejima, S. (1975) Biochim. Biophys. Ada 388, 413-422 10. Oi, S., Sawada, A., & Sotomura, Y. (1967) Agr. Biol. Chem. 31, 1357-1366 11. Tsujisaka, Y., Iwai, M., & Tominaga, Y. (1973) Agr. Biol. Chem. 37, 1457-1464 12. Liu, W.-H., Beppu, T., & Arima, K. (1973) Agr. Biol. Chem. 37, 157-163 13. Lawrence, R.C., Fryer, T.F., & Reiter, B. (1967) / . Gen. Microbiol. 48, 401-418 14. Rottem, S. & Razin, S. (1964) / . Gen. Microbiol. 37, 123-134 15. Finkelstein, A.E., Strawich, E.S., & Sonnino, S. (1970) Biochim. Biophys. Ada 206, 380-391 16. Troller, J.A. & Bozeman, M.A. (1970) Appl. Microbiol. 20, 480-484 17. O'leary, W.M. & Weld, J.T. (1964) / . Baderiol. 88, 1356-1363 18. Vadehra, D.V. & Harmon, L.G. (1967) Appl. Microbiol. 15, 292-295 19. Renshaw, E.C. & San Clemente, C.L. (1967) Develop. Ind. Microbiol. 8, 214-216 20. Oterholm, A., Ordal, Z.J., & Witter, L.D. (1970) Appl. Microbiol. 20, 16-22 21. Chorvath, B. & Fried, M. (1970) / . Baderiol. 102, 879-880 22. Patel, V., Goldberg, H.S., & Blenden, D. (1964) / . Baderiol. 88, 877-884 23. Henderson, C. (1971) / . Gen. Microbiol. 65, 81-89 24. Hassing, G.S. (1971) Biochim. Biophys. Ada 242, 381-394 25. Fulton, J.E., Noble, N.L. Bradley, S., & Atwad, W.M. (1974) Biochemistry 13, 2320-2377 26. Toraizuka, N., Ota, Y., & Yamada, K. (1966) Agr. Biol. Chem. 30, 576-584 27. Motai, H., Ichishima, E., & Yoshida, F. (1966) Nature 210, 308-309 28. Ota, Y, Nakamiya, T., & Yamada, K. (1970) Agr. Biol. Chem. 34, 1368-1374 29. Breuil, C. & Kushner, D.J. (1975) Can. J. Microbiol. 21, 434-441 30. Yamaguchi, T., Muroya, N., Isobe, M., & Sugiura, M. (1973) Agr. Biol. Chem. 37, 999-1005 31. Sugiura, M., Isobe, M., Muroya, N., & Yamaguchi, T. (1974) Agr. Biol. Chem. 38, 947-952 32. Sugiura, M. & Isobe, M. (1974) Biochim. Biophys. Ada 341, 195-2C0 33. Sugiura, M. & Isobe, M. (1975) Chem. Pharm. Bull. 23, 1226-1230 / . Biochetn.
