Agricultural and Biological Chemistry Alkaline ...

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Alkaline Cellulase for Laundry Detergents: Production by Bacillus sp. KSM-635 and Enzymatic Properties a

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Susumu Ito , Shitsuw Shikata , Katsuya Ozaki , Shuji Kawai , a

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Kikuhiko Okamoto , Shigeo Inoue , Akira Takei , Yu-ichi Ohta & Tomokazu Satoh

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Tochigi Research Laboratories of Kao Corporation, 2606 Akabane, Ichikai, Haga, Tochigi 321–34, Japan Published online: 09 Sep 2014.

To cite this article: Susumu Ito, Shitsuw Shikata, Katsuya Ozaki, Shuji Kawai, Kikuhiko Okamoto, Shigeo Inoue, Akira Takei, Yu-ichi Ohta & Tomokazu Satoh (1989) Alkaline Cellulase for Laundry Detergents: Production by Bacillus sp. KSM-635 and Enzymatic Properties, Agricultural and Biological Chemistry, 53:5, 1275-1281 To link to this article: http://dx.doi.org/10.1080/00021369.1989.10869489

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Agric. Bioi. Chem., 53 (5),

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Alkaline Cellulase for Laundry Detergents: Production by Bacillus sp. KSM-635 and Enzymatic Properties Susumu ITo,t Shitsuw SHIKATA, Katsuya OZAKI, Shuji KAWAI, Kikuhiko OKAMOTO,* Shigeo INoUE,** Akira TAKEI,*** Yu-ichi OHTA*** and Tomokazu SATOH** Tochigi Research Laboratories of Kao Corporation. 2606 Akabane. Ichikai. Haga. Tochigi 321-34. Japan

Downloaded by [220.219.245.125] at 05:37 23 March 2015

Received November 7, 1988 Bacillus sp. KSM-635, which was isolated from soil, constitutively produced an alkaline carboxymethyl cellulase (CMCase) in quantity. Maximum cell growth was observed at an initial pH between 9 and 10, and no growth occurred below pH 8, thus illustrating the alkalophilic nature of this organism. Higher production of the CMCase required the presence of sugars (e.g., fructose, sucrose, maltose and mannitol) and complex nitrogen, with an initial pH of 8 - 9. The CMCase, partially purified by precipitation with ethanol, showed an optimum pH for activity of 9.5 and an optimum temperature of 40°C, in glycine-NaOH buffer. It showed strong activity toward CMC, but very little activity toward p-nitrophenyl-p-D-glucoside or cellulosic substrates showing high crystallinity. Activity toward p-nitrophenyl-p- D~cellobioside was detected at pH 7, but it amounted to less than 2 % of the maximum CMCase activity. Characteristically, the alkaline CMCase activity was not affected by various laundry components, such as surfactants, chelating agents and alkaline proteinases.

Enzymes have been added to laundry detergents for over 50 years to facilitate the release of proteinaceous materials in stains such as those of grime, blood and milk. 1 ) Over the past several years, proteolytic and amylolytic enzymes have frequently been used in laundry products, mostly because of the decreased use of phosphate in detergents and as a means of compensating for the poorer performance at lower washwater temperatures. 2 • 3 ) Recently, the use of cellulase in toileteries was found to be possible in our laboratories, particularly as an additive for improving the efficacy of laundry detergent products. However, the great majority of cellulases, so far reported, have pH optima in the acidic or neutral range,4) which is no good because of the alkalinity of detergent compositions. In addition, their activities were inhibited seriously by surfactants, chelating agents (buil-

ders) and proteinases under washing conditions. Therefore, we screened soil bacteria, and an isolate, alkalophilic Bacillus sp. KSM635, was found to produce an alkaline carboxymethyl cellulase (CMCase; endo-fJ-I,4glucanase, EC 3.2.1.4) that fulfills the essential requirements for use in laundry detergents. In this paper, we describe the isolation and identification of Bacillus sp. KSM-635, and some properties of the alkaline CMCase, named alkaline cellulase K, produced by it. Materials and Methods Isolation of CMCase-producing microorganisms. MPCMC-medium was used for isolating CMCaseproducing bacteria: CMC, 20 g; meat extract (LABLEMCO powder; Oxoid Co., Ltd.), 10 g; Bacto peptone, 10 g; NaC!, 10 g; KH 2 P04 , 1 g; Na 2 C0 3 (separately sterilized), 5 g; and I I of tap water. The MPCMC-medium was solidified by the addition of Bacto agar (1.5%, w/v), when

t To whom all correspondence should be addressed. Present addresses: * Research and Development Division of Kao Co., ** Kashima Plant of Kao Co., and *** Kashima Research Laboratories of Kao Co.

1276

S. ITO et al.


necessary. A soil sample (0.5 g) was suspended in 10 ml of saline and then treated at 80°C for 30 min. The thermally treated solution was suitably diluted and then spread on MPCMC-agar paltes, followed by incubation at 30°C for 3 days. Bacillus sp. KSM-635, thus obtained, was used in this study. The stock culture was maintained by periodical transfer on MPCMC-agar slants supplemented with 0.5% (wjv) Bacto yeast extract. Culture conditions. Bacillus sp. KSM-635 was grown on either NY-medium or MY-medium, containing 1.0% (wjv) carbon source. NY-medium consisted (in wjv %) of 0.8% nutrient broth, 0.1 % KH zP0 4 and 0.5% Na 2 C0 3 . MY-medium was composed (in wjv %) of 1.5% meat extract, 0.5% yeast extract, 0.1 % KH zP04 and 0.5% Na 2 C03 . The MY -medium was the result of optimization of the nutritional requirements for the production of alkaline cellulase K. The carbon source and Na 2 C0 3 were autoclaved separately and then added aseptically to the basal media. Fifty-ml portions of the complete medium were placed in 500-ml Sakaguchi flasks, followed by inoculation with the organism and shaking culture at 30°C for I to 33 days. After completion of the culture, the degree of bacterial growth and the CMCase activity were measured. Bacterial growth. The degree of growth was estimated by measuring the absorbance of the culture broth at 600nm (A6oo) with a Hitachi 220A spectrophotometer. Enzyme preparation. Bacillus sp. KSM-635 was cultivated at 30°C for 2 days with shaking in Sakaguchi flasks containing 50 ml of MY -medium, supplemented with 1% (wjv) fructose. Three parts of ethanol (-10°C) were gradually added to one part of the supernatant of the culture broth. The precipitate formed was collected by centrifugation (12,000 x.g), redissolved in a minimum volume of deionized water and then neturalized with dilute acetic acid. The solution was dialyzed overnight against deionized water at 5°C and the resultant dialyzate was used as the enzyme preparation, alkaline cellulase K. When necessary, the protein concentration in the dialyzate was standarized by ultrafiltration with an Amicon ultrafiltration cell containing a PM-IO Diaflo membrane (10,000M, cutoff). Enzyme assays. CMCase activity (CMC-saccharifying activity). The reaction mixture consisted of 0.9 ml of 1.1 % (wjv) CMC with a DS of 0.68 (equilibrated with an appropriate buffer) and 0.1 ml of the suitably diluted enzyme solution. After the mixture had been incubated at 40°C for an appropriate period, the reducing power was determined by the method of Summer and Somers S ) with 0.5% (wjv) 3,5-dinitrosalicylic acid. The activity was measured at pH 9.5 in 0.1 M glycine-NaOH buffer, unless otherwise specified.

Hydrolytic activities toward cellulosic substrates. The hydrolysis of Avicel, filter paper, cellulose powder, and NaOH- and H 3 P04 -swollen celluloses was determined at 30°C with shaking in 2 ml of 0.1 M glycine-NaOH buffer (pH 9.5). The swollen celluloses were prepared according to Tomita et al. 6 ) A piece of a filter paper (0.5 cm x 5 cm) or 50 mg of a cellulosic substrate was used as the substrate, and the reducing sugar liberated was estimated. Hydrolytic activities toward polysaccharides. The possible hydrolysis of xylan (from larch wood), pectin (from citrus fruit), inulin (from dahlia tubers), curdlan (from Alkaligenes faecalis var. myxogenes), laminarin (from Laminaria digitata), dextrin (from corn) and soluble starch (from potato) was examined. To 0.9ml of 1.1% (wjv) ofa substrate in 0.1 M glycine-NaOH buffer (pH 9.5) was added 0.1 ml of the enzyme solution. The reaction was carried out at 30°C for an appropriate period with shaking and then the reducing power was determined. p-Nitrophenyl-f3-D-glucoside (PN PG) and p-nitrophenylf3-D-cellobioside (PNPC)-hydrolyzing activities. The activity was measured at 30°C with 2mM of a substrate in 50 mM phosphate buffer, pH 7.0. The p-nitrophenol liberated was determined at 400 nm, using an E value of 15,000. One unit (U) of each enzyme activity was defined as the amount of protein which produced 1.0/lmol of product per min under the above standard assay conditions. Protein was determined by the dye-binding assay method 7 ) with bovine plasma albumin as a standard, using a BioRad protein assay kit.

G+ C content of DNA. The G+C content of DNA was estimated from the Tm value by the method of Marmur and Doty,8) using a Beckman DU-8B spectrophotometer. Electron microscopy. Scanning electron microscopy: Cells were grown on an NY -agar slant at 30°C for several days. The resultant cell paste was fixed with glutaraldehyde, postfixed with OS04 and then dehydrated in a graded ethanol series (from 50 to 100%).9) Specimens were critical-point dried, and then mounted on stubs and coated with gold for several min by vacuum evaporation. Specimens were examined under a JSM-35C electron microscope (JEOL Lt., Japan) operated at 15kY. Transmission electron microscopy: Cells were grown at 25°C for 18hr on MY-medium containing 1% (wjv) glucose. The washed cells were soaked in 10% (vjv) formaldehyde and then platinum-shadowed, according to the method of Kay.IO) Specimens were examined under a Hitachi H-300 electron microscope operated at 80 kV. Chemicals. CMC (AOIMC, DS=0.68) was obtained from Sanyo Kokusaku Pulp Ltd. (Tokyo, Japan). All other chemicals used were of the highest quality available commercially, most of which were described in a previous paper. l l )

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Alkaline Cellulase for Detergents


Results

Identification of the isolated strain Morphological and taxonomic characteristics of the isolated strain, KSM-635, were studied by the established methods of Gordon et al. 12 ) and Sneath.13) Unless stated otherwise, the media used for identification were supplemented with 0.5% (w/v) Na 2 C03 and 0.05% (w/v) yeast extract. The results obtained are summarized in Table I and the electron microscopic features are shown in Fig. 1. The isolate, Bacillus sp. KSM-635, was an aerobic, sporeforming (cylindrical, terminal endospores), Gram positive, motile, rod-shaped bacterium with peritrichous flagella. The G+C content of DNA was 39.5 mol%. The outstanding characteristic of this organisms was that it grew well in alkaline MY-medium at an initial pH between 9 and 10 (pH adjustTable I. Morphology Form Size (11m) Motility Gram staining Spores (11m)

PROPERTIES OF

Rods 0.5 -1.2 x 1.5 -4.0

+ Positive 0.7 -1.2 x 1.0 -2.0 Central to terminal

Acid-fastness Characteristics of cultures On nutrient agar plates: Circular colonies, flat surface, white or yellow semi-transparant gloss Gelatin Litmus milk

Not liquefied Not liquefied; color unchanged

In nutrient broth with 7% NaCl:

+

Biochemical properties Reduction nitrate to nitrite + Denitrification Color unchanged MR test VP test + Formation of indole Formation of hydrogen sulfide

ed with Na 2 C03 ), when I % (w/v) glucose was used as the carbon source. Below pH 8, no growth was observed on this medium. Biotin (or desthiobiotin) was essential for the growth of the isolate. Effects of carbon and nitrogen sources on CMCase production Media containing different carbon sources were examined in order to obtain the culture showing the highest alkaline cellulase K activity. The enzyme was produced almost constitutively, in quantity, on NY-medium (supplemented with 0.5% (w/v) Na 2 C03 , pH 9.3), in which no apparent production pattern, as to either the structure or composition of the sugar used, was observed (1.2 to 4.8 U/ml, Table II). Only xylose repressed the enzyme synthesis, the level of production being lower than those in cultures containing unutilizable Bacillus sp. KSM-635 Hydrolysis of starch Hydrolysis of casein Utilization of citrate Utilization of inorganic nitrogen Formation of pigment Urease Oxidase Catalase Temperature for growth pH for growth Behavior on oxygen Anaerobic growth Utilization of sugars L-Arabinose D-Xylose D-Ribose D-Glucose D-Fructose D-Mannose D-Galactose Sucrose Lactose Maltose Trehalose Raffinose D-Sorbitol D-Mannitol Inositol Glycerol

+ Unclear

+ 20-45°C 8-11 Aerobic

+ + + + + +

+ + + + + +

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S. ITo et at. Table II.

EFFECTS OF CARBON SOURCES ON ALKALINE CELLULASE K PRODUCTION

Bacillus sp. KSM -635 was cultivated at 30°C for 3 days in duplicate flasks, in which was placed 50ml of NYmedium containing 1% (w/w) of a carbon source. Assays were perfotmed at 40°C in glycine-NaOH buffer, pH 9.5.


Carbon source

None Cellulose powder Avicel CMC Cellobiose Xylose Ribose Glucose Fructose Sucrose Maltose Glycerol Mannitol •

Fig. 1. Electron Micrographs of the Isolate, Bacillus sp. KSM-635.

Experiment I

Experiment 2

Degree of CMCase growth produced (Vlml) (A600)

Degree of CMCase growth produced (Vlml) (A600)

3.3

0.8

4.0 Nm a

1.3 1.4

Nm 3.3 3.3 4.9 2.9 6.0 6.0 4.8 3.9 5.1 4.0

0.8 1.5 1.6 0.1 1.2 1.0 3.0 2.4 4.8 1.4 3.8

Nm 4.0 4.1 6.1 3.7 5.5 7.5 5.8 4.5 5.9 4.7

1.5 1.8 2.2 0.1 1.5 1.4 2.7 3.2 2.5 2.9 2.1

Not measurable because of the nature of the substrate.

A: Transmission electron micrograph of the isolate. Bar= I !lm. B: Scanning electron micrograph of the isolate. Bar= I !lm.

mic phase, then the enzyme level rose rapidly, maximum activity being observed after 40 to 45 hr, corresponding to the early stationary phase.

cellulosic substrates or no carbon source at all. No clear-cut pattern emerged, as far as the degree of growth was concerned, as regards all carbon sources which yielded the higher enzyme levels. Higher production of alkaline cellulase K required the presence of complex nitrogen, such as that in meat extract and yeast extract. When examined with the optimized MYmedium (supplemented with 0.5% Na 2 C03 , pH 9.0), fructose, mannitol and sucrose gave the highest yields after incubation at 30°C for 3 days (5 to 7 U jml), while ribose, glucose, glycerol and maltose gave less than half this activity. In all cases, the enzyme activity was not initially detected until after 20 hr, by which time growth had reached the middle logarith-

Effect of pH on the activity and stability The effect of pH on the CMCase activity of alkaline cellulase K was examined in buffers of various pHs. As shown in Fig. 2, the enzyme was active over a broad pH range, from 4 to 12, and most active at pH 9.5. Even at pH 11, more than 80% of the maximum activity was observed. The stability of alkaline cellulase· K was investigated at various pHs. After preincubation at 40°C for 10 min, the residual activity was determined at pH 9.5. The enzyme was not inactivated over the pH range of 4.5 to 10.5. Residual activity was minimal or nil after incubation at pH 13. When the enzyme was incubated for 30 min at 40°C, residual activity of more than 95% of the initial activity was


1279

various temperatures for 20 min (Fig. 3B). The activity was not abolished at all at 40°C, and about 50% of the original activity was retained after heat treatment at 65°C.

100

.... >-

+"'

.;::

Effects oj metal ions on the enzyme activity Individual metal ions and CMC were mixed 50 .;:: with an alkaline cellulase K solution, and then +"' 0 the CMCase activities were measured at pH c::. 9.5 and 40°C. Among the various metal ions tested, at 1 mM, Hg2 +, Cu2+ and Cd2+ ino hibited the activity slightly, whereas the other 3 5 7 9 11 13 divalent cations showed either no effect or a pH moderate level of stimulation. Potassium ferFig. 2. Effect of pH on the Activity of Alkaline Cellulase ricyanide, hydroxylamine, iodoacetate, pK. chloromercuribenzoate and N-ethylmaleimide, The following buffers were used: MacIlvaine, e; 50mM each added at 1 mM, did not have any lllsodium phosphate, A; 0.1 Mglycine--NaOH, .6,; and 0.1 M hibitory effect on the enzyme activity. KCl~NaOH, o. +"' U

0

Cl.l


Cl.l

A

B

100

100

B

B

~

~

~

'" E

E ~

u

c

50

50 "0 :!:!

'"

c

~

~

'"'" 10 20 30 40 50

60 20 30 40 50 60 70

80

Temperature (oel

Fig. 3. Effect of Temperature on the Activity (A) and Stability (8) of Alkaline Cellulase K.

obtained over the pH range of pH 7 to 10 (data not shown). Optimum temperature and the thermal stability The optimum temperature for the activity toward CMC was determined by varying the incubation temperature. As shown in Fig. 3A, alkaline cellulase K was active in a wide temperature range, 10 to 65°C, at pH 9.5. The optimum temperature was about 40°C, which is lower than those observed for other extracellular enzymes from Bacillus strains. The thermal stability of the enzyme was examined by heating it in glycine-NaOH buffer (pH 9.5) at

Effects oj chelating agents and surJactants on the enzyme activity The following chelating agents and surfactants used in laundry detergents were examined; sodium tripolyphosphate, zeolite 4A, sodium citrate, sodium ethylenediaminetetraacetate, sodium ethyleneglycol-bis-(f3aminoethyl ether) N,N,N',N'-tetraacetate, alkyl sulfate, a-olefin sulfonate, polyoxyethylene alkyl sulfonate, a-carboxymethyl alkylphosphate, linear-alkylbenzene sulfonate, polyoxyethylene alkyl ether, secondary alkyl sulfonate, sodium laurate and sodium dodecyl sulfate. The enzyme solution was mixed with an additive and then CMCase activity was measured at pH 9.5 and 40°C. Although linear-alkylbenzene sulfonate (0.05%, wjv) inhibited the enzyme activity by 25%, the enzyme was not affected by other chelating agents (1 mM) or surfactants (0.05%, wjv) examined. Effects oj laundry proteinases The activity of alkaline cellulase K was measured in the presence of commerciallaundry alkaline proteinases at pH 9.5 and 40°C. The amount of proteinase examined was 30,uU per ml, under the standard CMCase assay conditions. The CMCase activity was not af-

1280

S. Table III.

ITO

ENZYME ACTIVITIES OF ALKALINE CELLULASE K.

The enzyme assay conditions are given under Materials and Methods. Assays were performed at 30°C in glycineNaOH buffer, pH 9.5. Activity (Ux 103 /mg protein)


Substrate Avicel Filter paper Cellulose powder Acid-swollen cellulose Alkali-swollen cellulose CMC Xylan Pectin Inulin Curdlan Laminarin Dextran Soluble starch PNPG PNPC

55 45

o 80

o 16,300

o o o o o o o 0" 304"

" The activity was measured in 50 mM phosphate buffer (pH 7.0).

fected by such alkaline proteinases as the alkalase from Bacillus licheniformis, maxatase from B. subtilis, savinase from B. subtilis, esperase from B. licheniformis and protease API-21 from Bacillus sp. NKS-21. Substrate specificity Alkaline cellulase K showed strong CMCase activity, as shown in Table III. It hydrolyzed PNPC slightly, liberating p-nitrophenol. Cellulosic substrates showing high crystallinity and PNPG were hydrolyzed to lesser extents. The contaminant activities toward xylan, pectin, inulin, curdlan, laminarin, dextran and soluble starch were minimal or nil. Discussion

For several decades, studies on cellulases have involved searches for sources of the enzymes among fungi, mostly from the viewpoint of the utilization of biomass resources. Celluloytic enzymes produced by bacteria, however, have found no industrial or commercial

et al.

use as yet. During the course of studies on the application of enzymes for household products, we found that cellulases improve the detergency of laundry detergent products (M. Murata et al., unpublished data). However, most cellulases of microbial origin have pH optima in the acidic or neutral range, which is no good because of the alkalinity of detergent compositions. Alkaline cellulases found so far have been very few. For instance, Horikoshi et af.1 4 ) partially purified two alkaline CMCase components from alkalophilic Bacillus sp. No. N4, which resembles B. pasteurii. Fukumori et al. 15 ) characterized an inducible alkaline CMCase from alkalophilic Bacillus sp. no. 1139, which is similar to B. firmus. Recently, we reported an alkaline cellulase from "neutrophilic" Bacillus sp. KSM-522, which is similar to B. pumilus. l l ) In the present study, we described the isolation of alkalophilic Bacillus sp. KSM-635 which secretes a constitutive alkaline cellulase K that fulfills the essential requirements for use in laundry detergents. This isolate could also be classified as B. circulans or B. pumilus, although it differs in the pH optimum for growth and also in several taxonomic properties. We have succeeded in obtaining mutant strains which produce several hundred times more cellulase than the parental KSM-635 strain, and in integrating this enzyme into a heavy duty laundry powder. To our knowledge, this is the first case of large scale industrial application of a bacterial cellulase. The molecular weight of alkaline cellulase K varied, depending on the cultural conditions (approx. 100,000 to 300,000-600,000), asjudged from its behavior on gel chromatography. Such multiple forms of cellulases have also been reported for cultures of various microorganisms, including Trichoderma viride,16) Irpex lacteus,17) Myrothecium verrucaria,18) Cellvibrio gilvus,19) Pseudomonas fluorescens var. cellulosa20 ) and genus Ruminococcus. 21 ) In order to characterize the multiplicity of cellulases, we are currently in the process of purifying alkaline cellulase K and cloning the


gene encoding it into Escherichia coli HBlOl, using pBR322 as the vector plasmid. Acknowledgments. We wish to thank Dr. K. Horikoshi, the Institute of Physical and Chemical Research, for the invaluable discussions. We also thank Drs. M. Saitoh and F. Masuda for the continuous encouragement during the course of this study. The technical assistance of Mrs. Y. Akiyama, Mr. M. Takaiwa and Mr. H. Mori is also gratefully acknowledged.


References I) C. A. Starace, 1. Am. Oil Chem. Soc., 60,1025 (1983). 2) J. G. Moffett, Jr. and D. H. von Henning, Soap. Cosmetics Chem. Spec., 57, 29 (1981). 3) F. W. J. L. Maase and R. van Tilburg, J. Am. Oil Chem. Soc., 60, 1672 (1983). 4) M. Mandels and J. Weber, Adv. Chem. Ser., 95, 391 (1969). 5) J. R. Summer and G. F. Somers, "Laboratory Experiments in Biological Chemistry," Academic Press, New York, 1944. 6) Y. Tomita, H. Suzuki and K. Nisizawa, 1. Biochem., 78, 499 (1975). 7) M. Bradford, Anal. Biochem., 72, 248 (1976). 8) J. Marmur and P. Doty, J. Mol. Bioi., 5, 109 (1962). 9) S. Ito, T. Kobayashi, K. Ozaki, T. Morichi and M.

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Saitoh, Food Microstructure, 6, 17 (1987). 10) D. Kay, "Techniques for Electron Microscopy," Blackwell Scientific Publication Co., New York, 1964. II) S. Kawai, H. Okoshi, K. Ozaki, S. Shikata, K. Ara and S. Ito, Agric. Bioi. Chem., 52, 1425 (1988). 12) R. E. Gordon, W. C. Haynes and C. H. Pang, "The Genus Bacillus," United States Department of Agriculture, Washington, D. c., 1973. 13) P. H. A. Sneath, in "Bergey's Manual of Systematic Bacteriology," Vol. 2., ed. by P. H. A. Sneath, N. S. Main, M. E. Sharpe and J. G. Holt, The Williams and Wilkins Company, Baltimore 1986, pp. 1105 ~ 1139. 14) K. Horikoshi, M. Nakao, Y. Kurono and N. Sashihara, Can. J. Microbiol., 30, 774 (\984). 15) F. Fukumori, T. Kudo and K. Horikoshi, J. Gen. Microbiol., 131, 3339 (1985). 16) G. Okada, K. Nisizawa and H. Suzuki, J. Biochem., 63, 591 (1968). \7) T. Kanda, K. Wakabayashi and K. Nisizawa, J. Ferment. Technol., 48, 607 (1970). 18) D. R. Whitaker, K. R. Hanson and P. K. Datta, Can. J. Biochem. Physiol., 41, 671 (1963). 19) W. O. Storvick, F. E. Cole and K. W. King, Biochemistry, 2, 1106 (1963). 20) T. Yoshikawa, H. Suzuki and K. Nisizawa, J. Biochem., 75, 531 (1974). 21) R. M. Gardner, K. C. Doerner and B. A. White, J. Bacterio!., 169,4581 (1987).