World Journal of Microbiology & Biotechnology
10, 551-555
Purification and properties of mannanase from Bacillus subtilis N.S. M e n d o z a ,
M. A r a i , * T. K a w a g u c h i ,
T. Y o s h i d a
and L.M. Joson
Extracellular mannanase from Bacillus subtilis NM-39, an isolate from Philippine soil, was purified about 240-fold with a yield of 7.3% by ammonium sulphate fractionation, DEAE-Toyopearl chromatography and Sephacryl S-200 gel filtration. Its Mr was 38 kDa and it had a pI of 4.8 and optimum activity at pH 5.0 and 55°C. It was stable at pH 4 to 9 and below 55°C. The amino acid composition of the enzyme was in the order Gly > Glx > Ser and Asx > Ala. Key words: Bacillus subtilis, mannan, fl-mannanase.
fl-D-Mannanases hydrolyse the (1--*4) -fl-D-mannopyranosyl linkages of (1--*4)-fl-D-mannans, namely mannan, galactomannan, glucomannan and galactoglucomannan. Hetero1,4-fl-mannan and hereto-I, 4-fl-xylan are the major constituents of hemicellulose, the second most abundant and renewable organic compound in nature. Many studies on the mannanases from bacteria, fungi and plant have been reported (Dekker & Richards I976) but not much information is available about their industrial applications. Enzymes with endo-fl-D-mannanase activity can be used for the treatment of high-protein by-products such as guar meal and copra meal, the high crude-fibre contents of which limit their digestibilities by man and non-ruminant animals (McCleary et al. I985). Possible applications of fl-mannases are in pulp biobleaching, clarification of fruit juices and processing of coffee wastes (Clark et al. 1990; Dekker 1979). in the Philippines, coconut is one of the biggest agricultural crops, not only for domestic use but also for export. The edible part of the coconut is a rich source of highly concentrated mannan, the carbohydrates of which comprise 89.4% mannose, 6.2% galactose, 2.6% arabinose and 1.8% glucose (Kusakabe et al. 1986). The large amounts of
coconut residues discharged by coconut oil industries can not be fully utilized for animal feed and/or human food because of their high fibre content. They must first be hydrolysed into monomers by acid or enzymatic methods. The enzymatic processes are more desirable because the mild conditions needed for the reactions lead to waste products that are more friendly to the environment than those from acid treatments. A soil bacterium producing extracellular mannanase was isolated from the soil in a coconut field in the Philippines by an enrichment technique using 1% coconut meat residue as carbon source. The bacterium was identified as Bacillus subtilis (Mendoza et al. 1994). The purification and some properties of the mannanase from this strain are the subjects of the present study.
Medium and Cultivation
N.S. Mendoza and L.M. Joson are with Industrial Technology Development Institute, Department of Science and Technology, Manila, Philippines. M. Arai and T. Kawaguchi are with Department of Agricultural Chemistry, College of Agriculture, University of Osaka Prefecture, Sakai, Osaka 593, Japan; fax: 72252 0341.T. Yoshidais with Faculty of Engineering, Osaka University, Suita, Osaka 565,Japan."Corresponding author.
The medium for mannanase production contained (g/f): locust bean galactomannan, 10; (NH,)zSO4, 0.2; FeSO4.7H~O, 0.01; CaClz, 0.05; K2HPO4, 7.54; KH2PO4, 2.32; and meat extract, 10; at pH 7.0. An overnight seed culture (1 d) was inoculated into I7 d, of the medium in a jar fermenter and incubated for 24 h with appropriate agitation and aeration. The culture fluid was then collected by centrifugation (I5,000 x g for I5 min at 4°C) and used for mannanase purification.
Materials and Methods Microorganism Bacillus subtilis NM-39 was maintained on an agar slant containing
the same mineral salts as used by Jones & Ballou (1909) and Pavlova & Tin'yanova (1981).
(~ 1994 Rapid Communications of Oxford Ltd
WorldJournalof Microbiology& Biotechnology,Vol 1O, 1994
5 $1
N,5. Mendoza et al. 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 35
/L
80
100 8O
_A
s
6O
/ _o------ /
=
i
i
,
40
45
50
55
40
2
2O '
,m
O
0 60
2O . Metal ion (at I mM)
Relative activity
(%) None Ca 2+
100" 100
Mg2 ÷ C& + Zn z+ Cu =+ Mn z+ Fe =+ Hg 2+ Ag + Ni 2+ Fe 3+ Cd z+ Sr 2+ Ba 2+ Pb 2+
100 100 100 100 88 100 22 77 94 95 100 100 100 100
> 4.i 0 ¢o
80
>
40
Amino acid
Asx* Thr Ser Glx l Gly Ala 1/2Cys Val Met lie Leu Tyr Phe Lys His Trp Arg Pro * Asp + A s n . 1"Glu + Gin.
Content (mol%) 9.00 4.75 14.45 14.31 14.74 7.83 0.79 5.11 1.66 3.11 4.85 3.01 2.45 3.21 2.34 2.07 1.42 4.90
b
i 8
I I I I ! I 30 40 ,50 60 70 80
60
n-
i 4
0
i 6
A
d
C v 100 >, ,1.8
20 0
Table 3. Amino acid composition of the mannanase from Bacillus subtilis NM-39.
a
20
•
* 100% = 0.015u
100
i
i
i
i
i
i
i
i
4
6
8
10
30
40
50
60
pH
Temperature
(°C)
Figure 4. Enzyme properties of the purified mannanase. (a) Effect of pH on mannanase activity. Enzyme solution was incubated with 0.5% substrate in various buffer solutions at 37°C for 10 min. (b) Effect of temperature on mannanase activity. Enzyme solution was incubated with 0.5% substrate in phosphate buffer, pH 6, at each temperature for 10 min. (c) Effect of pH on the stability of mannanase. Enzyme solution was incubated at various pH values at 37°C for 30 min and remaining activity was measured at pH 5. (d) Effect of temperature on the stability of mannanase. Enzyme solution was incubated in phosphate buffer, pH 8, at various temperatures for 10 min and the remaining activity was measured.
dures. The mannanase was purified 240-fold, with a final yield of 7.3% of the activity in the culture supematant. A typical elution pattern from the Sephacryl column is shown in Figure 1. Homogeneity of the Enzyme
The purified enzyme appeared as a single band of protein in SDS-PAGE (Figure 2) and isoelectrofocusing indicated a
WorldJournalof Microbiology& Biotechnology,Vol 10, 1994
553
N.S. Mendoza et al. pI of 4.8 (Figure 3). The M r was calculated as 38 kDa from the SDS-PAGE results.
Enzyme Propeffies The purified mannanase had optimal activity at pH 5 (Figure 4a) and was stable from pH 4 to 9 (Figure 4c). Maximum mannanase activity was at 55°C and the enzyme was stable below 55°C (Figure 4b and 4d). The mannanase was strongly inhibited by Hg + and slightly by Ag + (Table 2).
NM-39 enzyme. The fl-mannanase from an anaerobic extreme thermophile, Caldocellum saccharolyticum, has also been characterized (Gibbs et. al. 1992). Mendoza et al. (1994) showed that the mannanase of Bacillus sp. NM-39 exhibited high saccharifying activity on mannan from coconut residue, with mannose as the major product of hydrolysis. Coconut residues and copra meal, therefore, constitute attractive substrates for single-cell protein or alcohol production.
References Amino Acid Analysis The amino acid composition (% mol) of the mannanase from B. subtilis NM-39 was calculated from the mean contents of amino acids other than threonine, tyrosine and serine after 24, 48 and 72 h hydrolysis. The threonine, tyrosine and serine contents were obtained by extrapolating to zero-time hydrolysis. As shown in Table 3, the enzyme contained glycine, glutamic acid, serine, aspartic acid and alanine in considerable amounts. The N-terminal amino acid sequence of the enzyme was determined as X-Thr-ValTyr-Pro-Val-Asn-Pro-Asn-Ala-Gln-Gln-Thr-Thr-Lys-AspIle-Met-Asn-Trp-Leu-Ala-His-Leu-Pro-Asn-Arg-Ser-GluAsn-Arg-Val-Met-Ser-Gly-Ala-Phe-Gly-Gly-Tyr-Ser-AspVal-Thr-Phe-, where X is an undefined residue.
Discussion Bacteria belonging to the genus Bacillus are prolific and versatile organisms capable of producing several metabolites and enzymes, including hemicellulolytic enzymes (Dekker & Richards 1976). A fl-mannanase system from a Streptomyces sp. was found to hydrolyse mannan from copra, the dried kernel of coconut meat (Takahashi et al. 1983). Bacillus subtilis NM-39 is also capable of utilizing the same mannan (Mendoza et al. 1994). Emi et al. (1972) first studied and crystallized a mannanase from a B. subtilis strain, initially isolated as an arabino-galactanase-producing bacterium. However, the enzyme differs in many aspects from that of B. subtilis NM-39, including being smaller (22 versus 38 kDa) and having different pH and temperature optima. The mannanase of NM-39 has high contents of glycine, glutamic acid, serine and aspartic acid. Interestingly, the same amino acids were also found to be present in large amounts in the mannanases of Aspergillus niger (Eriksson & Winell 1968) and Streptomyces sp. (Takahashi et al. 1984). The mannanase of another B. subtilis strain was characterized by McClearly (1979); although the molecular weight of this enzyme is similar to that o f NM-39, the pI and enzymatic properties are slightly different. Three mannanase components have been purified from an alkaliphilic Bacillus sp. by Akino et al. (1988) but the properties of each component were significantly different from those of the
554
Worldlournalof Microbiology& Biotechnology,Vo110,1994
Akino, T., Nakamura, N. & Horikoshi, K. 1988 Characterization of fl-mannanase of an alkalophilic Bacillus sp. Agricultural and Biological Chemistry 52, 1459-1464. Clark, T.A., McDonald, A.G., Senior, D.J. & Mayer, P.R. 1990 Mannanase and xylanase treatments of chemical pulp: effects of pulp properties and bleach ability. In Biotechnology in Pulp and Paper Manufacture, eds Kirk, K. & Chang, H.H.M. pp. 153167. Boston: Butterworth-Heinemann. Davis, B.J. 1964 Disc electrophoresis II. Method and application to human serum proteins. Annals of the New York Academy of Science 120, 404-427. Dekker, R.F.H. 1979 The hemicellulase group of enzymes. In Polysaccharides in Food, eds Blanchard, J.M.V. & Mitchell, J.R., pp. 93-108. London: Butterworth. Dekker, R.F.H. & Richards, G.N. 1976 Hemicellulases: their occurrence, purification, properties, and mode of action. Advances in Carbohydrate Chemistry and Biochemistry 32, 277-352. Emi, S., Fukumoto, J. & Yamamoto, T. 1972 Crystallization and some properties of mannanase. Agricultural and Biological Chemistry 36, 991-1001. Eriksson, K.E. & Winell, M. 1968 Purification and characterization of a funga] fl-mannanase. Acta Chemica Scandinavica 22, 19241934. Gibbs, M.D., Saul, D.J., L6thi, E. & Bergquist, P.L. 1992 The flmannanase from Caldocellum saccharolyticum is part of multidomain enzyme. Applied and Environmental Microbiology 58, 3864-3867. Jones, G.H. & Ballou, C.E. 1969 Studies on the structure of yeast mannan. I. Purification and some properties of an ~-mannosidase from an Arthrobacter. Journal of Biological Chemistry 244, 10431051. Kusakabe, I., Zamora, A.F., Kusama, S., Fernandez, W.L. & Murakami, K. 1986 Studies on the mannanase of Streptomyces. Japanese Journal of Tropical Agriculture 30, 264-271. McCleary, B.V. 1979 Modes of action of fl-mannanase enzymes of diverse origin on legume seed galactomannan. Phytochemistry, 18, 757-763. McCleary, B.V., Clark, A.H., Dea, I.C.M. & Rees, D.A. I985 The fine structure of carob and guar galactomannans. Carbohydrate Research 139, 237-260. Mendoza, N.S., Arai, M., Kawaguchi, T., Cubol, F.S., Panerio, E.G., Yoshida, T. & Joson, L.M. 1994 Isolation of mannanutilizing bacteria and the culture conditions for mannanase production. World Journal of Microbiology and Biotechnology 10, 51-54. Pavlova, I.N. & Tin'yanova, N.Z. 1981 Isolation and characterization of microorganisms with mannanase activity. Applied Biochemistry and Microbiology 16, 427-440. Somogyi, N. 1952 Notes on sugar determination. Journal of Biological Chemistry 195, 19-23. Takahashi, R., Kusakabe, I., Kobayashi, H., Murakami, K., Maekawa,
Mannanase from Bacillus subtilis A. & Suzuki, T. 1984 Purification and some properties of mannanase from Streptomycessp. Agriculturaland BiologicalChemistry 48, 2189-2195. Takahashi, R., Kusakabe, I., Maekawa, A., Suzuki, T. & Murakami, K. 1983 Studies on mannanase of ActinomycetesI. Some properties of extra-cellular mannanase. Japanese Journal of Tropical Agriculture 27, 140--148. Vesterberg, O. & Svesson, H. 1966 Isoelectric fractionation, analy-
sis, and characterization of ampholytes in natural pH gradients. IV. Resolving power connection with separation of myoglobins. Acta ChemicaScandinavica20, 820-834. Weber, K. & Osbom, M. 1969 The reliability of molecular weight determination by dodecyl sulfate polyacrylamide gel electrophoresis. Journalof BiologicalChemish3y244, 4406--4412.
(Received in revisedform 29 April 1994; accepted 3 May 1994)
World ]ournal of Microbiology & Biotechnology, Vol W, 1994
555
10, 551-555
Purification and properties of mannanase from Bacillus subtilis N.S. M e n d o z a ,
M. A r a i , * T. K a w a g u c h i ,
T. Y o s h i d a
and L.M. Joson
Extracellular mannanase from Bacillus subtilis NM-39, an isolate from Philippine soil, was purified about 240-fold with a yield of 7.3% by ammonium sulphate fractionation, DEAE-Toyopearl chromatography and Sephacryl S-200 gel filtration. Its Mr was 38 kDa and it had a pI of 4.8 and optimum activity at pH 5.0 and 55°C. It was stable at pH 4 to 9 and below 55°C. The amino acid composition of the enzyme was in the order Gly > Glx > Ser and Asx > Ala. Key words: Bacillus subtilis, mannan, fl-mannanase.
fl-D-Mannanases hydrolyse the (1--*4) -fl-D-mannopyranosyl linkages of (1--*4)-fl-D-mannans, namely mannan, galactomannan, glucomannan and galactoglucomannan. Hetero1,4-fl-mannan and hereto-I, 4-fl-xylan are the major constituents of hemicellulose, the second most abundant and renewable organic compound in nature. Many studies on the mannanases from bacteria, fungi and plant have been reported (Dekker & Richards I976) but not much information is available about their industrial applications. Enzymes with endo-fl-D-mannanase activity can be used for the treatment of high-protein by-products such as guar meal and copra meal, the high crude-fibre contents of which limit their digestibilities by man and non-ruminant animals (McCleary et al. I985). Possible applications of fl-mannases are in pulp biobleaching, clarification of fruit juices and processing of coffee wastes (Clark et al. 1990; Dekker 1979). in the Philippines, coconut is one of the biggest agricultural crops, not only for domestic use but also for export. The edible part of the coconut is a rich source of highly concentrated mannan, the carbohydrates of which comprise 89.4% mannose, 6.2% galactose, 2.6% arabinose and 1.8% glucose (Kusakabe et al. 1986). The large amounts of
coconut residues discharged by coconut oil industries can not be fully utilized for animal feed and/or human food because of their high fibre content. They must first be hydrolysed into monomers by acid or enzymatic methods. The enzymatic processes are more desirable because the mild conditions needed for the reactions lead to waste products that are more friendly to the environment than those from acid treatments. A soil bacterium producing extracellular mannanase was isolated from the soil in a coconut field in the Philippines by an enrichment technique using 1% coconut meat residue as carbon source. The bacterium was identified as Bacillus subtilis (Mendoza et al. 1994). The purification and some properties of the mannanase from this strain are the subjects of the present study.
Medium and Cultivation
N.S. Mendoza and L.M. Joson are with Industrial Technology Development Institute, Department of Science and Technology, Manila, Philippines. M. Arai and T. Kawaguchi are with Department of Agricultural Chemistry, College of Agriculture, University of Osaka Prefecture, Sakai, Osaka 593, Japan; fax: 72252 0341.T. Yoshidais with Faculty of Engineering, Osaka University, Suita, Osaka 565,Japan."Corresponding author.
The medium for mannanase production contained (g/f): locust bean galactomannan, 10; (NH,)zSO4, 0.2; FeSO4.7H~O, 0.01; CaClz, 0.05; K2HPO4, 7.54; KH2PO4, 2.32; and meat extract, 10; at pH 7.0. An overnight seed culture (1 d) was inoculated into I7 d, of the medium in a jar fermenter and incubated for 24 h with appropriate agitation and aeration. The culture fluid was then collected by centrifugation (I5,000 x g for I5 min at 4°C) and used for mannanase purification.
Materials and Methods Microorganism Bacillus subtilis NM-39 was maintained on an agar slant containing
the same mineral salts as used by Jones & Ballou (1909) and Pavlova & Tin'yanova (1981).
(~ 1994 Rapid Communications of Oxford Ltd
WorldJournalof Microbiology& Biotechnology,Vol 1O, 1994
5 $1
N,5. Mendoza et al. 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 35
/L
80
100 8O
_A
s
6O
/ _o------ /
=
i
i
,
40
45
50
55
40
2
2O '
,m
O
0 60
2O . Metal ion (at I mM)
Relative activity
(%) None Ca 2+
100" 100
Mg2 ÷ C& + Zn z+ Cu =+ Mn z+ Fe =+ Hg 2+ Ag + Ni 2+ Fe 3+ Cd z+ Sr 2+ Ba 2+ Pb 2+
100 100 100 100 88 100 22 77 94 95 100 100 100 100
> 4.i 0 ¢o
80
>
40
Amino acid
Asx* Thr Ser Glx l Gly Ala 1/2Cys Val Met lie Leu Tyr Phe Lys His Trp Arg Pro * Asp + A s n . 1"Glu + Gin.
Content (mol%) 9.00 4.75 14.45 14.31 14.74 7.83 0.79 5.11 1.66 3.11 4.85 3.01 2.45 3.21 2.34 2.07 1.42 4.90
b
i 8
I I I I ! I 30 40 ,50 60 70 80
60
n-
i 4
0
i 6
A
d
C v 100 >, ,1.8
20 0
Table 3. Amino acid composition of the mannanase from Bacillus subtilis NM-39.
a
20
•
* 100% = 0.015u
100
i
i
i
i
i
i
i
i
4
6
8
10
30
40
50
60
pH
Temperature
(°C)
Figure 4. Enzyme properties of the purified mannanase. (a) Effect of pH on mannanase activity. Enzyme solution was incubated with 0.5% substrate in various buffer solutions at 37°C for 10 min. (b) Effect of temperature on mannanase activity. Enzyme solution was incubated with 0.5% substrate in phosphate buffer, pH 6, at each temperature for 10 min. (c) Effect of pH on the stability of mannanase. Enzyme solution was incubated at various pH values at 37°C for 30 min and remaining activity was measured at pH 5. (d) Effect of temperature on the stability of mannanase. Enzyme solution was incubated in phosphate buffer, pH 8, at various temperatures for 10 min and the remaining activity was measured.
dures. The mannanase was purified 240-fold, with a final yield of 7.3% of the activity in the culture supematant. A typical elution pattern from the Sephacryl column is shown in Figure 1. Homogeneity of the Enzyme
The purified enzyme appeared as a single band of protein in SDS-PAGE (Figure 2) and isoelectrofocusing indicated a
WorldJournalof Microbiology& Biotechnology,Vol 10, 1994
553
N.S. Mendoza et al. pI of 4.8 (Figure 3). The M r was calculated as 38 kDa from the SDS-PAGE results.
Enzyme Propeffies The purified mannanase had optimal activity at pH 5 (Figure 4a) and was stable from pH 4 to 9 (Figure 4c). Maximum mannanase activity was at 55°C and the enzyme was stable below 55°C (Figure 4b and 4d). The mannanase was strongly inhibited by Hg + and slightly by Ag + (Table 2).
NM-39 enzyme. The fl-mannanase from an anaerobic extreme thermophile, Caldocellum saccharolyticum, has also been characterized (Gibbs et. al. 1992). Mendoza et al. (1994) showed that the mannanase of Bacillus sp. NM-39 exhibited high saccharifying activity on mannan from coconut residue, with mannose as the major product of hydrolysis. Coconut residues and copra meal, therefore, constitute attractive substrates for single-cell protein or alcohol production.
References Amino Acid Analysis The amino acid composition (% mol) of the mannanase from B. subtilis NM-39 was calculated from the mean contents of amino acids other than threonine, tyrosine and serine after 24, 48 and 72 h hydrolysis. The threonine, tyrosine and serine contents were obtained by extrapolating to zero-time hydrolysis. As shown in Table 3, the enzyme contained glycine, glutamic acid, serine, aspartic acid and alanine in considerable amounts. The N-terminal amino acid sequence of the enzyme was determined as X-Thr-ValTyr-Pro-Val-Asn-Pro-Asn-Ala-Gln-Gln-Thr-Thr-Lys-AspIle-Met-Asn-Trp-Leu-Ala-His-Leu-Pro-Asn-Arg-Ser-GluAsn-Arg-Val-Met-Ser-Gly-Ala-Phe-Gly-Gly-Tyr-Ser-AspVal-Thr-Phe-, where X is an undefined residue.
Discussion Bacteria belonging to the genus Bacillus are prolific and versatile organisms capable of producing several metabolites and enzymes, including hemicellulolytic enzymes (Dekker & Richards 1976). A fl-mannanase system from a Streptomyces sp. was found to hydrolyse mannan from copra, the dried kernel of coconut meat (Takahashi et al. 1983). Bacillus subtilis NM-39 is also capable of utilizing the same mannan (Mendoza et al. 1994). Emi et al. (1972) first studied and crystallized a mannanase from a B. subtilis strain, initially isolated as an arabino-galactanase-producing bacterium. However, the enzyme differs in many aspects from that of B. subtilis NM-39, including being smaller (22 versus 38 kDa) and having different pH and temperature optima. The mannanase of NM-39 has high contents of glycine, glutamic acid, serine and aspartic acid. Interestingly, the same amino acids were also found to be present in large amounts in the mannanases of Aspergillus niger (Eriksson & Winell 1968) and Streptomyces sp. (Takahashi et al. 1984). The mannanase of another B. subtilis strain was characterized by McClearly (1979); although the molecular weight of this enzyme is similar to that o f NM-39, the pI and enzymatic properties are slightly different. Three mannanase components have been purified from an alkaliphilic Bacillus sp. by Akino et al. (1988) but the properties of each component were significantly different from those of the
554
Worldlournalof Microbiology& Biotechnology,Vo110,1994
Akino, T., Nakamura, N. & Horikoshi, K. 1988 Characterization of fl-mannanase of an alkalophilic Bacillus sp. Agricultural and Biological Chemistry 52, 1459-1464. Clark, T.A., McDonald, A.G., Senior, D.J. & Mayer, P.R. 1990 Mannanase and xylanase treatments of chemical pulp: effects of pulp properties and bleach ability. In Biotechnology in Pulp and Paper Manufacture, eds Kirk, K. & Chang, H.H.M. pp. 153167. Boston: Butterworth-Heinemann. Davis, B.J. 1964 Disc electrophoresis II. Method and application to human serum proteins. Annals of the New York Academy of Science 120, 404-427. Dekker, R.F.H. 1979 The hemicellulase group of enzymes. In Polysaccharides in Food, eds Blanchard, J.M.V. & Mitchell, J.R., pp. 93-108. London: Butterworth. Dekker, R.F.H. & Richards, G.N. 1976 Hemicellulases: their occurrence, purification, properties, and mode of action. Advances in Carbohydrate Chemistry and Biochemistry 32, 277-352. Emi, S., Fukumoto, J. & Yamamoto, T. 1972 Crystallization and some properties of mannanase. Agricultural and Biological Chemistry 36, 991-1001. Eriksson, K.E. & Winell, M. 1968 Purification and characterization of a funga] fl-mannanase. Acta Chemica Scandinavica 22, 19241934. Gibbs, M.D., Saul, D.J., L6thi, E. & Bergquist, P.L. 1992 The flmannanase from Caldocellum saccharolyticum is part of multidomain enzyme. Applied and Environmental Microbiology 58, 3864-3867. Jones, G.H. & Ballou, C.E. 1969 Studies on the structure of yeast mannan. I. Purification and some properties of an ~-mannosidase from an Arthrobacter. Journal of Biological Chemistry 244, 10431051. Kusakabe, I., Zamora, A.F., Kusama, S., Fernandez, W.L. & Murakami, K. 1986 Studies on the mannanase of Streptomyces. Japanese Journal of Tropical Agriculture 30, 264-271. McCleary, B.V. 1979 Modes of action of fl-mannanase enzymes of diverse origin on legume seed galactomannan. Phytochemistry, 18, 757-763. McCleary, B.V., Clark, A.H., Dea, I.C.M. & Rees, D.A. I985 The fine structure of carob and guar galactomannans. Carbohydrate Research 139, 237-260. Mendoza, N.S., Arai, M., Kawaguchi, T., Cubol, F.S., Panerio, E.G., Yoshida, T. & Joson, L.M. 1994 Isolation of mannanutilizing bacteria and the culture conditions for mannanase production. World Journal of Microbiology and Biotechnology 10, 51-54. Pavlova, I.N. & Tin'yanova, N.Z. 1981 Isolation and characterization of microorganisms with mannanase activity. Applied Biochemistry and Microbiology 16, 427-440. Somogyi, N. 1952 Notes on sugar determination. Journal of Biological Chemistry 195, 19-23. Takahashi, R., Kusakabe, I., Kobayashi, H., Murakami, K., Maekawa,
Mannanase from Bacillus subtilis A. & Suzuki, T. 1984 Purification and some properties of mannanase from Streptomycessp. Agriculturaland BiologicalChemistry 48, 2189-2195. Takahashi, R., Kusakabe, I., Maekawa, A., Suzuki, T. & Murakami, K. 1983 Studies on mannanase of ActinomycetesI. Some properties of extra-cellular mannanase. Japanese Journal of Tropical Agriculture 27, 140--148. Vesterberg, O. & Svesson, H. 1966 Isoelectric fractionation, analy-
sis, and characterization of ampholytes in natural pH gradients. IV. Resolving power connection with separation of myoglobins. Acta ChemicaScandinavica20, 820-834. Weber, K. & Osbom, M. 1969 The reliability of molecular weight determination by dodecyl sulfate polyacrylamide gel electrophoresis. Journalof BiologicalChemish3y244, 4406--4412.
(Received in revisedform 29 April 1994; accepted 3 May 1994)
World ]ournal of Microbiology & Biotechnology, Vol W, 1994
555
