Chapter 5
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Mannan-Degrading Enzymes Produced by Bacillus Species AM-001 Koki Horikoshi The Riken Institute, Wako-shi, Saitama 351-01, Japan
An alkaliphilic Bacillus sp. AM-001 producing high amounts of βmannan-degrading enzymes was isolated. Three mannanases were purified from the culture broth and characterized. A mannanase gene was cloned into E. coli and sequenced. Two mannanases having different C-terminal fragments were expressed from this gene in E. coli carrying pMAH3. Beta-mannosidase and P-mannanase cleave the P-mannoside linkages in P-l,4-Dmannans to yield D-mannose and manno-oligosaccharides, respectively. Bacterial Pmannanases have been found in Bacillus (1), Aeromonas (2), in Streptomyces (3). Fungal P-mannanase have been reported by Hashimoto and Fukumoto (4), Eriksson and Winnel (5) and Civas etal (6). Aspergillus niger (7,8) and Aeromonas sp. (9) are known to produce P-mannosidase. Mannans have been found in some kinds of plants such as endosperms of copra and ivory palm nuts, guar beans, locust beans, coffee beans and roots of konjak (Amorphophallus konjac). Most of these saccharides are used only in the food and feed processing industries. Recently, it has been reported that manno-oligosaccharides are useful as one of the best growth factors for Bifidobacterium sp. and Lactobacillus sp. Although much works has been presented on the characterization of microbial Pmannanases, little information is available about the industrial application of these enzymes. There is no information so far available about mannan degrading enzymes of alkaliphiles. In this paper, a P-mannan degrading alkaliphile (Bacillus sp. AM-001) with high productivities of cell-associated P-mannosidase and three extracellular Pmannanases was isolated from soil. These three (3-mannanases differed in several enzymatic properties including optimum pH, optimum temperature, pH stability, isoelectric point and molecular weight. To elucidate the genetic basis for production of multiple forms of P-mannanase by the strain, we cloned one P-mannanase gene of this strain into Escherichia coli using pUC19 as a vector, and characterized two different P-mannanases expressed from this gene in E. coli. 0097-6156/91/0460-0052$06.00/0 © 1991 American Chemical Society
Leatham and Himmel; Enzymes in Biomass Conversion ACS Symposium Series; American Chemical Society: Washington, DC, 1991.
5.
HORIKOSHI
Mannan-Degrading Enzymes
53
Isolation of Mannan Degrading Microorganisms (10) A small amount of soil was spread on agar plates containing 1% (3-mannan from larchwood, 1% polypeptone, 0.2% yeast extract, 0.1% K H P 0 , 0.02% MgS0 7H20 and 0.5% sodium carbonate. The plates were incubated at 37°C for 4872 h. Strain AM-001 with a large clear zone around the colony was selected as the enzyme producer. The isolate grew at temperatures from 20°C to 45°C, with an optimum at 37°C in the medium described above. The pH range for growth was from pH 7.5 to 11.5 with an optimum at pH 8.5 to 9.5. The bacterium was aerobic, motile and gram-variable; the rod-shaped cells (0.6-0.8 um x 3.0-6.0 um) had peritrichous flagella and terminal swollen sporangia containing oval spores (1-1.2 um x 1.5-2.0 um). The taxonomical characteristics of this alkaliphilic Bacillus strain were almost the same as those of Bacillus circulans except for the pH range for growth. 2
4
4
Culture Conditions for Enzyme Production Bacillus sp. AM-001 was cultivated aerobically under various conditions, and activities of P-mannanase in the culture broth and P-mannosidase extracted from the cells treated with 0.1% Triton X-100 were monitored. Both enzymes were formed when the bacterium was grown under alkaline conditions in the presence of optimal concentrations of 0.5% Na C0 or 0.5-1.0% NaHC0 . Various carbohydrates were also tested and the best carbohydrate for enzyme production was konjak powder (1% w/V). The optimal cultivation temperature for enzyme production was 31 °C for Pmannosidase and 37°C for P-mannanase in a production medium composed of 1% konjak powder, 0.2% yeast extract, 2% Polypeptone, 0.1% K H P 0 , 0.02% MgS047H 0 and 0.5% sodium carbonate. The crude enzyme preparation showed optimum activity of P-mannosidase at pH 7.0 and 55°C and of P-mannanase at pH 9.0 and65°C. 2
3
3
2
4
2
Purification of P-mannanase and Its Properties(l 1) Three extracellular P-mannanases (M-1, M i l and M-III) were purified by ammonium sulfate precipitation (80% saturation) followed by chromatography on a DEAEToyopearl 650 M column (4.6 x 35 cm) equilibrated and eluted with 0.01 M phosphate buffer (pH 7.0 ) and by a hydroxylapatite column (1.6 x 25 cm). As shown in Fig. 1, two active fractions (Fraction 1 and 2) were detected after hydroxylapatite chromatography. Each fraction 1 and 2 was applied onto a Sephacryl S-200 column (2.6 x 90 cm) equilibrated with 0.01 M phosphate buffer (pH 7.0) containing 0.1M NaCl and eluted with the same buffer. Mannanase-I and -II were isolated from fraction 1 and mannanase-III was from fraction 2. Polyacrylamide gel electrophoresis revealed that these three mannanases were electrophoretically homogeneous. The molecular weights estimated by SDS-PAGE were 58,500 for M-I, 59,500 for M-II and 42,000 for M-III. As shown in Fig. 2, pmannanase M-I and M-II were most active at pH 9.0 and M-III demonstrated optimal enzyme action at pH 8.5. Each of these enzymes hydrolyzed 0-1,4mannooligosaccharides larger than mannotriose and the major components in the digest were di-, tri- and tetra-saccharides.
Leatham and Himmel; Enzymes in Biomass Conversion ACS Symposium Series; American Chemical Society: Washington, DC, 1991.
54
ENZYMES IN BIOMASS CONVERSION
2000 ~ ID
M000
0
o CO — - - — — — +
- +
3.1
pMAH 5 -+
- +
4.7
1 — — 4
Bgl Hindlll .Pstl L H pMAH 1 rev
Xbal
H i n d
|„
^ ^ ^ ^ H H L H H M H ^ -
0.4
Fig. 3. Restriction Maps of pMAHl and Its Derivatives. Deleted areas are indicated with dotted lines.
Xbal -219
TCTAGACTCCAAAGGTTACTATCAACCTGTCTATTTATT
TAACTGTACAGTAGATGGGGTAGAATCAAACCATCATCATCCCTGCCATCTAAATTCATTATATGAACTCCTCAATAGAGAACAACAAAT CATAATCCAACCATATTTTTCTAATCAATCACTATGTTAAGATAAAAAATGTAATCGCTTACAATTAAAAGGATAGAGGAGGATTATGTA ATGAAGGTGTACAAGAAGGTCGCTTTTGTTATGGCTTTTATTATGTTTTTTTCGGTCCTGCCGACGATCTCAATGTCGTCAGAAGCAAAC HetLysValTyrLysLysYalAlaPheVatHetAUPhelleMetPhePheSerValLeuProThrlleSerrtetSei^SerGluAI&Asn 0
C VF
)
100 150 GGTGCTGCATTATCGAATCCTAATGCGAACCAAACGACAAAAAACGTGTATAGTTGGTTAGCCAATCTACCAAACAAGAOTAATAAACGT GI y A l a A I a L e u S e r A s n P r o A s T l A l a A s n G I n T h r T h r L y a A s n V a l T y r S e r T r p L e u A l a A s n L e u P r o A s n L y f e S e r A s n L y s A r g
__
M
s
t
I
___
200 250 GTGGTGTCGGGACACTTCGGAGGGTACAGTGATTCTACCTTAGCCTGGATCAAACAATGCGCAAGGGAGCTGACAGGAAAAATGCCAGGA ValValSaraiyHlsPheOlyGlyTyrSerAapSerThrLeuAlaTrpIleLyaainCysAlaAr^GluLeuThrGlyLysMetProGly n
n
n
FnuDII
_
BA
300 350 ATATTATCTTGTGATTATAAGAATTGGCAGACGCGATTGTATGTAGCCGATCAAATTAGCTATGGCTGCAATCAAGAATTAATAAACTTT 11 e L e u S e r C y s A s p T y r L y s A s n T r p G l n T h r A r g L e u T y r V a ) A ) a A a p G l n l l e S e r T y r G l y C y s A s n Q l n a i u L e u I l e A s n P h e (100) 400 450 TGGAACCAAGGAGGTTTGGTCACGATCAGTGTACACATGCCAAATCCAGGGTTTCATTCGGGGGAAAACTACAAAACAATTTTGCCTACT TrpAsnGI nGl y G l y L e u V a l T h r I 1 eSerValHlaMe IProAsnProGl y P h e H l a S e r G l yGl u A t n T y r L y s T h r I I e L e u P r o T h r 500 TCACAGTTCCAAAATCTAACCAATCACAGGACAACAGAGGGTAGAAGGTGGAAGGATATCCTGGATAAGATGGCAGATGGGTTGGACGAG S e r G l n P h e G l n A s n L e u T h r A s n H l s A r ^ T h r T h r G l uG) y A r g A r g T r p L y a A s p M e t L e u A a p L y a M e t A l a A s p G I y L e u A s p G l u
Fig. 4. Nucleotide sequence of the Xbal-PstI fragment containing /?mannanase gene(s). Symbols: (B), N-terminal of three /?-mannanases produced by alkaliphilic Bacillus sp. A-001; (E) N-terminal of two /?mannanases isolated from E. Coli carrying pMAH3; (MA), C-terminal end of the mannanase A ; (MB), C-terminal end of the mannanase B. (Continued on the next page.)
Leatham and Himmel; Enzymes in Biomass Conversion ACS Symposium Series; American Chemical Society: Washington, DC, 1991.
5.
Mannan-Degrading Enzymes
HORIKOSHI
59
550 600 CTACAOAACAATGCAGTGACGGTTCTTTTCCGTCCTTTACATGAAATGAATGGAGAATGGTTCTGGTCGGGAGCAGAAGGTTACAATCAA L«uOlnA«nAsnGlyValThrValLeuPheAr«ProLeuHl«GluMetA8nGlyGluTrpPheTrpTrpGlyAlaGluGlyTyrAanGln (200) Bell 650 700 TTTGATCAAACACGTGCCAATGCCTATATCAGCGCATGGAGAGATATGTATCAATATTTTACTCATGAGCGTAAGCTGAATAACCTTATT PheAspGlnThrArgAlaAsnAlaTyrlleSerAlaTrpArgAspMetTyrGlnTyrPheThrHlsGluAr^LysLeuAsnAsnLeuIle 750 800 TGGGTTTACTCACCTGATGTTTACAGAGATCATGTAACAAGTTACTACCCAGGAGCAAATTATGTAGATATTGTGGCTCTTGATTCCTAC TrpValTyrScrProAspYalTyrArgAspMisValThrSerTyrTyrProGlyAlaAsnTyrValAspIleYalAlaLeuAspSerTyr 850 900 CATCCTGATCCACATAGCCTTACTGACCAATATAATCGAATGATCGCTTTAGATAAACCTTTTGCTTTTGCTGAAATCGGTCCTCCTGAA HlaProAspProHlsSerLcuThrAspGlnTyrAsnArgMet lleAULeuAspLysProPheAlaPheAlaGlulleGlyProProGlu (300) 950 AGCATGGCTGGTTCCTTTGATTATTCAAATTATATTCAAGCAATTAAACAAAAATATCCACGTACTGTCTATTTCCTAGCTTGGAATGAT SerMetAlaGlySerPheAspTyrSerAsnTyrlleGlnAlal IeLysGlnLysTyrProArgThrValTyrPheLeuAlaTrpAsnAsp H
f
l
1
1000 1050 AAATGGAGTCCACATAACAACAGAGGAGCATGGGATCTATTTAATGATTCATGGGTTGTAAATAGGCGAGAGATTGATTATGGTCAATCA LysTrpSerProHlsAsnA8nArgGlyAlaTrpA8pLeuPheAsnAspSerTrpValVaUsnArgpiyGlul l e A s p T y r G l y G l n S e r
1100
1150
N
c
o
1
Ns
V
P
AATCCAGCCACTGTTCTCl^TGATTTTOAAAACAATACGCTATCGTGGTCCGGGTGTGAATTTACGGACCGAGGACCATGGACTICGAAT AanProAlBThrVallLeuTyrAspPheOluAsnABnThrLeuSerTrpSerGlyCyaGruPlieThrAspOlyGlyProTrpThrSerAan M
d
1200 1250 GAATGGTCGGCAAATGOTACTCAATCGTTGAAAGCAOATGTCGTTCTOGOCAATAATAGCTACCATTTOCAAAAAACAGTGAATCGAAAT G l u T r p S e r A l aAsnGl yThrG 1 nSerLeuLysAl aAspVal YalLeuGl yAsnAsnSerTyrHi sLeuGl nLysThrVal AsnArgAtn (400) 1300 1350 CTTAGTTCATTCAAAAACCTAGAAATTAAAGTGAGCCATTCTTCGTGGGGAAATGTAGGAAGTGGCATGACAGCAAGAGTTTTCGTCAAA LcuSerSerPheLysAsnLeuGluIleLysYalSerHlsSerSerTrpGlyAsnValGlySerGlyMetThrAlaArgValPheValLys 1400 ACAGGGAGTCCTTGGACATGGAATGCAGGTGAATTTTGTCAGTTTGCAGGCAAACGAACAACCGCACTATCTATTGATTTGACGAAAGTA ThrGlyScrAlaTrpAr«TrpAsnAla0lyGluPheCy8GlnPheAlaGlyLysAr«ThrThrAlaLeuSerneA8pLeuThrLy8Yal 1450 1500 AGTAATCTGCATGATGTTCGAGAGATAGQTGTAGAGTATAAAGCACCAGCAAATAGCAACGGGAAGACGGCGATTTACTTAGATCATGTG SerAsnLeuHi sAspValArgGluI ItOlyValOluTyrLytAlaProAlaAsnSerAsnGlyLyaThrAlal 1 eTyHLeuAtpHlaVal
Mel
500
< >
ACCGTAAGATAATACAAAAAAAAGTGGTTGAAAGCGGTAACATATCTAGCATATGATGATAGGGACTAGATAATAATAGACTGTCAGACT T h r V t l Ani)*** ^ ^ MA AOGACGTAAOTCATAATGAAAAAAAGTCTGATCCTCTTGCTCGOACTTTTATTAGCTTTCTCCATGCTATTAATAGCCTATCTATCATTC — T T ' / V
PstI ACCCCTGCAG
1720
Fig. 4 (continued). Nucleotide sequence of the Xbal-PstI fragment containing /7-mannanase gene(s). Symbols: (B), N-terminal of three /?mannanases produced by alkaliphilic Bacillus sp. A-001; (E) N-terminal of two /^-mannanases isolated from E. Coli carrying pMAH3; (MA), Cterminal end of the mannanase A; (MB), C-terminal end of the mannanase B.
Leatham and Himmel; Enzymes in Biomass Conversion ACS Symposium Series; American Chemical Society: Washington, DC, 1991.
60
ENZYMES IN BIOMASS CONVERSION
Table IV. Properties of the Purified P-mannanases from Bacillus sp. AM-001 and E. coli carrying pMAH3
Optimum temp. Optimum pH Thermal stability pH stability Molecular weight
M-I 60 9.0 50 8-9 58,500
AM-001 M-II 60 9.0 50 8-9 59,500
M-III 65 8.5 60 7-9 42,000
E. co7/(pMAH3) A B 60 65 9.0 8.5 50 60 8-9 7-9 58,000 43,000
Leatham and Himmel; Enzymes in Biomass Conversion ACS Symposium Series; American Chemical Society: Washington, DC, 1991.
5.
HORIKOSHI
Mannan-Degrading Enzymes
61
messenger RNAs) are produced from one open reading frame. We have not analyzed the m-RNA, but two transcripts may be produced as shown in other microorganisms. Literature cited 1. Emi, S.; Fukumoto, J.; Yamamoto, T.Agric.Biol.Chem. 1972, 36, 991-1001. 2. Araki, T. J. Fac. Agr. Kyushu Univ. 1983, 27, 89-98. 3. Takahashi, R.; Kusakabe, I.; Kobayashi, H.; Murkami, K.; Maekawa, Α.; Suzuki, I.Agric.Biol. Chem. 1984, 48, 2189-2195 4. Hashimoto, Y.; Fukumoto, J. Nippon Nogeikagaku Kaishi, 1969, 43, 317-322. 5. Eriksson, Κ. E.; Winnel, M . Acta Chem. Scand, 1968, 22, 1924-1934. 6. Civas, Α.; Bverhard, D. P. L.; Petek, F. Biochem.J.,1984, 219, 857-863. 7. Hashimoto, Y.; Fukumoto, J. Nippon Nogeikagaku Kaishi, 1969, 43, 564-569. 8. Bouquelet, S.; Spik, G.; Montreuil, J. Biochim. Biophys. Acta, 1978, 552, 521530. 9. Araki, T.; Kitamikado, M . J. Biochem., 1982, 91, 1181-1186. 10. Akino, T.; Nakamura, N.; Horikoshi, K. Appl. Mircobiol. Biotechnol., 1987, 26, 323-327. 11. Akino, T.; Nakamura, N.; Horikoshi, K. Agric. Biol. Chem., 1988, 52, 773779. 12. Akino, t.; Nakamura, N.; Horikoshi, K. Agric. Biol. Chem., 1988, 52, 14591464. 13. Akino, T.; Kato, C.; Horikoshi, K. Arch. Microbiol., 1989, 152, 10-15. 14. Saito, H.; Miura, K. Biochim. Biophys. Acta, 1963. 72, 619-626. RECEIVED September 26, 1990
Leatham and Himmel; Enzymes in Biomass Conversion ACS Symposium Series; American Chemical Society: Washington, DC, 1991.
