Purification and characterization of four keratinases ... - ResearchGate

F. Xie et al. / Bioresource Technology 101 (2010) 344–350 349
Table 4
N-terminal sequences of Streptomyces sp. strain 16 keratinases and Alignment of keratinase KIV, SFase-2, 17.6 kDa protease and SGPA.
Microorganism Keratinase N-terminal residues
1 2 3 4 5 6 7 8 9 10
Streptomyces sp. strain 16 KI a A G N S A S E I R V
KII a A A P G D K D V T A
KIII a A P D I I L A N A
KIV a I A G G E A I Y A A
S. fradiae ATCC 14544 SFase-2 b I A G G E A I Y A A
S. fradiae 17.6 kDa c I A G G E A I Y A A
S. griseus SGPA d I A G G E A I T T G
a
This study.
b (Kitadokoro et al., 1994).
c (Sinha et al., 1991).
d (Johnson and Smillie, 1974).
keratinases of Streptomyces sp. strain 16 are apparently different
peptidase. From the identical molecular weight and N-terminal sequence,
KIV may be the same peptidase as SFase-2 in S. fradiae, and
a homologue of the 17.6 kDa protease in S. fradiae and SGPA in S.
griseus.
Base on the fact that no enzyme among the four keratinases
could solubilize NHFS alone, there must be some cooperation
among them or with some other components in the culture filtrate
in the process of degrading NHFS. Therefore, it will be interesting
to study the cooperation among the keratinases in further
research.
5. Conclusions
In this work, four keratinases were purified from the culture
broth of Streptomyces sp. strain 16 simultaneously, and they differed
from each other in terms of molecular weight, oligomeric
nature, and some physicochemical characteristics. All four keratinases
showed strong keratinolytic activity toward NHFS. The N-terminal
sequences of KI, KII, and KIII showed no similarity to
sequences of known microbial peptidases, whereas keratinase
KIV shared an identical N-terminal sequence with two other peptidases
of Streptomyces. Keratinase KI is a homo-octamer with a
molecular weight of 203.2 kDa. Based on the properties of the four
keratinases, they might become attractive for potential applications
in laundry detergents.
Acknowledgement
This work was financially supported by the National High Technology
Research and Development Program of China (863;
2006AA020204).
References
Anbu, P., Hilda, A., Sur, H.W., Hur, B.K., Jayanthi, S., 2008. Extracellular keratinase
from Trichophyton sp. HA-2 isolated from feather dumping soil. Int. Biodeterior.
Biodegrad. 62, 287–292.
Awad Jr., W.M., Soto, A.R., Siegel, S., Skiba, W.E., Bernstrom, G.G., Ochoa, M.S., 1972.
The proteolytic enzymes of the K-1 strain of Streptomyces griseus obtained from
a commercial preparation (Pronase). I. Purification of four serine
endopeptidases. J. Biol. Chem. 247, 4144–4154.
Bockle, B., Muller, R., 1997. Reduction of disulfide bonds by Streptomyces pactum
during growth on chicken feathers. Appl. Environ. Microbiol. 63, 790–792.
Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principle of protein-dye binding.
Anal. Biochem. 72, 248–254.
Bressollier, P., Letourneau, F., Urdaci, M., Verneuil, B., 1999. Purification and
characterization of a keratinolytic serine proteinase from Streptomyces
albidoflavus. Appl. Environ. Microbiol. 65, 2570–2576.
Cai, C., Zheng, X., 2009. Medium optimization for keratinase production in hair
substrate by a new Bacillus subtilis KD-N2 using response surface methodology.
J. Ind. Microbiol. Biotechnol. 36, 875–883.
Chao, Y.P., Xie, F.H., Yang, J., Lu, J.H., Qian, S.J., 2007. Screening for a new
Streptomyces strain capable of efficient keratin degradation. J. Environ. Sci. 19,
1125–1128.
Cheng, S.W., Hu, H.M., Shen, S.W., Takagi, H., Asano, M., Tsai, Y.C., 1995. Production
and characterization of keratinase of a feather-degrading Bacillus licheniformis
PWD-1. Biosci. Biotechnol. Biochem. 59, 2239–2243.
De Toni, C.H., Richter, M.F., Chagas, J.R., Henriques, J.A., Termignoni, C., 2002.
Purification and characterization of an alkaline serine endopeptidase from a
feather-degrading Xanthomonas maltophilia strain. Can. J. Microbiol. 48, 342–
348.
Essien, J.P., Umoh, A.A., Akpan, E.J., Eduok, S.I., Umoiyoho, A., 2009. Growth,
keratinolytic proteinase activity and thermotolerance of dermatophytes
associated with alopecia in Uyo. Nigeria Acta Microbiologica Et Immunologica
Hungarica 56, 61–69.
Gradisar, H., Friedrich, J., Krizaj, I., Jerala, R., 2005. Similarities and specificities of
fungal keratinolytic proteases: comparison of keratinases of Paecilomyces
marquandii and Doratomyces microsporus to some known proteases. Appl.
Environ. Microbiol. 71, 3420–3426.
Gradisar, H., Kern, S., Friedrich, J., 2000. Keratinase of Doratomyces microsporus.
Appl. Microbiol. Biotechnol. 53, 196–200.
Ignatova, Z., Gousterova, A., Spassov, G., Nedkov, P., 1999. Isolation and partial
characterisation of extracellular keratinase from a wool degrading thermophilic
actinomycete strain Thermoactinomyces candidus. Can. J. Microbiol. 45, 217–
222.
Johnson, P., Smillie, L.B., 1974. The amino acid sequence and predicted structure of
Streptomyces griseus protease A. FEBS Lett. 47, 1–6.
Kim, J.S., Kluskens, L.D., de Vos, W.M., Huber, R., van der Oost, J., 2004. Crystal
structure of fervidolysin from Fervidobacterium pennivorans, a keratinolytic
enzyme related to subtilisin. J. Mol. Biol. 335, 787–797.
Kitadokoro, K., Tsuzuki, H., Nakamura, E., Sato, T., Teraoka, H., 1994. Purification,
characterization, primary structure, crystallization and preliminary
crystallographic study of a serine proteinase from Streptomyces fradiae ATCC
14544. Eur. J. Biochem. 220, 55–61.
Kluskens, L.D., Voorhorst, W.G., Siezen, R.J., Schwerdtfeger, R.M., Antranikian, G., van
der Oost, J., de Vos, W.M., 2002. Molecular characterization of fervidolysin, a
subtilisin-like serine protease from the thermophilic bacterium
Fervidobacterium pennivorans. Extremophiles 6, 185–194.
Kumar, A.G., Swarnalatha, S., Gayathri, S., Nagesh, N., Sekaran, G., 2008.
Characterization of an alkaline active-thiol forming extracellular serine
keratinase by the newly isolated Bacillus pumilus. J. Appl. Microbiol. 104, 411–419.
Laemmli, U.K., 1970. Cleavage of structural proteins during assembly of head of
bacteriophage T4. Nature 227, 680–685.
Ledoux, M., Lamy, F., 1986. Determination of proteins and sulfobetaine with the
folin phenol reagent. Anal. Biochem. 157, 28–31.
Macedo, A.J., da Silva, W.O., Gava, R., Driemeier, D., Henriques, J.A., Termignoni, C.,
2005. Novel keratinase from Bacillus subtilis S14 exhibiting remarkable
dehairing capabilities. Appl. Environ. Microbiol. 71, 594–596.
Margesin, R., Schinner, F., 1991. Characterization of a metalloprotease from
phychrophilic Xanthomonas maltophilia. FEMS Microbiol. Lett. 79, 257–262.
Morihara, K., Oka, T., Tsuzuki, H., 1967. Multiple proteolytic enzymes of
Streptomyces fradiae. Production, isolation, and preliminary characterization.
Biochimica et Biophysica Acta 139, 382–397.
Nam, G.W., Lee, D.W., Lee, H.S., Lee, N.J., Kim, B.C., Choe, E.A., Hwang, J.K., Suhartono,
M.T., Pyun, Y.R., 2002. Native-feather degradation by Fervidobacterium
islandicum AW-1, a newly isolated keratinase-producing thermophilic
anaerobe. Arch. Microbiol. 178, 538–547.
Onifade, A.A., Al-Sane, N.A., Al-Musallam, A.A., Al-Zarban, S., 1998. A review:
potentials for biotechnological applications of keratin-degrading
microorganisms and their enzymes for nutritional improvement of feathers
and other keratins as livestock feed resources. Bioresour. Technol. 66, 1–11.
Pillai, P., Archana, G., 2008. Hide depilation and feather disintegration studies with
keratinolytic serine protease from a novel Bacillus subtilis isolate. Appl.
Microbiol. Biotechnol. 78, 643–650.
Simpson, R.J., 2003. Proteins and Proteomics: A Laboratory Mannual. Cold Spring
Harbor Laboratory Press, New York.