Manabu FUKUDA, Yasushi HASEGAWA

Protease activity of a 90-kDa protein Isolated from scallop shells

We have previously reported the free radical scavenging activity of a protein with a molecular weight of 90 kDa (90- kDa protein) isolated from the scallop shell. In this study, we found that the 90-kDa protein also shows protease activity. The protein was most active at an alkali pH and at 60°C, and its activity was inhibited by serine protease inhibitors, phenylmethylsulfonyl fluoride and diisopropyl fluorophosphate. Its activity was maintained at approximately 90% of the initial activity, even in the presence of denaturants such as 1% sodium dodecyl sulfate (SDS) and 6 M urea. Substrate specificity analysis performed using synthetic peptides showed that the 90-kDa protein cleaves preferentially at Lys-X and Arg-X bonds. A portion of Phe-X bond was also cleaved by the 90-kDa protein. When casein was treated with the 90-kDa protein, it was digested at the Arg-X, Lys-X, and Phe-X bonds. The 90-kDa protein may be useful for proteome analysis because it retains its activity even in the presence of 1% SDS. To the best of our knowledge, this is the first report of a protease found in scallop shell.

PDF

___

Anwar, A. and Saleemuddin, M. 1998. Alkaline proteases: a review. Bioresource Technol., 64: 175 -183. doi: 10.1016/S0960-8524(97)00182-X

Aranishi, F. 1999. Lysis of pathogenic bacteria by epidermal cathepsin L and B in the Japanese eel. Fish Physiol. Biochem., 20: 37-41. doi: 10.1023/A:1007763711158

Banik, R.M. and Prakash, M. 2004. Laundry detergent compatibility of the alkaline protease from Bacillus cereus. Microbiol. Res., 159: 135-140.

Delmar, E.G., Largman, C., Brodrick, J.W. and Geokas, M.C. 1979. A sensitive new substrate for chymotrypsin. Anal. Biochem., 99: 316-320. doi: 10.1016/S0003-2697(79)80013 -5

Green, M.R., Pastewka, J.V. and Peacock, A.C. 1973. Differential staining of phosphoproteins on polyacrylamide gels with a cationic carbocyanine dye. Anal. Biochem., 56: 43 -51. doi: 10.1016/0003 - 2697(73)90167-X

Gupta, R., Beg, Q.K. and Lorenz, P. 2002. Bacterial alkaline protease: molecular approaches and industrial applications. Appl. Microbiol. Biotechnol., 59: 15 -32. doi: 10.1007/s00253 -002-0975-y

Hjelmeland, K., Christie, M. and Raa, J. 1983. Skin mucus protease from rainbow trout Salmo gairdneri Richardson and its biological significance. J. Fish. Biol., 23: 13-22. doi: 10.1111/j.1095 - 8649.1983.tb02878.x

Johnvesly, B. and Naik, G.R. 2001. Studies on production of thermostable alkaline protease from thermophilic and alkaliphilic Bacillus sp JB-99 in a chemically defined medium. Process Biochem., 37: 139-144. doi: 10.1016/S0032-9592(01)00191 -1

Joo, H.S., Kumar, C.G., Park, G.C., Paik, S.R. and Chang, C.S. 2003. Oxidant and SDS-stable alkaline protease from Bacillus clausii I-52 production and some properties. Appl. Microbiol., 95: 267-272. doi: 10.1046/j.1365-2672.2003.01982.x

Khan, N.A., Jarroll, E.L., Panjwani, N., Cao, Z. and Paget, T.A. 2000. Proteases as markers for differentiation of pathogenic and nonpathogenic species of Acanthamoeba. J. Clin. Microbiol., 38: 2858 -2861.

Kumar, C.G. and Takagi, H. 1999. Microbial alkaline proteases from a bioindustrial viewpoint. Biotechnol. Adv., 17: 561 -594. doi: 10.1016/S0734-9750(99)

Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227: 680-685. doi: 10.1038/227680a0

Liu, Y.C., Uchiyama, K., Natsui, N. and Hasegawa, Y. 2002. In vitro activities of the components from scallop shells. Fish. Sci., 68: 1330-1336. doi: 10.1046/j.1444-2906.2002.00572.x

Liu, Y.C. and Hasegawa, Y. 2006. Reducing effect of feeding powdered scallop shell on the body fat mass of rats. Biosci. Biotechnol. Biochem., 70: 86-92. doi: 10.1271/bbb.70.86

Miyamoto, H., Miyashita, T., Okushima, M., Nakano, S., Morita, T. and Matsushiro, A. 1996. A carbonic anhydrase from the nacreous layer in oyster pearls. Proc. Natl. Acad. Sci. U.S.A., 93: 9657-9660. doi: 10.1073/pnas.93.18.9657

Mitsuhashi, T., Ono, K., Fukuda, M. and Hasegawa, Y. 2013. Free radical scavenging ability and structure of a 90-kDa protein from scallop shell. Fish. Sci., 79: 495-502. doi: 10.1007/s12562-013 -0616-7

Nagai, K., Yano, M., Morimoto, K. and Miyamoto, H. 2007. Tyrosinase localization in mollusc shell. Comp. Biochem. Physiol., 146: 207-214. doi: 10.1016/j.cbpb.2006.10.105

Oberoi, R., Beg, Q.K., Puri, S., Saxena, R.K. and Gupta, R. 2001. Characterization and wash performance analysis of an SDS-stable alkaline protease from a Bacillus sp. World J. Microbiol. Biotechnol., 17: 493 - 497. doi: 10.1023/A:1011911109179

Perkins, D.N., Pappin, D.J., Creasy, D.M. and Cottrell, J.S. 1999. Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis, 20: 3551 -3567. doi: 10.1002/(SICI)1522-2683(19991201)20:18_____3551 ::AID-ELPS3551>3.0.CO;2-2

Rahman, R.N.Z.R.A., Geok, L.P., Basri, M. and Salleh, A.B. 2006. An organic solvent-stable alkaline protease from Pseudomonas aeruginosa strain K: Enzyme purification and characterization. Enzyme Microb. Technol., 39: 1484-1491. doi: 10.1016/j.enzmictec.2006.03.038

Sarashina, I. and Endo, K. 2001. The complete primary structure of molluscan shell protein-1 (MSP-1), an acidic glycoprotein in the shell matrix of the scallop Patinopecten yessoensis. Mar. Biotechnol., 3: 362- 369. doi: 10.1007/s10126-001 -0013-6

Takahashi, K., Satoh, K., Katagawa, M., Torita, A. and Hasegawa, Y. 2012. Scallop shell extract inhibits 3T3-L1 preadipocyte differentiation. Fish. Sci., 78: 897-903 . doi: 10.1007/s12562-012-0515 -3

Torita, A., Liu, Y.C. and Hasegawa, Y. 2004. Photoprotective activity of scallop shell water-extract in keratinocyte cells. Fish. Sci., 70: 910-915. doi: 10.1111/j.1444-2906.2004.00886.x(D)