Sains Ma1aysiana 25(4) 1996: 131-141                                                                  Sains Hayat/

Life Sciences

Partial Purification and Characterization of Lipases from

Thermophilic Rhizopus Rhizopodiformis

Abu Bakar Salleh, C.N.A. Razak, M.Y.A. Samad

K. Ampon, W.M.Z.W. Yunus & M. Basri

Enzyme and Microbial Technology Research

Fakulti Sains dan Pengajian Alam Sekitar

Universiti Pertanian Malaysia

43400 UPM Serdang Selangor D.E. Malaysia

ABSTRAK

Lipase daripada satu stren Rhizopus rhizopodifonnis yang baru dipencilkan telah diseparatulen dan diciri. Melalui fraksi asiton, enzim telah ditulen lebih kurang 2.8 ganda, dengan pengutipan 62.5% dan aktiviti spesifik 3.2 U/mg. Dengan penurasan gel melalui Sephadex G-l00, enzim ditulenkan lagi hingga 9.7 ganda dengan aktiviti spesifik mencapai 11.1 U/mg. Dengan elektroforesis gel poliakritamida, 5 jalur protein didapati selepas fraksi asetone, sementara 2 jalur protein dilihati selepas penurasan gel Sephadex G-10. Enzim ini mempunyai pH optimum pada pH 6.0 dan suhu optimumnya ialah 45°C Enzim ini paling stabit pada pH 7.0 dan suhu 50°C Enzim mempunyai kepilihan pada trigliserida rantai pendek dan dapat menghidrolisiskan beberapa ester metit. Lipase ini spesifik pada posisi 1,3.

ABSTRACT

Lipase from a newly isolated strain of Rhizopus rhizopodifonnis was partially purified and characterized. By acetone fractionation, the enzyme was purified to about 2.8 fold, with 62.5% recovery and with specific activity of 3.2 U/mg. By gel filtration through Sephadex G-100, the enzyme was further purified to 9.7 fold and had a specific activity of 11.1 U/mg. By polyacrylamide gel electrophoresis, five protein bands were observed after acetone fractionation, white two protein bands were observed after the preparation was passed through Sephadex G-100. It has a pH optimum at 6.0 and a temperature optimum at 45°C. The enzyme is most stable at pH 7.0 and temperature of 50°C. The enzyme has a preference for short chain triglycerides and can also hydrolyse some methyl esters. The lipase is specific for 1,3 positions.

RUJUKAN/REFERENCES

Benzonona. 1974. Some properties of extracellular lipase from Rhizopus arrhizus. Lipids 9: 166-172.

Borgstrom, B. & Brockman, H.L. 1984. Lipases. Amsterdam: Elsevier Science Publisher.

Davis, B.T. 1964. Disc-electrophoresis method and application to human serum proteins. Annals New York Academy of Sciences 121: 404.

Fukumoto, J., Iwai, M., and Tsujisaka, Y. 1963. Studies on lipase 1. Purification and            crystallisation of a lipase secreted by Aspegillus niger. Journal of General and Applied Microbiology 9: 353-361.

Haas, M.J., Cichowicz & Bailey, D.G. 1992. Purification and characterisation of an extracellular lipase from the fungus Rhizopus delemar. Lipids 27: 571-576.

Ibrahim, C.O., Hayashi, M. & Nagai, S. 1987. Purification and some properties of a thermostable from Humicola Inuginos No.3. Journal of Agriculture Biological Chemistry 51: 37-45.

Iwai, M., Tsujisaka, Y., Haya, K., Okamoto, Y. & Fukumoto, J. 1966. The culture conditions of Rhizopus delemar and its lipase. Kagaku to Kogyo (Osaka) 40:      80­-89.

Iwai, M., & Tsujisaka, Y. 1984. Fungal lipase. In Lipases, (Borgstrom, B. and Brockman, HL) eds. Amsterdam, Elsevier. pp. 443-469.

Kilara, A 1985. Enzyme modified lipid food ingredients. Journal of Process Biochemistry 20: 35-41.

Liu, W.H., Beppu, T. & Arima, K. 1973. Substrate specificity and mode of activity of the lipase of thermophilic fungus Humicola lanuginosa S-36. Journal of Agriculture Biological Chemistry 37: 1349-1355.

Lobyreva, L.B., & Marchenkova, A.I. 1981. Characteristics of lipase in the culture growth of Penicillum raqeuforti. Microbiologiya 50: 90-95.

Lowry, O.H. Rosebrough, N.J. Farr, AL. and Randall, R.J. 1951. Protein measurement with folin phenol reagent. Journal of Biological Chemistry 193: 265-275.

Mozaffar, Z. & Weete, J.D. 1993. Purification and properties of an extracellular lipase from Pythium ultimum. Lipids 28: 377-382.

Nishio, T., Chikano, T. & Kamimura, M. 1987. Purification and some properties of lipase production by Pseudomonas fragii 22.39B. Journal of Agriculture Biological Chemistry 51: 181-186.

Okumura, S., Iwai, M. & Tsujisaka, Y. 1980. Purification and properties of partial glyceride hydrolase of Penicillum cytopium M1. Journal of Biochemistry 87:   205-211.

Okumura, S., Iwai, M. & Tsujisaka, Y. 1976. Positional specificities of kinds of microbial lipases. Journal of Agriculture Biological Chemistry 40: 655-660.

Ota, Y., Miyairi, S. & Yamada, K. 1968. Sterol requirement for the lipase production by Candida cyclindraceae. Journal of Agriculture Biological Chemistry 32: 1476-1478.

Pal, N., Dal, S. & Kundu, A.K 1978. Influence of culture and nutritional conditions on the production of lipase by submerged culture of Aspegillus niger. Journal of Fermentation Technology 56: 593-598.

Samad, M.Y., Razak, C.N.A., Salleh, A.B., Ampon, K, Yunus, W.M.Z. & Basri, M. 1991. A lipase from a newly isolated thermophilic Rhizopus rhizopodiformis. World Journal of Microbiology and Biotechnology 6: 390-394.

Salleh, A.B., Razak, C.N.A., Rosailis, B., Ampon, K, Yunus, W.M.Z. & Basri, M. 1989. Purification of lipase from Rhizopus oryzae. Proceeding of Malaysian Biochemical Society Conference 14: 155-157.

Sugiura, M. & Isobe, M. 1975. Studies on Mechanism of lipase reaction 1. Inhibition of lipase activity by emulsion by organic solvent. Chemical and Pharmaceutical Bulletin 23: 1226-1230.

Sugihara, A., Shimada, Y. & Torninaga, Y. 1988. Purification and characterisation of Aspegillus niger lipase. Journal of Agriculture Biological Chemistry 52: 1591-1592.

Vadehra, D.V. & Harmon, L.G. 1969. Factors affecting production of Staphylococcol lipase. Journal of Applied Bacteriology 32: 147-150.