Production of Milk Clotting Protease by Rhizopus Stolonifer through Optimization of Culture Conditions
The present study describes the biosynthesis of a milkclotting
protease by solid state fermentation (SSF) of a locally
isolated mould, Rhizopus stolonifer. The production medium was
prepared using wheat bran at 50% (w/v). The production conditions
are optimized by varying 7 parameters: carbon and nitrogen sources,
medium moisture, temperature, pH, fermentation time and
inoculum-s size. The maximum enzyme synthesis was measured after
96 h of incubation time at temperature of 28°C. The optimum pH
determined was 6 and the inoculum size was 3.106spores/ml. The
optimum initial moisture content is comprised between 50 to 70%.
The formation of milk clotting protease is enhanced when galactose
and peptone are used at 10% (w/v) and 1% (w/v) concentrations
respectively. The maximum production of milk clotting protease is
120 US/ml.
[1] A.L. Abdullah, and M.V.G. Tengerdy, "Optimization of solid substrate
fermentation of wheat bran", Biotech. Bioengin. vol. 27, pp. 20-27,
1985.
[2] C.N. Aguilar, G.G. Sanchez, P.L.A.R. Barragan, R.R. Herrera, J.L.M.
Hernandez, and J.C. Esquivel, "Perspectives of solid state fermentation
for production of food enzymes", Americ. J. Biochem .Biotechno., vol 4,
no 4, pp. 354-366, 2008.
[3] D. Anandan, W.N. Marmer, and R. L. Dudley,"Isolation characterization
and optimization of culture parameters for production of an alkaline
protease isolated from Aspergillus tamari ", J . Ind. Microbiol.
Biotechnol., vol. 34, pp. 339-347, 2007.
[4] K. Arima, J. Yu, and S. Iwasaki, "Milk-clotting enzyme from Mucor
pusillus var.Lindt", Metho. Enzymo., vol. 19, pp. 446-459, 1970.
[5] M.J. Bailey, M. Siika-aho, "Production of microbial rennin", Biotech.
Letters., vol. 10, no. 3, pp. 161-166, 1988.
[6] B. Botton, A. Breton, M. Fevre, S. Gauthier, P. Guy, J.P. Larpent, P.
Reymond, J.J. Sanglier, Y. Vayssier, and P. Veau, « Moisissures utiles et
nuisibles importance industrielle », Collec Biotech., pp. 512, 1990.
[7] M.M Bradford, "A rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principle of protein-dye
binding", Anal. Biochem. Vol. 72, no. 1-27, pp. 248-254, 1976.
[8] M. T. H. Cavalcanti, C. R. Martinez, V. C. Furtado, B. B. Neto, M. F.
Teixeira, F. J. L. Lima, and A. L. F. Porto, "Milk-clotting protease
production by Nocardiopsis sp. in an inexpensive medium", World. J.
Microb. Biotech., vol. 21, pp. 151-154, 2005.
[9] P.S. Channe, and J.G. Shewale, "Influence of culture conditions on the
formation of milk-clotting protease by Aspergillus niger MC4", World.
J. Microbio. Biotech., vol. 14, pp. 11-15, 1998.
[10] S. Chazarra, L. Sidrach, D. Lopez-Molina, and J. N. Rodr─▒guez-Lopez,
"Characterization of the milk-clotting properties of extracts from flowers
artichoke (Cynara scolymus, L.)", Intern. Dairy. J., vol. 17, pp. 1393-
1400, 2007.
[11] S.R. Couto, and M. A. Sanroma, "Application of solid-state fermentation
to food industry", J. Food. Engineer., vol. 76, pp. 291-302, 2006.
[12] M.U. Dahot, M.Y. Khan, and A. N. Memon, "Screening of some
Pakistani plants for milk clotting activity", J. Islamic. Acad. Scienc., vol.
3, no. 4, pp. 284-286, 1990.
[13] R. Davidson, A. Gertler, and T. Hofmann, "Aspergillus oryzae acid
proteinase purification and properties and formation of 7r-
Chymotrypsin", J. Biochem., vol. 147, pp. 45-53, 1975.
[14] K. Dutt, P. Gupta, S. Saran, M. Swati, and R. Kumar Saxena,
"Production of milk-clotting protease from Bacillus subtilis". Appl.
Biochem. Biotech., vol. 158, no.3, pp. 761-772, 2009.
[15] A.M. Jurus, and W.J. Sundberg, "Penetration of Rhizopus oligosporus
into soybeans in tempeh", Appl. Environ. Microbio. Vol. 32, no. 2, pp.
284-287, 1976.
[16] E.M. El-Safey, and U.M. Abdul-Raouf, "Production, purification and
characterization of protease enzyme from Bacillus subtilis". Interna.
Confer. Develop. Environ. In The Arab World., pp. 14, 2004.
[17] J. Escobar, and S. M. Barnett, "Effect of agitation speed on the synthesis
of Mucor miehei acid protease", Enzym. Microbio. Techno. ,vol. 15, pp.
1009-1013, 1993.
[18] H. M. Fernandez-Lahore, R. M. Auday, E. R. Fraile, O, Cascone, M.
Biscoglio de jimenez.Bonino, L. Pirpignani and C. Machalinski, «
Purification and characterization of an acid proteinase from mesophilic
Mucor sp on solid state cultures", J. Pept. Res., vol. 53, pp. 1-7, 1997.
[19] H. Hashimoto, Y. Kaneko, T. Iwaasa, and T. Yokotsuka,. "Production
and Purification of Acid Protease from the Thermophilic Fungus
Penicillium duponti K1014", Appl. Microbio., vol. 25, no. 4, pp. 584-
588, 1973
[20] C.W. Hasseltine, "Solid state fermentation". Biotech. Bioeng., vol. 14,
pp. 517-532, 1972
[21] G. V. González, E.F. Torres, C.N. Aguilar, S.J.R. Gomez, G. D.
God─▒nez, and C. Augur, "Advantages of fungal enzyme production in
solid state over liquid fermentation systems", Biochem. Engin. Journal.,
vol. 13, pp. 15-167, 2003.
[22] A. Kazemi-Vaysari, A. Kheirolomoom, M. Arjmand and M.
Habibollahi,. Optimization of Mucor miehei rennin production and
recovery. Scientia. Iranica. 9 (1): 99-104. 2002.
[23] S. Kumar, N.S. Sharma, M.R. Saharan, and R. Singh, "Extracellular acid
protease from Rhizopus oryzae purification and characterization",
Process. Biochem., vol. 40, pp. 1701-1705, 2005.
[24] S. Kumar, J. Sharma, A. Kumar Mohanty, S. Grover, V. Kumar Batish,
"Purification and characterization of milk clotting enzyme from goat
(Capra hircus)", Comparative. Biochem. Physio., vol. 145, pp. 108-113,
2006.
[25] O. A. Odeniyi, A. A. Onilude, and M. A. Ayodele, "Growth and
substrate utilization patterns of a Rhizopus stolonifer strain isolated from
depolymerising rice husk", World. Appl. Scien. J., vol. 6, no. 5, pp. 595-
599, 2009.
[26] A. Pandey, "Solid-state fermentation", Biochem. Engin., vol. 13, pp.
127-135, 2003.
[27] S. U. Phadatare, M. C. Srinivasan, and V. V. Deshpande, "High activity
alkaline protease from Conidiobolus coronatus (NCL 86.8.20):enzyme
production and compatibility with commercial detergents", Enzym.
Microbio. Techno., vol. 15, pp. 72-76, 1993
[28] J. L. Sardinas, "Rennin enzyme of Endothia parasitica". Appl.
Microbiol., vol. 16, pp. 248-53, 1968.
[29] R. Sathya, B.V. Pradeep, J. Angayarkanni, and M. Palaniswamy.
Production of milk clotting protease by a local isolate of Mucor
circinelloides under SSF using agro-industrial wastes. Biotechn.
Bioprocess. Engin., vol. 14, pp. 788-794, 2009
[30] R. A. Sparringa, M. Kendall, A. Westby, and J.D. Owens, "Effects of
temperature, pH, water activity and CO2 concentration on growth of
Rhizopus oligosporus NRRL 2710", J. Appl. Microbio., vol. 92, pp.
329-337, 2002.
[31] A. Sumantha, C. Larroche, and A. Pandey, "Micorbiology and
Industrial Biotechnology of food-grade proteases: A perspective". Food.
Technol.Biotechnol., vol. 44, no. 2, pp. 211-220, 2006.
[32] A. Sumantha, P. Deepa, C. Sandhya, C. Szakacs, R. C. Soccol, and A.
Pandey, "Rice bran as a substrate for proteolytic enzyme production",
Brazilian . Arch . Biol.Technol., vol. 49, no.5, pp. 843-851, 2006.
[33] M. Z. Sternberg, "Crystalline Milk-Clotting Protease from Mucor
miehei and some of its Properties". J. Dairy. Scien., vol. 53, no. 2, pp.
159-167, 1970.
[34] I. Ul-Haq, H. Mukhtar, S. Daudi, S. Ali, and M.A. Qadeer, "Production
of protease by locally isolated mould culture under lab conditions".
Biotechno., vol. 2, no. 1, pp. 30-36, 2003.
[35] I. Ul-Haq, and H. Mukhtar, "Biosynthesis of protease by Rhizopus
oligosporus IHS13 in low-cost medium by solid state fermentation", J.
Basic.Microbiol., vol. 44, pp. 280-287, 2004.
[36] Z. A. Tubesha, and K. S. AL-Delaimy, "Renin-like milk coagulant
enzyme produced by local isolated of Mucor", Intern. J. Dairy. Techno.,
vol. 56, no. 4, pp. 237-241, 2003.
[37] R. Tunga, "Influence of Temperature on Enzyme Production", Tech.M.
Thesis, II. T. Kharagpur, India, 1995.
[38] R. Tunga, R. Banerjee, and B. C. Bhattacharyya, "Optimizing some
factors affecting protease production under solid state fermentation",
Bioprocess . Engin., vol. 19, pp. 187-190, 1998.
[1] A.L. Abdullah, and M.V.G. Tengerdy, "Optimization of solid substrate
fermentation of wheat bran", Biotech. Bioengin. vol. 27, pp. 20-27,
1985.
[2] C.N. Aguilar, G.G. Sanchez, P.L.A.R. Barragan, R.R. Herrera, J.L.M.
Hernandez, and J.C. Esquivel, "Perspectives of solid state fermentation
for production of food enzymes", Americ. J. Biochem .Biotechno., vol 4,
no 4, pp. 354-366, 2008.
[3] D. Anandan, W.N. Marmer, and R. L. Dudley,"Isolation characterization
and optimization of culture parameters for production of an alkaline
protease isolated from Aspergillus tamari ", J . Ind. Microbiol.
Biotechnol., vol. 34, pp. 339-347, 2007.
[4] K. Arima, J. Yu, and S. Iwasaki, "Milk-clotting enzyme from Mucor
pusillus var.Lindt", Metho. Enzymo., vol. 19, pp. 446-459, 1970.
[5] M.J. Bailey, M. Siika-aho, "Production of microbial rennin", Biotech.
Letters., vol. 10, no. 3, pp. 161-166, 1988.
[6] B. Botton, A. Breton, M. Fevre, S. Gauthier, P. Guy, J.P. Larpent, P.
Reymond, J.J. Sanglier, Y. Vayssier, and P. Veau, « Moisissures utiles et
nuisibles importance industrielle », Collec Biotech., pp. 512, 1990.
[7] M.M Bradford, "A rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principle of protein-dye
binding", Anal. Biochem. Vol. 72, no. 1-27, pp. 248-254, 1976.
[8] M. T. H. Cavalcanti, C. R. Martinez, V. C. Furtado, B. B. Neto, M. F.
Teixeira, F. J. L. Lima, and A. L. F. Porto, "Milk-clotting protease
production by Nocardiopsis sp. in an inexpensive medium", World. J.
Microb. Biotech., vol. 21, pp. 151-154, 2005.
[9] P.S. Channe, and J.G. Shewale, "Influence of culture conditions on the
formation of milk-clotting protease by Aspergillus niger MC4", World.
J. Microbio. Biotech., vol. 14, pp. 11-15, 1998.
[10] S. Chazarra, L. Sidrach, D. Lopez-Molina, and J. N. Rodr─▒guez-Lopez,
"Characterization of the milk-clotting properties of extracts from flowers
artichoke (Cynara scolymus, L.)", Intern. Dairy. J., vol. 17, pp. 1393-
1400, 2007.
[11] S.R. Couto, and M. A. Sanroma, "Application of solid-state fermentation
to food industry", J. Food. Engineer., vol. 76, pp. 291-302, 2006.
[12] M.U. Dahot, M.Y. Khan, and A. N. Memon, "Screening of some
Pakistani plants for milk clotting activity", J. Islamic. Acad. Scienc., vol.
3, no. 4, pp. 284-286, 1990.
[13] R. Davidson, A. Gertler, and T. Hofmann, "Aspergillus oryzae acid
proteinase purification and properties and formation of 7r-
Chymotrypsin", J. Biochem., vol. 147, pp. 45-53, 1975.
[14] K. Dutt, P. Gupta, S. Saran, M. Swati, and R. Kumar Saxena,
"Production of milk-clotting protease from Bacillus subtilis". Appl.
Biochem. Biotech., vol. 158, no.3, pp. 761-772, 2009.
[15] A.M. Jurus, and W.J. Sundberg, "Penetration of Rhizopus oligosporus
into soybeans in tempeh", Appl. Environ. Microbio. Vol. 32, no. 2, pp.
284-287, 1976.
[16] E.M. El-Safey, and U.M. Abdul-Raouf, "Production, purification and
characterization of protease enzyme from Bacillus subtilis". Interna.
Confer. Develop. Environ. In The Arab World., pp. 14, 2004.
[17] J. Escobar, and S. M. Barnett, "Effect of agitation speed on the synthesis
of Mucor miehei acid protease", Enzym. Microbio. Techno. ,vol. 15, pp.
1009-1013, 1993.
[18] H. M. Fernandez-Lahore, R. M. Auday, E. R. Fraile, O, Cascone, M.
Biscoglio de jimenez.Bonino, L. Pirpignani and C. Machalinski, «
Purification and characterization of an acid proteinase from mesophilic
Mucor sp on solid state cultures", J. Pept. Res., vol. 53, pp. 1-7, 1997.
[19] H. Hashimoto, Y. Kaneko, T. Iwaasa, and T. Yokotsuka,. "Production
and Purification of Acid Protease from the Thermophilic Fungus
Penicillium duponti K1014", Appl. Microbio., vol. 25, no. 4, pp. 584-
588, 1973
[20] C.W. Hasseltine, "Solid state fermentation". Biotech. Bioeng., vol. 14,
pp. 517-532, 1972
[21] G. V. González, E.F. Torres, C.N. Aguilar, S.J.R. Gomez, G. D.
God─▒nez, and C. Augur, "Advantages of fungal enzyme production in
solid state over liquid fermentation systems", Biochem. Engin. Journal.,
vol. 13, pp. 15-167, 2003.
[22] A. Kazemi-Vaysari, A. Kheirolomoom, M. Arjmand and M.
Habibollahi,. Optimization of Mucor miehei rennin production and
recovery. Scientia. Iranica. 9 (1): 99-104. 2002.
[23] S. Kumar, N.S. Sharma, M.R. Saharan, and R. Singh, "Extracellular acid
protease from Rhizopus oryzae purification and characterization",
Process. Biochem., vol. 40, pp. 1701-1705, 2005.
[24] S. Kumar, J. Sharma, A. Kumar Mohanty, S. Grover, V. Kumar Batish,
"Purification and characterization of milk clotting enzyme from goat
(Capra hircus)", Comparative. Biochem. Physio., vol. 145, pp. 108-113,
2006.
[25] O. A. Odeniyi, A. A. Onilude, and M. A. Ayodele, "Growth and
substrate utilization patterns of a Rhizopus stolonifer strain isolated from
depolymerising rice husk", World. Appl. Scien. J., vol. 6, no. 5, pp. 595-
599, 2009.
[26] A. Pandey, "Solid-state fermentation", Biochem. Engin., vol. 13, pp.
127-135, 2003.
[27] S. U. Phadatare, M. C. Srinivasan, and V. V. Deshpande, "High activity
alkaline protease from Conidiobolus coronatus (NCL 86.8.20):enzyme
production and compatibility with commercial detergents", Enzym.
Microbio. Techno., vol. 15, pp. 72-76, 1993
[28] J. L. Sardinas, "Rennin enzyme of Endothia parasitica". Appl.
Microbiol., vol. 16, pp. 248-53, 1968.
[29] R. Sathya, B.V. Pradeep, J. Angayarkanni, and M. Palaniswamy.
Production of milk clotting protease by a local isolate of Mucor
circinelloides under SSF using agro-industrial wastes. Biotechn.
Bioprocess. Engin., vol. 14, pp. 788-794, 2009
[30] R. A. Sparringa, M. Kendall, A. Westby, and J.D. Owens, "Effects of
temperature, pH, water activity and CO2 concentration on growth of
Rhizopus oligosporus NRRL 2710", J. Appl. Microbio., vol. 92, pp.
329-337, 2002.
[31] A. Sumantha, C. Larroche, and A. Pandey, "Micorbiology and
Industrial Biotechnology of food-grade proteases: A perspective". Food.
Technol.Biotechnol., vol. 44, no. 2, pp. 211-220, 2006.
[32] A. Sumantha, P. Deepa, C. Sandhya, C. Szakacs, R. C. Soccol, and A.
Pandey, "Rice bran as a substrate for proteolytic enzyme production",
Brazilian . Arch . Biol.Technol., vol. 49, no.5, pp. 843-851, 2006.
[33] M. Z. Sternberg, "Crystalline Milk-Clotting Protease from Mucor
miehei and some of its Properties". J. Dairy. Scien., vol. 53, no. 2, pp.
159-167, 1970.
[34] I. Ul-Haq, H. Mukhtar, S. Daudi, S. Ali, and M.A. Qadeer, "Production
of protease by locally isolated mould culture under lab conditions".
Biotechno., vol. 2, no. 1, pp. 30-36, 2003.
[35] I. Ul-Haq, and H. Mukhtar, "Biosynthesis of protease by Rhizopus
oligosporus IHS13 in low-cost medium by solid state fermentation", J.
Basic.Microbiol., vol. 44, pp. 280-287, 2004.
[36] Z. A. Tubesha, and K. S. AL-Delaimy, "Renin-like milk coagulant
enzyme produced by local isolated of Mucor", Intern. J. Dairy. Techno.,
vol. 56, no. 4, pp. 237-241, 2003.
[37] R. Tunga, "Influence of Temperature on Enzyme Production", Tech.M.
Thesis, II. T. Kharagpur, India, 1995.
[38] R. Tunga, R. Banerjee, and B. C. Bhattacharyya, "Optimizing some
factors affecting protease production under solid state fermentation",
Bioprocess . Engin., vol. 19, pp. 187-190, 1998.
@article{"International Journal of Biological, Life and Agricultural Sciences:64169", author = "S. Gais and F. Fazouane and A. Mechakra", title = "Production of Milk Clotting Protease by Rhizopus Stolonifer through Optimization of Culture Conditions", abstract = "The present study describes the biosynthesis of a milkclotting
protease by solid state fermentation (SSF) of a locally
isolated mould, Rhizopus stolonifer. The production medium was
prepared using wheat bran at 50% (w/v). The production conditions
are optimized by varying 7 parameters: carbon and nitrogen sources,
medium moisture, temperature, pH, fermentation time and
inoculum-s size. The maximum enzyme synthesis was measured after
96 h of incubation time at temperature of 28°C. The optimum pH
determined was 6 and the inoculum size was 3.106spores/ml. The
optimum initial moisture content is comprised between 50 to 70%.
The formation of milk clotting protease is enhanced when galactose
and peptone are used at 10% (w/v) and 1% (w/v) concentrations
respectively. The maximum production of milk clotting protease is
120 US/ml.", keywords = "Milk clotting activity, protease production,Rhizopus stolonifer, Solid state fermentation.", volume = "3", number = "6", pages = "343-5", }
