Arthrobacter arilaitensis Re117 as a Source of Solvent-Stable Proteases: Production, Characteristics, Potential Application in the Deproteinization of Shrimp Wastes and Evaluation in Liquid Laundry Commercial Detergents
Author(s)
Rayda Siala1*,
Ines Hammemi2,
Sabrine Sellimi1,
Tatiana Vallaeys3,
Alya Sellami Kamoun1,
Moncef Nasri1
Affiliation(s)
1 Laboratory of Enzyme Engineering and Microbiology, National School of Engineering of Sfax (ENIS), University of Sfax, Sfax, Tunisia. 2 Unité de Recherche Protection des Plantes Cultivées et Environnement, Institut de l’Olivier, Sfax, Tunisia. 3 Laboratoire Ecosystèmes Lagunaires, UMR 5119, Université de Montpellier, Montpellier, France.
1 Laboratory of Enzyme Engineering and Microbiology, National School of Engineering of Sfax (ENIS), University of Sfax, Sfax, Tunisia. 2 Unité de Recherche Protection des Plantes Cultivées et Environnement, Institut de l’Olivier, Sfax, Tunisia. 3 Laboratoire Ecosystèmes Lagunaires, UMR 5119, Université de Montpellier, Montpellier, France.
ABSTRACT
The present study describes the characterization of crude protease extract from Arthrobacter arilaitensis Re117 and its evaluation in solid and liquid detergent. One caseinolytic protease clear band was observed in zymogram. The crude alkaline protease showed optimum activity at pH 9.0 and 50°C, and it was highly stable over a wide range of pH from 8.0 to 9.0. Proteolytic enzymes showed extreme stability towards non-ionic surfactants (Tween 80, Tween 20 and Triton X-100) and stimulate activity towards oxidizing agents such as sodium perborate. They also showed high stability and compatibility with various laundry solid detergents from Tunisian market. The protease of A. arilaitensis Re117, was also tested for shrimp waste deproteinization to produce chitin. The protein removal with a ratio E/S of 20 was about 83%. The novelties of the Re117 protease include its high stability to organic solvents and surfactants. These unique properties make it an ideal choice for application in detergent formulations and enzymatic peptide synthesis. In addition, the enzyme may find potential applications in the deproteinization of shrimp wastes to produce chitin.
The present study describes the characterization of crude protease extract from Arthrobacter arilaitensis Re117 and its evaluation in solid and liquid detergent. One caseinolytic protease clear band was observed in zymogram. The crude alkaline protease showed optimum activity at pH 9.0 and 50°C, and it was highly stable over a wide range of pH from 8.0 to 9.0. Proteolytic enzymes showed extreme stability towards non-ionic surfactants (Tween 80, Tween 20 and Triton X-100) and stimulate activity towards oxidizing agents such as sodium perborate. They also showed high stability and compatibility with various laundry solid detergents from Tunisian market. The protease of A. arilaitensis Re117, was also tested for shrimp waste deproteinization to produce chitin. The protein removal with a ratio E/S of 20 was about 83%. The novelties of the Re117 protease include its high stability to organic solvents and surfactants. These unique properties make it an ideal choice for application in detergent formulations and enzymatic peptide synthesis. In addition, the enzyme may find potential applications in the deproteinization of shrimp wastes to produce chitin.
KEYWORDS
A. arilaitensis, Shrimp Wastes, Enzymatic Deproteinization, Organic Solvent-Stable Protease, Laundry Detergents Compatibility
A. arilaitensis, Shrimp Wastes, Enzymatic Deproteinization, Organic Solvent-Stable Protease, Laundry Detergents Compatibility
Cite this paper
Siala, R. , Hammemi, I. , Sellimi, S. , Vallaeys, T. , Kamoun, A. and Nasri, M. (2015) Arthrobacter arilaitensis Re117 as a Source of Solvent-Stable Proteases: Production, Characteristics, Potential Application in the Deproteinization of Shrimp Wastes and Evaluation in Liquid Laundry Commercial Detergents. Advances in Bioscience and Biotechnology, 6, 105-119. doi: 10.4236/abb.2015.62011.
Siala, R. , Hammemi, I. , Sellimi, S. , Vallaeys, T. , Kamoun, A. and Nasri, M. (2015) Arthrobacter arilaitensis Re117 as a Source of Solvent-Stable Proteases: Production, Characteristics, Potential Application in the Deproteinization of Shrimp Wastes and Evaluation in Liquid Laundry Commercial Detergents. Advances in Bioscience and Biotechnology, 6, 105-119. doi: 10.4236/abb.2015.62011.
References
[1] Gupta, R., Beg, Q. and Lorenz, P. (2002) Bacterial Alkaline Proteases: Molecular Approaches and Industrial Applications. Applied Microbiology and Biotechnology, 59, 15-32.
http://dx.doi.org/10.1007/s00253-002-0975-y [2] Cowan, D. (1996) Industrial Enzyme Technology. Trends Biotechnology, 4, 177-178.
http://dx.doi.org/10.1016/0167-7799(96)30009-7 [3] Kumar, C.G. and Takagi, H. (1999) Microbial Alkaline Proteases from a Bioindustrial Viewpoint. Biotechnology Advances, 17, 561-594.
http://dx.doi.org/10.1016/S0734-9750(99)00027-0 [4] Ben Khaled, H., Ktari, N., Ghorbel-Bellaaj, O., Jridi, M., Lassoued, I. and Nasri, M. (2011) Composition, Functional Properties and in Vitro Antioxidant Activity of Protein Hydrolysates Prepared from Sardinelle (Sardinella aurita) Muscle. Journal of Food Science Technology, 51, 622-633.
http://dx.doi.org/10.1007/s13197-011-0544-4 [5] Ktari, N., Fakhfakh, N., Balti, R., Ben Khaled, H., Nasri, M. and Bougatef, A. (2012) Effect of Degree of Hydrolysis and Protease Type on the Antioxidant Activity of Protein Hydrolysates from Cuttlefish (Sepia officinalis) By-Products. Journal of Food Science Technology, 22, 436-448. [6] Anwar, A. and Saleemuddin, M. (1998) Alkaline Proteases: A Review. Bioresource Technology, 64, 175-183.
http://dx.doi.org/10.1016/S0960-8524(97)00182-X [7] Samal, B.B., Kara, B. and Stabinsky, Y. (1990) Stability of Two Novel Serine Proteinases in Commercial Laundry Detergent Formulations. Biotechnology Bioengeiniring, 35, 650-652.
http://dx.doi.org/10.1002/bit.260350611 [8] Banerjee, U.C., Sani, R.K., Azmi, W. and Soni, R. (1999) Thermostable Alkaline Protease from Bacillus brevis and Its Characterization as a Laundry Detergent Additive. Process Biochemestry, 35, 213-219.
http://dx.doi.org/10.1016/S0032-9592(99)00053-9 [9] Siala, R., Fakhfakh, N., Hamza-Mnif, I., Nasri, M., Vallaeys, T. and Sellami-Kamoun, A. (2012) Arthrobacter arilaitensis Re117 Oxidant-Stable Alkaline Metalloprotease: Purification and Biochemical Characterization. Biotechnology and Bioprocess Engineering, 17, 556-564.
http://dx.doi.org/10.1007/s12257-011-0478-8 [10] Shahidi, F. and Synowiecki, J. (1991) Isolation and Characterization of Nutrients and Value-Added Products from Snow Crab (Chinoecetes opilio) and Shrimp (Pandalus borealis) Processing Discards. Journal of Agriculture and Food Chemestry, 39, 1527-1532.
http://dx.doi.org/10.1021/jf00008a032 [11] Bhaskar, N., Suresh, P.V., Sakhare, P.Z. and Sachindra, N.M. (2007) Shrimp Biowaste Fermentation with Pediococcus acidolactici CFR2182: Optimization of Fermentation Conditions by Response Surface Methodology and Effect of Optimized Conditions on Deproteination/Demineralization and Carotenoid Recovery. Enzyme and Microbial Technology, 40, 1427-1434.
http://dx.doi.org/10.1016/j.enzmictec.2006.10.019 [12] Sini, T.K., Santhosh, S. and Mathew, P.T. (2007) Study on the Production of Chitin and Chitosan from Shrimp Shell by Using Bacillus subtilis Fermentation. Carbohydrate Research, 342, 2423-2429.
http://dx.doi.org/10.1016/j.carres.2007.06.028 [13] Rinaudo, M. (2006) Chitin and Chitosan: Properties and Applications. Progress in Polymer Science, 31, 603-632.
http://dx.doi.org/10.1016/j.progpolymsci.2006.06.001 [14] Ghorbel-Bellaaj, O., Hmidet, N., Jellouli, K., Younes, I., Maalej, H., Hachicha, R. and Nasri, M. (2011) Shrimp Waste Fermentation with Pseudomonas aeruginosa A2: Optimization of Chitin Extraction Conditions through Plackett-Burman and Response Surface Methodology Approaches. International Journal of Biological Macromolecules, 48, 596-602.
http://dx.doi.org/10.1016/j.ijbiomac.2011.01.024 [15] Jo, G.H., Jung, W.J., Kuk, J.H., Oh, K.T., Kim, Y.J. and Park, R.D. (2008) Screening of Protease-Producing Serratia marcescens FS-3 and Its Application to Deproteinization of Crab Shell Waste for Chitin Extraction. Carbohydrate Polymers, 74, 504-508.
http://dx.doi.org/10.1016/j.carbpol.2008.04.019 [16] Ghorbel-Bellaaj, O., Jellouli, K., Younes, I., Manni, L., Ouled Salem, M. and Nasri, M. (2011) A Solvent-Stable Metalloprotease Produced by Pseudomonas aeruginosa A2 Grown on Shrimp Shell Waste and Its Application in Chitin Extraction. Applied Biochemistry and Biotechnology, 164, 410-425.
http://dx.doi.org/10.1007/s12010-010-9144-4 [17] Manni, L., Jellouli, K., Ghorbel-Bellaaj, O., Agrebi, R., Haddar, A., Sellami-Kamoun, A. and Nasri, M. (2010) An Oxidant- and Solvent-Stable Protease Produced by Bacillus cereus SV1: Application in the Deproteinization of Shrimp Wastes and as a Laundry Detergent Additive. Applied Biochemistry and Biotechnology, 160, 2308-2321.
http://dx.doi.org/10.1007/s12010-009-8703-z [18] Laemmli, U.K. (1970) Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4. Nature, 227, 680-685.
http://dx.doi.org/10.1038/227680a0 [19] Garcia-Carreno, F.L., Dimes, L.E. and Haard, N.F. (1993) Substrate-Gel Electrophoresis for Composition and Molecular Weight of Proteinases or Proteinaceous Proteinase Inhibitors. Analytical Biochemistry, 214, 65-69.
http://dx.doi.org/10.1006/abio.1993.1457 [20] Kembhavi, A.A., Kulkarni, A. and Pant, A. (1993) Salt-Tolerant and Thermostable Alkaline Protease from Bacillus subtilis NCIM No.64. Applied Biochemistry and Biotechnology, 38, 83-92.
http://dx.doi.org/10.1007/BF02916414 [21] Rao, M.S., Muñoz, J. and Stevens, W.F. (2000) Critical Factors in Chitin Production by Fermentation of Shrimp Biowaste. Applied Microbiology and Biotechnology, 54, 808-813.
http://dx.doi.org/10.1007/s002530000449 [22] Maurer, K.H. (2004) Detergent Proteases. Current Opinion in Biotechnology, 15, 330-334.
http://dx.doi.org/10.1016/j.copbio.2004.06.005 [23] Male, R., Lorens, L.B., Smalas, A.O. and Torrissen, K.R. (1995) Molecular Cloning and Characterization of Anionic and Cationic Variants of Trypsin from Atlantic Salmon. European Journal of Biochemistry, 232, 677-685.
http://dx.doi.org/10.1111/j.1432-1033.1995.677zz.x [24] Bezerra, R.S., Lins, E.J.F., Alencar, R.B., Paiva, P.M.G., Chaves, M.E.C., Coelho, L.C.B.B. and Carvalho Jr., L.B. (2005) Alkaline Proteinase from Intestine of Nile Tilapia (Oreochromis niloticus). Process Biochemistry, 40, 1829-1834.
http://dx.doi.org/10.1016/j.procbio.2004.06.066 [25] El-Hadj Ali, N., Hmidet, N., Ghorbel-Bellaaj, O., Fakhfakh-Zouari, N., Bougatef, A. and Nasri, M. (2011) Solvent-Stable Digestive Alkaline Proteinases from Striped Seabream (Lithognathus mormyrus) Viscera: Characteristics, Application in the Deproteinization of Shrimp Waste, and Evaluation in Laundry Commercial Detergents. Applied Biochemistry and Biotechnology, 164, 1096-1110.
http://dx.doi.org/10.1007/s12010-011-9197-z [26] Klomklao, S., Benjakul, S. and Visessanguan, W. (2004) Comparative Studies on Proteolytic Activity of Splenic Extracts from Three Tuna Species Commonly Used in Thailand. Journal of Food Biochemistry, 28, 355-372.
http://dx.doi.org/10.1111/j.1745-4514.2004.05203.x [27] Gupta, R., Gupta, K., Saxena, R.K. and Khan, S. (1999) Bleach-Stable Alkaline Protease from Bacillus sp. Biotechnology Letters, 21, 135-138.
http://dx.doi.org/10.1023/A:1005478117918 [28] Vulfson, E.N., Halling, P.J. and Holland, H.L. (2001) Methods in Biotechnology: Enzymes in Nonaqueous Solvents. Part II, Vol. 532, 241-422. [29] Oh, K.T., Kim, Y.J., Nguyen, V.N., Jung, W.J. and Park, R.D. (2007) Demineralization of Crab Shell Waste by Peudomonas aeruginosa F722. Process Biochemistry, 42, 1069-1074.
http://dx.doi.org/10.1016/j.procbio.2007.04.007 [30] Wang, S.L., Hsu, W.T., Liang, T.W., Yen, Y.H. and Wang, C.L. (2008) Purification and Characterization of Three Novel Keratinolytic Metalloproteases Produced by Chryseobacterium indologenes TKU014 in a Shrimp Shell Powder Medium. Bioresource Technology, 99, 5679-5686.
http://dx.doi.org/10.1016/j.biortech.2007.10.024 [31] Bustos, R.O. and Healy, M.G. (1994) Microbial Deproteinization of Waste Prawn Shell. Institution of Chemical Engineers Symposium Series, Institution of Chemical Engineers, Rugby, 13-15. [32] Oh, Y.S., Shih, I.L., Tzeng, Y.M. and Wang, S.L. (2000) Protease Produced by Pesudomonas aeroginosa K-187 and Its Application in the Deproteinization of Shrimp and Crab Shell Wastes. Enzyme and Microbial Technology, 27, 3-10.
http://dx.doi.org/10.1016/S0141-0229(99)00172-6
[1] Gupta, R., Beg, Q. and Lorenz, P. (2002) Bacterial Alkaline Proteases: Molecular Approaches and Industrial Applications. Applied Microbiology and Biotechnology, 59, 15-32.
http://dx.doi.org/10.1007/s00253-002-0975-y [2] Cowan, D. (1996) Industrial Enzyme Technology. Trends Biotechnology, 4, 177-178.
http://dx.doi.org/10.1016/0167-7799(96)30009-7 [3] Kumar, C.G. and Takagi, H. (1999) Microbial Alkaline Proteases from a Bioindustrial Viewpoint. Biotechnology Advances, 17, 561-594.
http://dx.doi.org/10.1016/S0734-9750(99)00027-0 [4] Ben Khaled, H., Ktari, N., Ghorbel-Bellaaj, O., Jridi, M., Lassoued, I. and Nasri, M. (2011) Composition, Functional Properties and in Vitro Antioxidant Activity of Protein Hydrolysates Prepared from Sardinelle (Sardinella aurita) Muscle. Journal of Food Science Technology, 51, 622-633.
http://dx.doi.org/10.1007/s13197-011-0544-4 [5] Ktari, N., Fakhfakh, N., Balti, R., Ben Khaled, H., Nasri, M. and Bougatef, A. (2012) Effect of Degree of Hydrolysis and Protease Type on the Antioxidant Activity of Protein Hydrolysates from Cuttlefish (Sepia officinalis) By-Products. Journal of Food Science Technology, 22, 436-448. [6] Anwar, A. and Saleemuddin, M. (1998) Alkaline Proteases: A Review. Bioresource Technology, 64, 175-183.
http://dx.doi.org/10.1016/S0960-8524(97)00182-X [7] Samal, B.B., Kara, B. and Stabinsky, Y. (1990) Stability of Two Novel Serine Proteinases in Commercial Laundry Detergent Formulations. Biotechnology Bioengeiniring, 35, 650-652.
http://dx.doi.org/10.1002/bit.260350611 [8] Banerjee, U.C., Sani, R.K., Azmi, W. and Soni, R. (1999) Thermostable Alkaline Protease from Bacillus brevis and Its Characterization as a Laundry Detergent Additive. Process Biochemestry, 35, 213-219.
http://dx.doi.org/10.1016/S0032-9592(99)00053-9 [9] Siala, R., Fakhfakh, N., Hamza-Mnif, I., Nasri, M., Vallaeys, T. and Sellami-Kamoun, A. (2012) Arthrobacter arilaitensis Re117 Oxidant-Stable Alkaline Metalloprotease: Purification and Biochemical Characterization. Biotechnology and Bioprocess Engineering, 17, 556-564.
http://dx.doi.org/10.1007/s12257-011-0478-8 [10] Shahidi, F. and Synowiecki, J. (1991) Isolation and Characterization of Nutrients and Value-Added Products from Snow Crab (Chinoecetes opilio) and Shrimp (Pandalus borealis) Processing Discards. Journal of Agriculture and Food Chemestry, 39, 1527-1532.
http://dx.doi.org/10.1021/jf00008a032 [11] Bhaskar, N., Suresh, P.V., Sakhare, P.Z. and Sachindra, N.M. (2007) Shrimp Biowaste Fermentation with Pediococcus acidolactici CFR2182: Optimization of Fermentation Conditions by Response Surface Methodology and Effect of Optimized Conditions on Deproteination/Demineralization and Carotenoid Recovery. Enzyme and Microbial Technology, 40, 1427-1434.
http://dx.doi.org/10.1016/j.enzmictec.2006.10.019 [12] Sini, T.K., Santhosh, S. and Mathew, P.T. (2007) Study on the Production of Chitin and Chitosan from Shrimp Shell by Using Bacillus subtilis Fermentation. Carbohydrate Research, 342, 2423-2429.
http://dx.doi.org/10.1016/j.carres.2007.06.028 [13] Rinaudo, M. (2006) Chitin and Chitosan: Properties and Applications. Progress in Polymer Science, 31, 603-632.
http://dx.doi.org/10.1016/j.progpolymsci.2006.06.001 [14] Ghorbel-Bellaaj, O., Hmidet, N., Jellouli, K., Younes, I., Maalej, H., Hachicha, R. and Nasri, M. (2011) Shrimp Waste Fermentation with Pseudomonas aeruginosa A2: Optimization of Chitin Extraction Conditions through Plackett-Burman and Response Surface Methodology Approaches. International Journal of Biological Macromolecules, 48, 596-602.
http://dx.doi.org/10.1016/j.ijbiomac.2011.01.024 [15] Jo, G.H., Jung, W.J., Kuk, J.H., Oh, K.T., Kim, Y.J. and Park, R.D. (2008) Screening of Protease-Producing Serratia marcescens FS-3 and Its Application to Deproteinization of Crab Shell Waste for Chitin Extraction. Carbohydrate Polymers, 74, 504-508.
http://dx.doi.org/10.1016/j.carbpol.2008.04.019 [16] Ghorbel-Bellaaj, O., Jellouli, K., Younes, I., Manni, L., Ouled Salem, M. and Nasri, M. (2011) A Solvent-Stable Metalloprotease Produced by Pseudomonas aeruginosa A2 Grown on Shrimp Shell Waste and Its Application in Chitin Extraction. Applied Biochemistry and Biotechnology, 164, 410-425.
http://dx.doi.org/10.1007/s12010-010-9144-4 [17] Manni, L., Jellouli, K., Ghorbel-Bellaaj, O., Agrebi, R., Haddar, A., Sellami-Kamoun, A. and Nasri, M. (2010) An Oxidant- and Solvent-Stable Protease Produced by Bacillus cereus SV1: Application in the Deproteinization of Shrimp Wastes and as a Laundry Detergent Additive. Applied Biochemistry and Biotechnology, 160, 2308-2321.
http://dx.doi.org/10.1007/s12010-009-8703-z [18] Laemmli, U.K. (1970) Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4. Nature, 227, 680-685.
http://dx.doi.org/10.1038/227680a0 [19] Garcia-Carreno, F.L., Dimes, L.E. and Haard, N.F. (1993) Substrate-Gel Electrophoresis for Composition and Molecular Weight of Proteinases or Proteinaceous Proteinase Inhibitors. Analytical Biochemistry, 214, 65-69.
http://dx.doi.org/10.1006/abio.1993.1457 [20] Kembhavi, A.A., Kulkarni, A. and Pant, A. (1993) Salt-Tolerant and Thermostable Alkaline Protease from Bacillus subtilis NCIM No.64. Applied Biochemistry and Biotechnology, 38, 83-92.
http://dx.doi.org/10.1007/BF02916414 [21] Rao, M.S., Muñoz, J. and Stevens, W.F. (2000) Critical Factors in Chitin Production by Fermentation of Shrimp Biowaste. Applied Microbiology and Biotechnology, 54, 808-813.
http://dx.doi.org/10.1007/s002530000449 [22] Maurer, K.H. (2004) Detergent Proteases. Current Opinion in Biotechnology, 15, 330-334.
http://dx.doi.org/10.1016/j.copbio.2004.06.005 [23] Male, R., Lorens, L.B., Smalas, A.O. and Torrissen, K.R. (1995) Molecular Cloning and Characterization of Anionic and Cationic Variants of Trypsin from Atlantic Salmon. European Journal of Biochemistry, 232, 677-685.
http://dx.doi.org/10.1111/j.1432-1033.1995.677zz.x [24] Bezerra, R.S., Lins, E.J.F., Alencar, R.B., Paiva, P.M.G., Chaves, M.E.C., Coelho, L.C.B.B. and Carvalho Jr., L.B. (2005) Alkaline Proteinase from Intestine of Nile Tilapia (Oreochromis niloticus). Process Biochemistry, 40, 1829-1834.
http://dx.doi.org/10.1016/j.procbio.2004.06.066 [25] El-Hadj Ali, N., Hmidet, N., Ghorbel-Bellaaj, O., Fakhfakh-Zouari, N., Bougatef, A. and Nasri, M. (2011) Solvent-Stable Digestive Alkaline Proteinases from Striped Seabream (Lithognathus mormyrus) Viscera: Characteristics, Application in the Deproteinization of Shrimp Waste, and Evaluation in Laundry Commercial Detergents. Applied Biochemistry and Biotechnology, 164, 1096-1110.
http://dx.doi.org/10.1007/s12010-011-9197-z [26] Klomklao, S., Benjakul, S. and Visessanguan, W. (2004) Comparative Studies on Proteolytic Activity of Splenic Extracts from Three Tuna Species Commonly Used in Thailand. Journal of Food Biochemistry, 28, 355-372.
http://dx.doi.org/10.1111/j.1745-4514.2004.05203.x [27] Gupta, R., Gupta, K., Saxena, R.K. and Khan, S. (1999) Bleach-Stable Alkaline Protease from Bacillus sp. Biotechnology Letters, 21, 135-138.
http://dx.doi.org/10.1023/A:1005478117918 [28] Vulfson, E.N., Halling, P.J. and Holland, H.L. (2001) Methods in Biotechnology: Enzymes in Nonaqueous Solvents. Part II, Vol. 532, 241-422. [29] Oh, K.T., Kim, Y.J., Nguyen, V.N., Jung, W.J. and Park, R.D. (2007) Demineralization of Crab Shell Waste by Peudomonas aeruginosa F722. Process Biochemistry, 42, 1069-1074.
http://dx.doi.org/10.1016/j.procbio.2007.04.007 [30] Wang, S.L., Hsu, W.T., Liang, T.W., Yen, Y.H. and Wang, C.L. (2008) Purification and Characterization of Three Novel Keratinolytic Metalloproteases Produced by Chryseobacterium indologenes TKU014 in a Shrimp Shell Powder Medium. Bioresource Technology, 99, 5679-5686.
http://dx.doi.org/10.1016/j.biortech.2007.10.024 [31] Bustos, R.O. and Healy, M.G. (1994) Microbial Deproteinization of Waste Prawn Shell. Institution of Chemical Engineers Symposium Series, Institution of Chemical Engineers, Rugby, 13-15. [32] Oh, Y.S., Shih, I.L., Tzeng, Y.M. and Wang, S.L. (2000) Protease Produced by Pesudomonas aeroginosa K-187 and Its Application in the Deproteinization of Shrimp and Crab Shell Wastes. Enzyme and Microbial Technology, 27, 3-10.
http://dx.doi.org/10.1016/S0141-0229(99)00172-6