Adaptation and Validation of a Proteolytic Activity Methodology Using Dye-Crosslinked Substrate Tablets


Enzymatic activities are important to be quantified in products as enzymatic cleaners, which are used in medical and surgical devices reprocessing. Enzymatic activities are critical for the proper chemical cleaning that intends to remove solid organic dirt from inaccessible sites. The most important enzyme for this purpose is the protease, which is able to dissolve the main dirt attached to medical and surgical instruments. In this context, this study contributes to the development of a new proteolytic activity quantification method and its validation. The methodology is based on colorimetry and uses a UV-Vis spectrophotometer to measure the substrate hydrolysis by the blue color intensity, employing Protazyme AK tablets as substrate.

Share and Cite:

Martins, F. and Ramires, E. (2014) Adaptation and Validation of a Proteolytic Activity Methodology Using Dye-Crosslinked Substrate Tablets. Advances in Enzyme Research, 2, 113-124. doi: 10.4236/aer.2014.23012.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] Rutala, W.A. and Weber, D.J. (2013) Disinfection and Sterilization: An Overview. American Journal of Infection Control, 41, S2-S5.
[2] Killeen, S. and McCourt, M. (2012) Decontamination and Sterilization. Surgery, 30, 687-692.
[3] Murdoch, H., Taylor, D., Dickinson, J., Walker, J.T., Perrett, D., Raven, N.D.H. and Sutton, J.M. (2006) Surface Decontamination of Surgical Instruments: An Ongoing Dilemma. Journal of Hospital Infection, 63, 432-438.
[4] Augustin, M., Ali-Vehmas, T. and Atroshi, F. (2004) Assessment of Enzymatic Cleaning Agents and Disinfectants against Bacterial Biofilm. Journal of Pharmaceutical Sciences, 7, 55-64.
[5] Ransjö, U., Engström, L., Håkansson, P., Ledel, T., Lindgren, L., Lindqvist, A.L., Marcusson, E. and Rudbäck, K. (2001) A Test for Cleaning and Disinfection Processes in a Washer-Disinfector. APMIS, 109, 299-304.
[6] Lopes, L.S., Silva, F.S.Q., Silva, A.S. and Delgado, I.F. (2013) Validation of a UV Spectrophotometric Method for Determination of Proteolytic Activity of Enzymatic Detergents. Journal of Surfactants and Detergents, 16, 445-448.
[7] Orgaz, B., Neufeld, R.J. and SanJose, C. (2007) Single-Step Biofilm Removal with Delayed Release Encapsulated Pronase Mixed with Soluble Enzymes. Enzyme and Microbial Technology, 40, 1045-1051.
[8] Agência Nacional de Vigilancia Sanitária (Anvisa) (2012) RDC no 55 de 14 de novembro de 2012—Dispõe sobre os detergentes enzimáticos de uso restrito em estabelecimentos de assistência à saúde com indicação para limpeza de dispositivos médicos e dá outras providências. Diário Oficial da Uniõo, Brasília.
[9] Valls, C., Pujadas, G., Garcia-Vallve, S. and Mulero, M. (2011) Characterization of the Protease Activity of Detergents. Biochemistry and Molecular Biology Education, 39, 280-290.
[10] Silva, C.J.S.M., Zhang, Q., Shen, J. and Cavaco-Paulo, A. (2006) Immobilization of Proteases with a Water Soluble- Insoluble Reversible Polymer for Treatment of Wool. Enzyme and Microbial Technology, 39, 634-640.
[11] Bendicho, S., Martí, G., Hernández, T. and Martín, O. (2002) Determination of Proteolytic Activity in Different Milk Systems. Food Chemistry, 79, 245-249.
[12] Sarath, G., Zeece, M.G. and Penheiter, A.R. (2001) Protease Assay Methods. In: Beynon, R. and Bond, J.S., Eds., Proteolytic Enzymes, Oxford University Press, New York, 45-51.
[13] Galo, A.L. and Colombo, M.F. (2009) Long Optical Path Length Spectrophotometry in Conventional Double-Beam Spectrophotometers: A Simple Alternative for Investigating Samples of Very Low Optical Density. Química Nova, 32, 488-492.
[14] Megazyme International Ireland Ltd. Assay of endo-Protease Using Protazyme AK Tablets [Internet].
[15] Agência Nacional de Vigilancia Sanitária (Anvisa) (2003) Resolução RE 899 de 29 de maio de 2003—Guia para Validação de Métodos Analíticos e Bioanalíticos. Diário Oficial da União, Brasília.
[16] International Organization for Standardization (1994) ISO 5725-3. Accuracy (Trueness and Precision) of Measurement Methods and Results—Part 3: Intermediate Measures of the Precision of a Standard Measurement Method. ISO/TC 69 Applications of Statistical Methods. Geneva.
[17] Polonini, H.C., Santos, F.C., Vaz, U.P., Brandão, M.A.F., Raposo, N.R.B. and Ferreira, A.O. (2011) Development and Validation of Analytical Method for Determination of Simvastatin in Capsules. Química Nova, 34, 516-519.

Copyright © 2023 by authors and Scientific Research Publishing Inc.

Creative Commons License

This work and the related PDF file are licensed under a Creative Commons Attribution 4.0 International License.