Caseinolytic activity

Bernard, D. et al. Analysis of proteins with caseinolytic activity in a human SC extract revealed a yet unidentified cysteine protease and identified the so called SC thiol protease as Cathepsin L2, J. Invest. Dermatol., 120, 592-600, 2003. 

It is irreversibly inhibited by 1-3 dibromoacetone, M-(4-dimethy 1 amino-3-5dinitrophenyl)maleimide, A/-ethylmaleimide, and iodoacetic acid. The chymotrypsin inhibitors W-tosyl-L-phenylalanyl-chloromethane (TPCK) and W-tosyl-L-lysylchloromethane (TLCK) alkylate the essential SH group [38]. Di-isopropylfluorophosphate (DFP) alkylates the Tyr residues of bromelain but does not react with its catalytic SH. It causes no inhibition of the caseinolytic activity, but the catalytic efficiency against BAEE is enhanced [75]. 

Nath, K. R., and Ledford, R. A. (1972). Caseinolytic activity of micrococd isolated from Cheddar cheese. J. Dairy Sci. 55, 1424-1427. 

The d.s. of macroporous cellulose /m/i5-2,3-carbonate obtained when cellulose reacts with ethyl chloroformate can be controlled by the addition of small quantities of water,225 This procedure produced a matrix that is suitable for the covalent binding of chymotrypsin the insolubilized enzyme is appreciably active towards high-molecular-weight substrates. The amount of chymotrypsin covalently bound to macroporous cellulose /mn5-2,3-carbonate and the caseinolytic activity of the immobilized enzyme are substantially improved by swelling the matrix in DMSO before the enzyme is coupled.22 The porosity of the insoluble support is increased by swelling, so that macromolecules can diffuse into it. The physical properties of cellulose /ra 5-2,3-carbonate are irreversibly destroyed unless it is properly stored. 

The Streptomyces aureofaciens extracellular proteolytic system has been split into four fractions by column chromatography giving three purely caseinolytic fractions and one fraction active toward both starch and casein. The caseinolytic and amylolytic fraction was further fractionated by DEAE-Sephadex A-50 chromatography into one purely amylolytic fraction and another showing both activities, was refractioned into four new fractions by DEAE-cellulose chromatography. These fractions were found to be heterogeneous by polyacrylamide gel electrophoresis, three of them acted on both starch and casein, and a fourth was only caseinolytic. 

The stock of 5% by weight trypsin solution consisted of the enzyme dissolved in 0.04M Tris-HCl buffer (pH=8.1) containing O.OIM CaCl2 (l.lg/1 1 of solution). Sodium azide (0.02% w/v) was added to incubation media to inhibit bacterial growth. The trypsin solution was prepared once before the experiment and the activity measured by caseinolytic method was 2.844 U/ccm. After six days of the experiment it decreased to 1.558 U/ccm The samples after treatment in trypsin were rinsed with deionized water. 

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