The purpose of this work was to review the recovery of proteolytic and collagenolytic activities from rayfish (< 0. most likely, insufficient linearity in the response at very long times for exhaustion of the 64862-96-0 supplier substrate (that could already happen at 20 min). Defining 1 EU of proteolytic activity as the enzyme concentration that generate 1 M (181.2 g) of tyrosine per min at 30 C and pH 8, using casein as standard, and taking into account the ponderal proportions in the homogenates it can be established that D section has 8 EU per g of dry weight. 2.2. Optimization of the experimental conditions for proteolytic activity quantification In order to improve the experimental conditions to measure proteolytic activities, a study of optimization of the enzymatic reaction progress was carried out. To establish the optimum test conditions firstly the reaction time during which tyrosine production maintains a reasonably constant slope with constant concentration of reagents should be known. Figure 2 shows the kinetics of tyrosine production using extracts from D section (< 0.05) 64862-96-0 supplier in the range 7C8. In all cases, Triton improves the extractions of enzymatic activity. Nevertheless, the effect of the Ca+2 ions concentration tested to pH 8 was not significant regarding non-Ca+2 reinforcement. Decreased activities were observed throughout the time for all experimental conditions. Similar results of stability were obtained for proteases isolated from Monterey sardine , sardinelle  and 64862-96-0 supplier Japanese sandfish . Figure 3. Proteolytic activities as % of the maximum value. ?: with 0.2% of Triton X-100; : without Triton X-100; ?: with 0.2% of Triton X-100 and Ca2+ 0.02 M. The corresponding confidence intervals of indie experiments aren’t proven … 2.4. Joint aftereffect of pH and temperatures on proteolytic activity In Body 4 combined aftereffect of pH and temperatures on proteolytic activity of are depicted. The experimental domains had been (7C10) for pH and (10C70 64862-96-0 supplier C) for temperatures. Response surface was fitted to the altered equation (3) of Rosso described in the Experimental section. The joint optimal pH (were 66.92 0.73 C and 11.69 2.44, respectively. The fitting of experimental data were statistically acceptable. The mathematical function was consistent (Fishers test; < 0.05) and the parametric estimations were significant (Students = 0.963). Physique 4. Combined effect of heat and pH on total proteolytic activity (Y) of rayfish extracts from D section (PRC). Response surface is the in shape of the experimental data (?) to the equation (3). 2.5. Partial purification of Procolax Firstly, precipitation with (NH4)2SO4 was studied. The experimental results are collected in Table 1. The increase of specific activity was low and, taking into account the recoveries of activity obtained, a 60% of saturation is the best option (see column F = C D). On the other hand, using overcooled ethanol for precipitation no more than 16% of protease and 23% of collagenase was obtained (data not shown). Similar values were released when PEG-4000 was employed as precipitant. However, high total proteolytic activities were recovered with overcooled acetone in high solvent proportions (5 volumes of acetone per residue weight). This last result was very similar to those reported by Michail et al.  using cold acetone on partial purification of 64862-96-0 supplier proteolytic enzymes from trout heads. Table 1. Effect of (NH4)2SO4 on total protein (A), total activity (B), specific activity (C), recovery of activity (D), purification factor (E) and combined response C D (F). Membrane technology working with ultrafiltration-diafiltration scheme was another resource tested in order to collect the enzymatic activity. A molecular cut-off of 675 kD filtrated the most of the protease and collagenase activity. In the retentate, obtained with a concentration factor of 4, only 13.6% (proteases) and 11.3% (collagenases) of the initial activity was recovered. The next step was to process the permeate from 675 kD with a membrane cut-off at 100 kD. In this way, a retentate with a concentration factor of Rabbit Polyclonal to Parkin 2, made up of 46.7% (proteases) and 47.5% (collagenases) of the initial activity, was performed. Furthermore, specific activities are increased around 15 occasions and no activity levels are lost in the permeate stream. It is therefore evident that shearing process due to recirculation and pump shear stress lead to enzyme deactivation. 2.6. Protocol for Procolax recovery Bearing in mind the results obtained in previous sections a simple protocol of extraction and purification could be proposed. Physique 5 shows the diagram for the process of recovery. The corresponding stages are as follows (in all cases working at 0C4 C): Initially, a Cvolumes (in litres) of phosphate buffer (0.05 M, pH 7.5) with Triton X-100 (0.2%) and KCl (0.08M)..