The aim of this work was to study some biochemical characteristics of crude alkaline protease extracts from the viscera of goby (
Proteases constitute the most important group of industrial enzymes used in the world today, accounting for approximately 50% of the total industrial enzyme market [
Today, there is an increasing demand for fish proteolytic enzymes in food processing. Fish viscera, one of the most important by-products of fishing industry, is known to be a rich source of digestive enzymes, especially proteases that have high activity over a wide range of pH and temperature conditions [
The most important digestive proteolytic enzymes from fish and aquatic invertebrates viscera are the aspartic protease pepsin secreted from gastric mucosa, and the serine proteases, trypsin, and chymotrypsin secreted from the pancreas, pyloric caeca, and intestine [
Chitin, a homopolymer of
The main sources of raw material for the production of chitin are cuticles of various crustaceans, principally crabs and shrimps. Chitin in biomass is closely associated with proteins, inorganic compounds (such as calcium carbonate), lipids, and pigments. They all have to be quantitatively removed to achieve the high purity of chitins necessary for biological applications [
Conventionally, to extract chitin from crustacean shells, chemicals processing for demineralization and deproteinization have been applied. Raw materials were first treated with dilute hydrochloric acid at room temperature to remove metal salts, particularly calcium carbonate, and then with strong bases to remove proteins [
In the present paper, we describe the extraction and characterization of alkaline proteases from
Casein sodium salt from bovine milk, trichloroacetic acid (TCA), ethylene diamine tetraacetic acid (EDTA), and bovine serum albumin were purchased from Sigma Company Co. (St Louis, Mo, USA). Hydrochloric acid and Tris(hydroxymethyl)aminomethane were procured from Panreac Quimica SA (Barcelone, Spain). Sodium dodecyl sulphate (SDS), acrylamide, ammonium persulphate,
Goby (
Viscera (20 g) were separated and rinsed with distilled water, and then homogenized for 5 minutes with 20 mL of extraction buffer (10 mM Tris-HCl, pH 8.0) with the use of tissue homogenizer. The resulting preparations were centrifuged at 8500 ×g for 30 minutes at 4°C. The pellets were discarded and the supernatants were collected and then frozen at −20°C and used as crude protease extracts. All enzymatic assays were conducted within a week after extraction.
Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out as described by Laemmli [
Protease activity staining was performed on SDS-PAGE according to the method of Garcia-Carreno et al. [
Protease activity in the crude alkaline enzyme extracts was measured by the method described by Kembhavi et al. [
The optimum pH of the crude protease extracts was studied over a pH range of 5.0–13.0, using casein as a substrate at 50°C. For the measurement of pH stability, the crude enzyme extracts were incubated for 1 hour at 4°C in different buffers and then the residual proteolytic activities were determined under standard assay conditions. The following buffer systems were used: 100 mM sodium acetate buffer for pH 5.0-6.0, Tris-HCl buffer for pH 7.0-8.0, glycine-NaOH buffer for pH 9.0–11.0, Na2HPO4-NaOH buffer for pH 12.0, and KCl buffer for pH 13.0.
To investigate the effect of temperature, the activity was tested using casein as a substrate at the temperature range from 30 to 80°C in 100 mM Tris-HCl buffer, pH 8.0 for
The influence of various metals ions, at a concentration of 5 mM, on enzyme activity was investigated by adding the monovalent (Na+ or K+) or divalent (Mg2+, Hg2+, Ca2+, Zn2+, Cu2+, Co2+, Ba2+, or Mn2+) metal ions to the reaction mixture. The activity of the crude enzyme extracts without any metallic ion was considered as 100%. The effect of NaCl concentrations on the activity of the alkaline crude protease extracts was studied, using casein as a substrate, by increasing NaCl concentrations in the reaction mixture.
The effects of some surfactants (Triton X-100, Tween 80, and SDS) and oxidizing agents (sodium perborate) on alkaline crude proteases stability were studied by preincubating enzymes for 1 hour at 30°C. The residual activities were measured at optimum conditions for each crude enzyme. The activity of the crude enzyme extract without any additive was taken as 100%.
The SWP was prepared in our laboratory. Briefly, shrimp waste, collected from the marine food processing industry, was washed thoroughly with tap water and then cooked 20 minutes at 90°C. The solid material obtained was dried, minced to obtain a fine powder, and then stored in glass bottles at room temperature. The chemical composition (proteins, chitin, lipids, and ash) was determined.
The moisture and ash content were determined according to the AOAC standard methods 930.15 and 942.05, respectively, [
Shrimp shell wastes (15 g) were mixed with 100 mM Tris-HCl buffer pH 8.0 at a ratio of 1 : 3 (w/v), minced and then cooked for 20 minutes at 90°C to inactivate endogenous enzymes. The cooked sample was then homogenized in a Moulinex blender for about 2 minutes. The pH of the mixture was adjusted to 8.0, and then the shrimp waste proteins were digested with proteolytic enzymes at 45°C using en enzyme/substrate ratio of 10/1 (unit of enzyme/mg of protein). After 3-hour incubation at 45°C, the reaction was stopped by heating the solution at 90°C during 20 minutes to inactivate enzymes. The shrimp waste protein hydrolysates were then centrifuged at 5000 ×g for 20 minutes to separate insoluble and soluble fractions. The solid phase was washed, pressed manually through four layers of gauze, and then dried for 1 hour at 60°C. The protein content was analyzed to measure the protein removal. The press cake was packed in a plastic bag and stored at −20°C until further processing.
Deproteinization percentage (%DP) was calculated by the following equation as described by Rao et al. [
All experiments were carried out in triplicate, and average values with standard deviation errors are reported. Mean separation and significance were analyzed using the SPSS software package (SPSS, Chicago, Ill). Correlation and regression analysis were carried out using EXCEL program.
In order to estimate the number of proteases in the alkaline crude enzyme extracts, samples were separated by SDS-PAGE, and then proteolytic activities were revealed by casein zymography activity staining. Casein zymography is a very sensitive and rapid assay method that detects nanogram of proteins, in contrast with SDS-PAGE which detects micrograms.
As can be observed in Figure
Activity staining of the crude alkaline protease extract from the viscera of
The activity of proteolytic enzymes was determined at different pH values from 5.0 to 13.0. The pH activity profiles of the crude alkaline proteases are shown in Figure
Effect of pH on activity (a) and stability (b) of alkaline crude protease extracts. The protease activity was assayed in the pH range 5.0–13.0 using buffers of different pH values at 50°C. The maximum activity of each crude enzyme extract was considered as 100%. The pH stability was determined by incubating the crude enzymes in different buffers for 1 hour at 4°C and the residual activities were measured at the optimum conditions of each enzyme preparation. The activity of the enzyme before incubation was taken as 100%. Buffer solutions used for pH activity and stability are presented in Section
The optimum pH for the crude protease of
With
The pH stability profiles of the three crude alkaline proteases are reported in Figure
The enzyme preparation from scorpionfish, which is highly active in the alkaline pH range, was also stable over a wide pH range. These results suggest that the viscera of scorpionfish would be a potential source of alkaline proteases for certain industrial applications that require high alkaline conditions, such as detergents. In fact, one of the most important parameters for selection proteases for detergents is the optimum pH. Since the pH of laundry detergents is commonly alkaline (in the range of 9.0–12.0) [
Optima temperatures for activity of crude alkaline proteases were determined in order to assess their suitability for biotechnological applications. The relative activities at various temperatures using casein as a substrate are reported in Figure
Effect of temperature on activity of alkaline crude protease extracts. The temperature profile was determined by assaying protease activity at temperatures between 30 and 80°C. The optimum activity was taken as 100%. Values are means of three independent experiments.
The relative activities of goby proteases at 40 and 60°C were 54% and 70%, respectively. However, an appreciable decrease in enzyme activity was observed above 65°C, due to thermal denaturation. Thornback ray proteases were more active at 60°C than the other crude proteases, retaining 90% of their activity after 1-hour incubation. However, the relative activities of
Thermal stability of crude alkaline proteases is depicted in Figure
Effect of temperature on thermal stability of the crude alkaline proteases from goby (a), thornback ray (b) and scorpionfish (c). The temperature stability was determined by incubating the crude extract at temperatures from 30 to 70°C for 1 hour. The residual enzyme activity was measured under the standard conditions assay at different times. The original activity before preincubation was taken as 100%. Values are means of three independent experiments.
The enzyme preparations from
The effects of various metal ions, at a concentration of 5 mM, on the activity of the crude alkaline proteases were studied at optimum conditions for each crude enzyme by the addition of the respective cations to the reaction mixture (Table
Effects of various metal ions (5 mM) on protease activity.
Metal ions | Relative activity (%) | ||
Goby | Scorpionfish | ||
Control | 100 | 100 | 100 |
Na+ | 100 | 91 | 110 |
K+ | 100 | 91 | 80 |
Mg2+ | 117 | 122 | 100 |
Mn2+ | 47.5 | 83 | 37.5 |
Zn2+ | 20 | 105 | 23 |
Cu2+ | 17.5 | 67 | 47.5 |
Hg2+ | 36 | 62 | 29.5 |
Fe2+ | 0 | 31 | 0 |
Ca2+ | 110 | 129 | 111 |
Ba2+ | 110 | 97 | 100 |
The addition of CaCl2 and MgSO4 increased the activity of crude protease extracts of goby and scorpionfish. Ca2+ increased the activity of crude proteases from goby and scorpionfish to 110% and 129%, respectively. These results indicated that Ca2+ was very effective in improving the activity of the crude proteases. The enhancement of protease activity in the presence of calcium may be explained by the strength of interactions inside protein molecules and the better stabilization of enzymes against thermal stabilization. However, the activity of
The ions Ba2+ affect partially the protease activity with a relative activity between 87% and 96%. However, Fe2+ and Hg2+ affect greatly the activity of all crude enzymes. The presence of 5 mM NaCl and KCl did not affect protease activity.
All the commercial detergents contain hydrolytic enzymes such as proteases. In addition to activity and stability at high pH range and various temperatures [
The suitability of crude alkaline proteases as detergent additive was investigated by testing their stability in the presence of some surfactants and oxidizing agents. As shown in Table
Stability of alkaline proteases in the presence of various surfactants and oxidizing agents.
Surfactants/oxidizing agents | Concentration (%) | Residual activity (%) | ||
Goby | Scorpionfish | Thornback ray | ||
None | 0 | 100 | 100 | 100 |
Triton X-100 | 5 (v/v) | 100 | 107 | 117 |
Tween 20 | 5 | 82 | 109 | 115 |
Tween 80 | 5 | 90 | 107 | 100 |
SDS | 0.1 (w/v) | 40 | 73.5 | 13 |
0.5 | 33 | 44 | 0 | |
1 | 14 | 16 | 0 | |
Sodium perborate | 0.2 | 92.3 | 106 | 88 |
1 | 66 | 100 | 70 |
Enzyme preparations were incubated with different surfactants and oxidizing agents for 1 hour at 30°C and the remaining activity was measured under standard conditions. The activity is expressed as a percentage of the activity level in the absence of additives.
In addition, we investigated the effects of oxidizing agents on the crude protease extract. Thornback ray and goby proteases were little influenced by oxidizing agents, retaining about 70% and 66% of their initial activity after incubation for 1 hour at 30°C in the presence of 1% (w/v) sodium perborate, respectively.
Interestingly, the crude enzyme of scorpionfish remains fully active after 1 hour incubation at 40°C. The stability of scorpionfish enzyme extract against sodium perborate was higher than A21 protease from
The effect of NaCl concentration on the activity of crude alkaline proteases is shown in Figure
Effect of NaCl concentration on the activity of alkaline crude protease extracts.
Chitin, a polysaccharide found in abundance in the shell of crustaceans, is closely associated with proteins. Therefore, deproteinization in chitin extraction process is crucial. Chemical treatment requires the use of HCl and NaOH, which can cause deacetylation and depolymerization of chitin.
Few studies on the use of proteolytic enzymes for the deproteinization of shrimp wastes have been reported. To the best of our knowledge, there are no available reports on the enzymatic deproteinization of shrimp wastes by fish proteases. Many factors, such as the specificity of the enzyme used for the proteolysis, E/S ratio and the conditions used during hydrolysis (initial temperature value and hydrolysis time) have been reported to influence the enzymatic deproteinization process.
In the present study, alkaline proteases from
Deproteinization degree of shrimp waste by the crude alkaline proteases.
The deproteinization activity of crude proteases used in this study was similar to many bacterial proteases reported in many previous studies [
In the present study, alkaline proteases were extracted from the viscera of
Crude alkaline proteases from
The alkaline crude proteases were found to be effective in the deproteinization of shrimp waste powder. The protein removals with a ratio E/S of 10 were more than 76%.
Considering their promising properties, crude protease extracts used in this study may find potential applications in the deproteinization of shrimp waste to produce chitin and chitosan. Further research is needed to purify alkaline proteases, and to determine their properties as a possible biotechnological tool in the fish processing and food industries.
This work was funded by the Ministry of Higher Education and Scientific Research, Tunisia.