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.
Proteases constitute one of the most important groups of industrial enzymes, and account for at least 60% of all global enzyme sales [
Shrimp by-products have been identified as an animal protein source of a great potential and also as an important source of chitin and asthaxanthin [
Over the years, techniques have been developed for the recovery and exploitation of these by products in valuable biopolymers such as chitin and chitosan [
The present paper describes some biochemical characterization of the alkaline crude enzyme preparation from A. arilaitensis as well as its compatibility with commercial laundry detergents, oxidants, surfactant agents and organic solvents and its application in the deproteinization of shrimp wastes.
Arthrobacter arilaitensis is one of the major bacterial species found at the surface of cheeses, especially in smear-ripened cheeses, where it contributes to the typical colour, flavour and texture properties of the final pro- duct. In model cheese experiments, the addition of small amounts of iron strongly stimulates the growth of A. arilaitensis, indicating that cheese is a highly iron-restricted medium. We suggest that there is a strong selective pressure at the surface of cheese for strains with efficient iron acquisition and salt-tolerance systems together with the abilities to catabolize substrates such as lactic acid, lipids and amino acids.
A. arilaitensis strain Re117 was previously isolated from the surface of a Reblochon cheese. This strain is the type strain of the species, and was deposited in the CIP public strain collection (Collection of Institut Pasteur) as strain CIP 108037. It was routinely grown at 25˚C in brain heart infusion broth (BHI, Biokar Diagnostics, Beauvais, France) under aerobic conditions (rotary shaker at 150 rpm, volume of broth equivalent to 20% of the volume of the conical flask).
The growth of the microorganism was estimated by the determination of colony-forming units (CFU/ml). All experiments were carried out in duplicate and repeated at least twice.
The initial medium M1 used for the production of proteases by A. arilaitensis Re117 consists of (g/l) Mirabilis Jalapa Tuber Powder (MJTP) 10.0, ammonium sulfate 2.0, CaCl2 0.5 and MgSO4・7H2O 0.5. Cultures were performed on a rotatory shaker (150 rpm) for 24 h at 25˚C in 250-ml conical flasks with a working volume of 25 ml. Media were autoclaved at 121˚C for 20 min. The growth of the microorganism was estimated by total plate count on nutrient agar. The culture medium was centrifuged at 12,000 × g for 15 min at 4˚C, and the cell-free supernatant was used for the estimation of proteolytic activity.
To study the relation between protease production and the growth profile of the bacterium, 100 ml of the optimized and unoptimized production media (MCD) was inoculated in 1-L flasks, and the growth was measured at regular intervals by viable count (spread plate method) determination. The protease production at different time intervals was determined using the standard protease assay.
Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out as described by Laemmli 1970 [
Zymography was performed in conjunction with SDS-PAGE according to the method described by Garcia- Carreno et al., [
Protease activity in the crude alkaline enzyme crude was measured by the method of Kembhavi et al. (1993) [
The reaction was stopped by the addition of 0.5 ml of 20% (w/v) trichloroacetic acid. The mixture was allowed to stand at room temperature for 15 min and then centrifuged at 10,000 × g for 15 min to remove the precipitate. The acid-soluble material was estimated spectrophotometrically at 280 nm. A standard curve was generated using solutions of 0 - 50 mg/l tyrosine. One unit of protease activity was defined as the amount of enzyme required to liberate 1 μg of tyrosine per min under the experimental conditions used. Values are the means of three independent experiments.
The optimum pH of the crude protease was studied over a pH range of 5.0 - 12.0 using casein as a substrate at 50˚C.
For the measurement of pH stability, the crude enzyme preparation was incubated for 1 h at 30˚C in different buffers, and then the residual proteolytic activity was determined under standard assay conditions. The following buffer systems were used: 100 mM acetate buffer, pH 5.0 - 6.0; 100 mM Tris-HCl buffer, pH 7.0 - 8.0; 100 mM glycine-NaOH buffer, pH 9.0 - 11.0; 100 mM Na2HPO4-NaOH buffer pH 12.0.
To investigate the effect of temperature, the activity was tested using casein as a substrate at the temperature range of 20˚C to 70˚C in 100 mM glycine-NaOH buffer, pH 9.0.
Thermal stability was examined by incubating the enzyme preparation for 60 min at different temperatures from 30˚C to 70˚C. Aliquots were withdrawn at desired time intervals to test the remaining activity at pH 9.0˚C and 50˚C. The non heated crude enzyme was taken as 100%.
The influence of various metal 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+, Ba2+ or Mn2+) metal ions to the reaction mixture.
The activity of the crude enzyme without any metallic ions was considered as 100%.
Enzyme activity was assayed in the presence of NaCl at various concentrations (0% - 15% (w/v)). The relative enzyme activity was determined at 50˚C for 15 min, using casein as a substrate.
The effects of enzyme inhibitors (5 mM), on protease activity were studied using phenylmethylsulfonyl fluoride (PMSF), β-mercaptoethanol, pepstatin A and ethylene-diaminetetraacetic acid (EDTA). The alkaline crude enzyme extract was preincubated with each inhibitor for 30 min at 25˚C, and then the remaining protease activity was tested using casein as a substrate. The activity of the enzyme assayed in the absence of inhibitors was taken as control.
The suitability of the crude protease as a detergent additive was determined by testing its stability towards some surfactants (SDS, Triton X-100, Tween 80) and oxidizing agents (sodium perborate). The enzyme preparation was incubated with different concentrations of additives for 1 h at 30˚C, and then the residual proteolytic activities were measured under standard assay conditions. The activity of the enzyme preparation, incubated under similar conditions without any additives, was taken as 100%.
The compatibility of the crude alkaline protease extract with liquid laundry detergents was studied using commercially available detergents: Dixan (Henkel-Spain), Nadhif (Henkel-Alki-Tunisia), Lav+ (STID-Tunisia), Carrefour (U.E/Geproduceerd-France) and Tex’til (U.E/Vervaardigd-Belgium). Commercial detergents were diluted 100-fold in tap water to simulate wash solution. The endogenous enzymes contained in these detergents were inactivated by heating the diluted detergents for 1 h at 65˚C prior to the addition of the enzyme preparation. The crude protease was incubated with different detergents for 1 h at 30˚C and 40˚C, and then the remaining activities were determined under the standard assay conditions.
The enzyme activity of a control, without detergent, incubated under the similar conditions, was taken as 100%.
The organic solvent stability of the enzyme was studied by incubating the crude enzyme with various organic solvents (50%; v/v; methanol, N,N-dimethylformamide (DMF), dimethylsulfoxide (DMSO), diethyl ether, hexane, acetone, and isopropanol) at 30˚C with shaking (150 rpm) for 30 days. Aliquots were withdrawn at desired time intervals to test the remaining protease activity. Crude enzyme diluted using buffer was considered as control [
Shrimp wastes (50%, w/v) were minced and cooked at 100˚C for 20 min to inactivate endogenous enzymes. The cooked sample was then homogenized in a Moulinex® blender for 2 min. The pH of the mixture was adjusted to 9.0, then, the shrimp wastes proteins were digested with the Re117 crude enzyme. After 3 hours incubation at 50˚C, the reaction was stopped by heating the solution during 20 min at 90˚C. The shrimp wastes protein hydrolysate was then centrifuged 20 min at 5000 × g to separate insoluble and soluble fractions. The solid phase was washed with distilled water and dried for 1 h at 60˚C.
Deproteinization (DP) was expressed as percentages and computed by the following equation as described by Rao et al. [
where PO and PR are protein concentrations (%) before and after hydrolysis, while O and R represent the mass (g) of original sample and hydrolyzed residue in dry weight basis, respectively.
Statistical analyses were performed with Statgraphics ver.5.1, professional edition (Manugistics Corp., USA) using ANOVA analysis. Differences were considered significant at P < 0.05. All tests were carried out in triplicate.
In this study, protease production by A. arilaitensis strain was first tested in M1 medium containing different carbon sources at a concentration of 10 g/l.
A. arilaitensis strain exhibited a higher production level of protease in culture media containing SHVF (181 U/ml) as carbon source followed by chicken feather meal (96 U/ml) and SWP (91 U/ml). On the contrary, protease production between 27 and 2.18 U/ml was obtained with the other carbon sources tested (casein, hulled grain of wheat, MJTP, glucose, starch) (
The effect of various organic and inorganic nitrogen sources, at a concentration of 2 g/l, was examined in M1 medium containing 45 g/l of SHVF (
Since pastone was the best nitrogen source for protease synthesis by A. arilaitensis, the effect of its concen- tration on the enzyme production was studied, and the maximum occurred at 5 g/l (2072 U/ml) (
The time course of protease activity and the growth of A. arilaitensis Re117 for the Chemical defined medium (MCD) and at different temperatures are shown in
The maximum activity was obtained at 24˚C after 24 h and decreased gradually while increasing the temperature. At temperature 30˚C and 37˚C, the proteolytic activities reached 49 and 30 U/ml, respectively.
The obtained results indicate that MCD medium is an excellent medium for growth and not for protease production by A. arilaitensis.
In order to estimate the number of proteases in the crude enzyme extract, sample was separated by SDS-PAGE, and then proteolytic activity was revealed by casein zymogram activity staining. As can be observed in
The relative activity values at various pHs from 3.0 to 12.0 are shown in
The pH stability profile, reported in
The residual activities at pH 10.0 and 11.0 were about 70% and 55%, respectively. These results suggest that the A. arilaitensis could be a potential source of proteases for certain industrial applications that require high alkaline conditions.
The effect of temperature on protease activity was determined by assaying enzyme activity at different temperatures (
The thermal stability of A. arilaitensis protease is depicted in
The effect of NaCl concentration on the activity of A. arilaitensis crude enzyme was studied at pH 9.0 and 50˚C by the addition of NaCl to the reaction mixture. As reported in
The effects of various enzyme inhibitors, such as chelating agents and group-specific reagents, on enzyme activity were studied and reported in
In addition to activity and stability at high pH range and various temperatures, enzymes incorporated into detergent formulations must be compatible and stable with all commonly used detergent compounds like surfactants, bleaches and other additives which might be present in the formulation (stability during storage and washing)
Chemicals | Concentration | Activity (%) |
---|---|---|
None | - | 100 |
PMSF | 5 mM | 80 ± 1.36 |
EDTA | 5 mM | 0 |
β-mercaptoethanol | 5 mM | 92 ± 1 |
DNTB | 5 mM | 100 |
Ca2+ | 5 mM | 84 ± 2 |
Ba2+ | 5 mM | 54 ± 1 |
Zn2+ | 5 mM | 89.5 ± 1.2 |
Cu2+ | 5 mM | 48.62 ± 1.62 |
Mg2+ | 5 mM | 84 ± 2.2 |
Mn2+ | 5 mM | 78.54 ± 2.4 |
K+ | 5 mM | 87.8 ± 2.1 |
Na+ | 5 mM | 85 ± 1.8 |
Hg2+ | 5 mM | 0 |
Fe2+ | 5 mM | 27 ± 2 |
Cu2+ | 71 ± 1.5 |
The crude enzyme was pre-incubated with various enzyme inhibitors for 30 min at 25˚C and the remaining activity was determined at pH 9.0 and 50˚C. Enzyme activity measured in the absence of any inhibitor was taken as 100%. The effect of metal ions on the activity of the crude enzyme was determined by incubating the enzyme in the presence of various metal ions for 15 min at 50˚C and pH 9.0.
[
The crude protease was highly stable in the presence of the non-ionic surfactants. It retained 80 and 79% of its initial activity in the presence of 1% Triton X-100 and Tween 80, respectively. In addition, the purified enzyme was slightly affected by oxidizing agents, and it retained more than 70% of its activity after incubation in the presence of H2O2. The Arthrobacter crude enzyme activity increased by 118% in the presence of 0.1% (w/v) sodium perborate compared to the control test.
The high stability of the enzyme in the presence of oxidising agents is a very important characteristic for its eventual use in detergent formulations.
Stability of the crude enzyme of A. arilaitensis in the presence of various commercial solid detergents.
The high activity and stability of the crude enzyme of the A. arilaitensis preparation in the pH range from 7.0 to 11.0, and its relative stability towards surfactants and oxidizing agents are very useful for its potential application as detergent additive. To check the compatibility of the crude protease with liquid and solid detergents, enzyme preparation was preincubated in the presence of various commercial laundry detergents for 1 h at different temperatures. Commercial protease Purafect® 2000 E was used under the same conditions as the crude enzyme.
The data presented in
The use of enzymes in organic media has been one of the novelties of catalysis in the last few years. One major concern in this regard has been their instability/low-activity in organic media, since proteases may be suitable for peptide and ester synthesis under non aqueous conditions [
These results are similar to earlier reports showing increased protease stability in the presence of organic solvent. In fact, the half-life of the B. cereus SV1 protease was approximately 34 days in the absence of organic solvent. However, in the presence of ethyl acetate and DMF, the half-lives were 51 and 48 days, respectively
Concentration (%) | Remaining activity (%) | |
---|---|---|
None | - | 100 |
Sodium perborate | 0.1 | 118 ± 1.3 |
1 | 67.16 ± 0.8 | |
SDS | 0.1 | 0 ± 0.4 |
0.5 | 0 ± 0.4 | |
1 | 0 ± 0.4 | |
Triton X-100 | 1 | 80 ± 1.4 |
5 | 77.2 ± 1.8 | |
Tween 80 | 1 | 79 ± 1.3 |
5 | 35.7 ± 0.7 | |
Tween 20 | 1 | 68.3 ± 1 |
5 | 40.9 ± 1.0 | |
H2O2 | 1 | 70.2 ± 0.3 |
5 | 68.5 ± 0.7 |
The alkaline crude enzyme extract was pre-incubated with surfactants and oxidizing agents for 1 h at 30˚C, and the remaining activity was measured at pH 9.0 and 50˚C. The activity is expressed as a percentage of the activity level in the absence of additives.
Organic solvent | Half-life (days) | Remaining activity (%) after 60 days |
---|---|---|
None | 5 | 27 ± 0.73 |
DMSO | >28 | 100 ± 0.8 |
Methanol | 4 | 62.8 ± 1.9 |
Diethyl ether | 20 | 34 ± 0.24 |
DMF | 17 | 25.7 ± 0.78 |
Hexane | 3 | 0 |
Acetone | 7 | 0 |
Isopropanol | 3 | 0 |
The alkaline crude enzyme was pre-incubated with various organic solvents (50%; v/v; dimethyl sulfoxide (DMSO), methanol, diethyl ether, n,n-dimethylformamide (DMF), hexane, acetone and isopropanol) at 30˚C with shaking (150 rpm) for 30 days. Aliquots were withdrawn at desired time intervals to test the remaining activity.
[
Chitin in the exoskeleton of shrimp shells 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 the deacetylation and depolymerization of chitin. Many reports have demonstrated the application of proteolytic microorganisms for the deproteinization of marine crustacean wastes to produce chitin [
Different E/S ratios (10, 20 and 30) were used to compare the deproteinization efficiency. As shown in
observed. The deproteinization activity of A. arilaitensis crude proteases was better than many bacterial proteases reported in many previous studies [
The obtained results demonstrated that the protease produced by A. arilaitensis Re117 could be used effectively in the deproteinization of shrimp wastes. Furthermore, the application of proteases or proteolytic bacteria for the deproteinization would be a good solution for the environmental problems associated with crustacean processing.
The present study reports the characterization and evaluation of proteases from A. arilaitensis Re117 as detergent additive and in shrimp waste deproteinisation. The crude protease was active in a broad range of pH 6.0 - 11.0 and exhibited a high stability in the presence of oxidizing agent and various laundry solid and liquid detergents, which made it an ideal choice for applications in liquid detergent formulation. Additionally, the crude protease was found to be relatively effective in the deproteinization of shrimp waste powder. The protein removal with an E/S ratio of 20 was about 83%. It was also stable in the presence of organic solvents.
This work was funded by the Ministry of Higher Education and Scientific Research, Tunisia and Pr. Hafedh Bejaoui a language expert at the Sfax Faculty of Sciences.