2Head of Animal Health Research Institution (AHRI-ARC), Sharkia, Egypt
Bacterial cultures were isolated from five soil samples which collected from rhizosphere zone of potato cultivated soils in Tanta and Zagazig City, Gharbia and Sharkia governorates using standard dilution plate technique (Johnson., et al. 1959).
The most potent tyrosinase producer isolate was traditionally identified and characterized according to Bergey's Manual of systematic bacteriology (Holt., et al. 1994 and Brenner., et al. 2005). Identification was molecularly confirmed by the analysis of 16S rRNA gene sequence (Altschul., et al. 1997).
Since tyrosinase catalysis two different oxidation reactions, the substrates used to determine its activity were divided into two groups, monophenols and diphenols. A continuous spectrophotometric rate determination method was used to monitor the change of the absorbance due to the transformation of the substrates to products (Espin., et al. 1997). Monophenol L-tyrosine was selected as the basis of the activity assay. This activity assay consists of 1 mM L-tyrosine, 0.1 M pH 6.5 sodium phosphate buffer, and 6 mg/mL tyrosinase reacting at 25ºC and pH 6.5 (Decker 1977). Tyrosinase oxidizes L-tyrosine to L-3, 4- dihydroxyphenylalanine (L-DOPA) which in turn was oxidized to dopaquinone.
Pseudomonas stutzer A3 was cultivated in tyrosinase production broth medium (Lelliott., et al. 1966). Enzyme production was tested under different cultured conditions; different incubation periods (12-72h); different temperatures (20-55°C); different pH-values (pH 5-11); different carbon sources (glucose, xylose, starch, sucrose, maltose, lactose, and mannitol) and different nitrogen sources (peptone, asparagine, glycine, tyrosine, glutamine, casein, yeast and gelatin) under shaking and static conditions. The culture was harvested and centrifuged at 10,000 rpm for 30 min and the obtained cell free filtrate was used as crude enzyme according to Arikan (2008).
The crude enzyme was prepared from three liters of optimized submerged culture of P. stutzeri A3 growing in L-tyrosinase producing medium. The crude enzyme preparation was subjected to slow addition of 70% ammonium sulfate with stirring at 4ºC. The precipitated protein was collected by centrifugation at 10.000 rpm at 4°C and dissolved in a minimum volume of phosphate buffer (0.01 M, pH 8.0) (Bollag., et al. 1996).
Different methods of tyrosinase immobilization described by Kumar., et al. (2012) were used during these experiments. The different carriers which used in this study were named as silica gel, Ca-alginate, agar–agar and polyvinyl alcohol (PVA). The activity of the immobilized enzyme was assessed as described previously. Immobilization efficiency (%) was expressed by the specific activity of immobilized L-tyrosinase per specific activity of the soluble enzyme.
Optimum pH and pH stability
Tyrosinase activity (free or immobilized) was assayed using 1mM tyrosine as substrate in 0.1M sodium phosphate buffer (pH 2.0–9.0) at 40°C. Stability of L-tyrosinase was examined after preincubation of the enzyme for 1h at pH from 5.0-11.0. After adding tyrosine (1mM) the reaction mixture was incubated at 40°C for 40 min. The residual tyrosinase activity was determined for each pH.
The study was carried out at various temperatures (30º–60°C) and tyrosinase activity was then assayed at the corresponding temperature in standard conditions. The thermal stability of the free and Ca-alginate immobilized enzymes were assessed by pre-incubation of enzyme without substrate at various temperatures (55, 60 and 65ºC) using 0.1M phosphate buffer for different incubation periods (20-150 min). The residual enzyme activity was determined for each temperature. The thermal inactivation rate kr (min) was calculated by the first-order kinetic model (Whitaker, 1972).
The substrate specificity of free and Ca-alginate immobilized enzymes were determined by measuring activity towards several monohydroxyphenol and dihydroxyphenol compounds like L-tyrosine, catechol, and hydroquinone. The activities of immobilized enzyme for this purpose were measured using solutions of these compounds prepared in 0.1M sodium phosphate buffer at concentrations of 1 to 5 mM for catechol, hydroquinone and L-tyrosine. The enzyme activity was assessed as described above.
The stability of free and Ca-alginate immobilized tyrosinase preparations were determined after storing in phosphate buffer (50 mM, pH 8) at -20oC for a predetermined period. Under the same storage conditions, the activities of free and immobilized tyrosinase were assessed as described above after 15, 30, 45, 60, 75 and 90 day.
Several oxidative cycles were determined using 1 mM tyrosine in order to assess the operational stability of the immobilized tyrosinase. At the end of each oxidation cycle, the immobilized tyrosinase pellets were washed three times with sodium phosphate buffer and the procedure repeated with a fresh aliquot of substrate, as described by Donato., et al. (2014).
The infra-red was carried out in Micro Analytical Center of Faculty of Science, Cairo University, Egypt. This analysis technique elucidated the types of functional groups on the surface of the free and Ca-alginate immobilized purified enzymes.
Six effluent samples (100 mL) were collected separately in sterile bottles from different Factories in Tenth of Ramadan City, Sharkia Governorate, Egypt. The working volume was prepared by adding 2 mL of each effluent sample with different weights of immobilized tyrosinase; 0.1, 0.2 and 0.3g. Then, 3.3 mg. mL-1 of chitosan was added to each sample. Chitosan was added to the reaction mixtures either before initiation or after completion of the reaction, to prevent color generation or to remove color solution. Chitosan solution (0.5% w/v) was prepared by dissolving chitosa in acetic acid 0.5 % (v/v). Reactions were stopped by adding 0.1mL of H3PO4 8.5 % (w/v). Phenolic concentration was analyzed at the beginning and after 20 hours reaction (Bevilaqua., et al. 2002).
The total phenolic content was determined by using Folin-ciocalteu reagent following a slightly modified method of Ainsworth (Ainsworth and Gillespie, 2007). Gallic acid was used as a reference standard for plotting calibration curve. A volume of 0.5 mL of sample was mixed with 2 mL of the Folin-ciocalteu reagent (diluted 1:10 with de-ionized water) and were neutralized with 4 mL of sodium carbonate solution (7.5%, w/v). The reaction mixture was incubated at room temperature for 30 min with intermittent shaking for color development. The absorbance of the resulting blue color was measured at 765 nm using double beam UV-VIS spectrophotometer (UV Analyst-CT 8200). The total phenolic content was determined from the linear equation of a standard curve prepared with gallic acid. The content of total phenolic compounds expressed as mg/mL gallic acid.
Tyrosinase enzymes and their genes have previously been characterized from bacteria, fungi, plants and mammals. Bacterial tyrosinases have been reported, of which Streptomyces tyrosinases are the most thoroughly characterized (Selinheimo., et al. 2006). In the present study, forty bacterial isolates were isolated from rhizosphere of potato cultivated soils in Tanta and Zagazig City, Egypt. All bacterial isolates were screened for tyrosinase production using medium containing tyrosine capable of forming melanin. One of all bacterial isolates, isolate no.3, attained the highest melanin production zone and was characterized morphologically and biochemically according to Bergey's key as a member of Pseudomonas genus.
Numerous investigations have revealed that the production of tyrosinase by a microorganism in a growth medium is regulated by genetics of the microorganism, the composition of the medium, the growth duration and temperature, pH, the presence of biosynthetic inhibitors, the density of tyrosinase-producing cells and the presence of enzyme inducers (Popa and Bahrim 2011). The present data revealed the maximum activity of extracellular tyrosinase by P. stutzeri A3 achieved after incubation for 48 h at 40°C in production medium adjusted at pH 8 and contained tyrosine as carbon and nitrogen sources under shaking condition (120 rpm) (Data not shown).
After optimizing the growth and enzyme productivity by P. stutzeri A3, the tyrosinase was purified to apparent homogeneity from the liquid state cultures by gel filtration. Fractional precipitation was carried out initially with 70% ammonium sulphate at 4.0°C (Bollag., et al. 1996). The obtained precipitated protein was suspended immediately (separately), in definite volume of 0.1M sodium phosphate buffer (pH 8.0). From the overall purification profile, the fine specific activity and purity of P. stutzeri A3 tyrosinase were increased to 45.6 Umg-1 and 6.5 fold respectively with 43% yield by Sephadex G100 (Table 1). In this connection, partial purification of thermophilic Bacillus sp. was performed by acetone precipitation and gel filtration chromatography with 35% yield and 1.24 purification fold (Güray 2009). The yield of purified tyrosinase enzyme from Streptomyces espinosus strain LK4 was 31.88% (Roy., et al. 2014).
|Total Enzyme activity (U)||Total Protein content (mg)||Specific activity Umg-1||Purification
|70% Amm. sulfate||72||5.4||13.3||1.9||75|
The purified homogeneity subunit structure of tyrosinase from culture of P. stutzeri A3 was analyzed using denaturing PAGE. From the profile of SDS-PAGE, a distinct band of 45 kDa for P. stutzeri A3 was appeared (Data not Shown). Similarly, Dalfard., et al. (2006) stated that the molecular weight of Bacillus sp. HR03 purified tyrosinase has 50 kDa.
The purified of P. stutzeri A3 tyrosinase was immobilized using different solid carriers. The main reason for enzyme immobilization is the anticipated increase in its stability to various deactivating force due to restricted conformational mobility of the molecules following immobilization (Estrada., et al. 1991). High yields of immobilization were defined as the activity ratio of immobilized enzyme to the activity of the free enzyme (Quiroga., et al. 2011).
|Immobilization method||Carrier||Specific Activity
(U mg-1 enzyme)
|Immobilization yield (%)||Enzyme loading (mg enzyme g-1 microbeads)||Activity
(U g-1 ̄ˡmicrobeads)
|Physical adsorption||Silica gel||32.7||71.7||1.5||49.1|
Optimal pH and pH stability
The present investigation was extended to study the biochemical properties of free and Ca-alginate immobilized P. stutzeri A3 tyrosinase. The optimum pH and temperature of free or immobilized tyrosinase achieved maximum oxidation of L-tyrosine to o-benzoquinone was pH 8 and 40°C respectively (Data not shown). Besides, in the whole investigated temperature and pH ranges, the curve profile of the immobilized enzyme was broader.
(A) pH stability profile. The enzyme was preincubated for 1h at various pH s (5.0 -11.0),
then measuring the residual activity
From the profile of thermal stability, the enzyme half-life times (T1/2) of Ca-alginate immobilized P. stutzeri A3 tyrosinase (4.93, 3.29 and 2.18h) were more than the free one (2.37, 1.40 and 0.96h) at incubation temperatures 55, 60 and 65°C, respectively (Figure 2B). From the data, an obvious acquired thermal stability of free and immobilized tyrosinase was thermal denaturation rate (kr). Its value for immobilized enzyme (2.37 x 10-3 min-1) was less than free tyrosinase (4.81 x 10-3 min-1) at 60°C.
Tm is temperature degree at which the enzyme retains half of its initial activity at 60 min.
The kinetic parameters of free and immobilized tyrosinase for P. stutzeri A3 as Vmax, Km and kcat were estimated using different concentrations of L-tyrosine, hydroquinone and catechol, separately (1-5 mM). From Line weaver-Burk plots, the maximum affinity of the free and immobilized tyrosinase was for catechol followed by hydroquinone and L-tyrosine. The affinity of immobilized enzyme (Km 0.033 mM and Vmax 27.77 U/mg) towards catechol was slightly reduced compared with free enzyme (Km 0.032 mM and Vmax 29.49 U/mg) (Table 3).
|Substrate||Free enzyme||Immobilized enzyme|
|Tyrosine||25.38 ± 0.38||0.040 ± 0.008||0.564 ± 0.004||24.15 ± 0.57||0.038 ± 0.01||0.536 ± 0.09|
|Hydroquinone||26.95 ± 0.20||0.041 ± 0.005||0.599 ± 0.005||25.25 ± 0.14||0.040 ± 0.01||0.561 ± 0.1|
|Catechol||29.49 ± 0.40||0.032 ± 0.002||0.655 ± 0.02||27.77 ± 0.56||0.033 ± 0.01||0.617 ± 0.08|
One of the most important parameters to be considered in enzyme immobilization is storage stability. The stability of the free and the immobilized P. stutzeri A3 tyrosinase preparations was determined after the preparations were stored in phosphate buffer (50 mM, pH 8) at -20°C for a predetermined period. Under the same storage conditions, the relative stability of the immobilized tyrosinase preparations decreased slower than that of the free tyrosinase with increasing the storage periods at -20°C (Figure 3).
Continuous activity of Ca-alginate immobilized P. stutzeri A3 tyrosinase was assessed for continuous elimination of tyrosine for five successive reactions under standard conditions. It was found that, the activity of immobilized enzyme retained about 80% and 50% by the second and fifth catalytic cycle respectively (Figure 4). Arica., et al. (2004) showed that, during the initial 24h, continuous operation the immobilized mushroom tyrosinase preserved all of its initial activity. After this period, a small decrease in enzyme activity was observed with time. After 40h, the immobilized enzyme lost about 3% of its initial activity, this would be possibly due to the inactivation of tyrosinase upon use.
The biomass of free and immobilized P. stutzeri A3 tyrosinase were subjected to IR analysis. It was appeared that, the bands in the spectra of the free and immobilized tyrosinase enzymes were assigned and the shift of the wave numbers or the intensity of the peaks for immobilized tyrosinase enzyme indicating to upload enzyme on Ca-alginate. Where, increasing in the molecular weight of the product changed the properties of vibration motions of groups.
The enzymatic polishing of phenolic effluent was the aim of this experiment. Phenol removal catalyzed by Ca-alginate immobilized P. stutzeri A3 tyrosinase was initially tested after adding 2 mL of different wastewaters separately with different weights of immobilized tyrosinase. From obtained results in (Table 4), the highest phenolic content was observed in sample 6 (72 mg/L) followed by sample 3 (29 mg/L) and sample 1 (17 mg/L).
|Immobilized enzyme (g)||Tyrosinase activity (U/mg)||Phenolic concentration (mg/L)|
|Sample 1||Sample 2||Sample 3||Sample 4||Sample 5||Sample 6|
|0.1||4.05 ± 0.028||17 ± 0.57*||6 ± 0.28*||2 ± 0.28*||-||29 ± 1.15*||8 ± 0.57*||5 ± 0.03||-||4 ± 0.57||-||72 ± 1.15*||3 ± 0.28|
|0.3||12.15 ± 0.08||17 ± 0.57*||2 ± 0.17*||2 ± 0.28*||-||29 ± 1.15*||1.1 ± 0.05*||5 ± 0.03*||-||4 ± 0.57*||-||72 ± 1.15*||0.1 ± 0.05*|
|0.5||20.25 ± 0.14||17 ± 0.57*||6 ± 0.23*||2 ± 0.28*||-||29 ± 1.15*||2 ± 0.28*||5 ± 0.03*||-||4 ± 0.57*||-||72 ± 1.15*||0.4 ± 0.05*|
*: The mean difference is significant at the 0.05 level.
Before: before treatment of effluent with Ca-alginate immobilized tyrosinase
After: after treatment of effluent with Ca-alginate immobilized tyrosinase.
Sample 1: Vegetable factory, Sample 2: Glass house factory, Sample 3: Factory of medication syrup,
Sample 4: Ampoules drugs factory, Sample 5: Pharmaceutical Factory of antibiotic,
Sample 6: Dyes factory, all factories located in Tenth of Ramadan City, Sharkia Governorate, Egypt.
There is no conﬂict of interests regarding the publication of this paper.
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