Abstract: One hundred and thirty actinomycetes soil-isolates were screened for their uricase activity. The most promising isolate, strain NEAE-25, was selected and identified on the basis of morphological, cultural, physiological and biochemical properties, together with 16S rRNA sequence as Streptomyces rochei NEAE-25 and the sequencing product was deposited in the database of GenBank under accession number HQ889312. The optimization of different process parameters for uricase production by Streptomyces rochei NEAE-25 and its validation using Plackett-Burman experimental design and response surface methodology was carried out during the present study. Fifteen variables were screened using Plackett-Burman experimental design. The most significant positive independent variables affecting enzyme production (incubation time, medium volume and uric acid concentration) were further optimized by central composite design. The maximum uricase production by Streptomyces rochei NEAE-25 after central composite design was 47.49 U mL–1 with a three-fold increase as compared to the unoptimized medium (16.1 U mL–1).
INTRODUCTION
Uric acid is the end product of purine metabolism in the human body and is excreted by the kidney out of the body. It is well authenticated that overproduction and accumulation of uric acid over than the normal value (hyperuricemia) in humans blood results in renal failure (Capasso et al., 2005) and may cause gout disease (Nakagawa et al., 2006), idiopathic calcium urate nephrolithiasis (Masseoud et al., 2005) and it was also reported that leukemia in children associated with an elevated uric acid level (Larsen and Loghman-Adham, 1996), toxemia of pregnancy (Kelly and Pelella, 1987). Uricase (urate oxidase, urate oxygen oxidoreductase, EC 1.7.3.3) is an enzyme that catalyzes the enzymatic oxidation of uric acid to allantoin, carbon dioxide and hydrogen peroxide (Brogard et al., 1972) which is more soluble and easily to be excreted than the uric acid:
The application for uricase as a diagnostic reagent for the determination of uric acid in biological fluids such as blood and urine was identified as the first important application of uricase in clinical biochemistry (Adamek et al., 1989). Uricase has been widely used for enzymatic determination of uric acid in routine clinical analysis by coupling it with a 4-amino-antipyrine-peroxidase system (Capasso et al., 2005; Gochman and Schmitz, 1971). This enzyme can be also used therapeutically as a protein drug to reduce toxic urate accumulation (Colloc’h et al., 1997). Immobilized uricase can be used as a uric acid biosensor (Arslan, 2008). Uricase is also used as an additive in commercial formulations of hair coloring agents (Nakagawa et al., 1995). Uricase is absent in humans but is widely present in most vertebrates.
Some microorganisms such as Gliocladium viride (Nanda et al., 2012), Pseudomonas putida (Poovizh et al., 2014) and Nocardia farcinica (Ishikawa et al., 2004) have been used to produce uricase. Although uricase has been produced using several microbial sources, due to its increasing importance in treatment and in diagnosis, new sources of uricase are sought aiming to produce better yield of the enzyme (Yazdi et al., 2006). The largest and most important genus in the order actinomycetales are Streptomyces. It comprises up to 90% of actinomycetes isolated from the soil samples, it is prolific producers of bioactive compounds such as antibiotics and enzymes which have important applications both in medicine and agriculture (Demain and Sanchez, 2009; El-Naggar and Abdelwahed, 2012, 2014; El-Naggar et al., 2014).
The composition of culture medium strongly influences the growth and enzyme production by microorganisms, thus optimization of cultural parameters and medium components can significantly affect product concentration, yield and the ease and cost of downstream product separation (Wang et al., 2008). The optimizations of cultural parameters and medium components have been performed traditionally using one-factor-at-a time method. The drawbacks of this method are that it ignores the combined interactions among different variables; it is time consuming especially for a large number of variables, laborious and it is expensive (Bandaru et al., 2006). Therefore, in recent years, full factorial or Plackett-Burman design and response surface methodology were used to search the factors rapidly from a multivariable system (Aghaie-Khouzani et al., 2012). Response surface methodology has eliminated the drawbacks of traditional optimization methods, it has the advantage of taking into account the interaction between the nutrients and is less time consuming (Deepak et al., 2008; Banik et al., 2007).
The aim of the present study was to screen some microbial isolates for uricase production, to identify the most potent producer isolate using a combination of phenotypic and genotypic characteristics, Plackett-Burman design was used in the first optimization step to screen the important variables that influence uricase production by Streptomyces rochei NEAE-25. The factors that had significant effects on uricase production were further optimized using a central composite design in the second step.
MATERIALS AND METHODS
Microorganisms and cultural conditions: Streptomyces spp. used in this study were isolated from various soil samples collected from different localities of Egypt. Actinomycetes had been isolated from the soil using standard dilution plate method procedure on Petri plates containing starch nitrate agar medium of the following composition (g L–1): Starch, 20; KNO3, 2; K2HPO4, 1; MgSO4.7H2O, 0.5; NaCl, 0.5; CaCO3, 3; FeSO4.7H2O, 0.01; agar, 20 and distilled water up to 1 L; then plates were incubated for a period of 7 days at 30oC. Nystatin (50 μg mL–1) was incorporated as an antifungal agent to minimize fungal contamination. The actinomycetes strains predominant on media were picked out, purified and maintained on starch-nitrate agar slants. These strains were stored as spore suspensions in 20% (v/v) glycerol at -20°C for subsequent investigation.
Primary and secondary screening of uricase production: The culture medium for screening consists of (g L–1): Uric acid, 5.0 g; glycerol, 30.0 g; NaCl, 5.0 g; K2HPO4, 1.0 g; MgSO4. 7 H2O, 0.2 g; CaCl2, 0.1 g; distilled water up to 1 L; pH 7. For plates 20.0 g agar was added (Azab et al., 2005). Preliminary screening for uricase production was done by conventional spot inoculation of pure actinomycetes strains on agar medium and incubated at 30°C for seven days. Uricase production by the microorganisms is indicated by the appearance of clear zone around the colonies.
The strains which forming bigger clear zones in a shorter time were selected for subsequent screening under submerged fermentation conditions. Fifty milliliter of fermentation medium were dispensed in 250 mL Erlenmeyer conical flasks, sterilized and inoculated. The fermentation media were incubated for at 30°C in a rotatory incubating shaker (200 rpm). The enzyme production was measured after 7 days. The strain which showed the most promising result was selected for further experiments.
Inoculum preparation: Two hundred fifty milliliter Erlenmeyer flasks containing 50 mL of yeast-malt extract broth (malt extract 1%; dextrose 0.4%; yeast extract 0.4%; pH 7.0) were inoculated with three disks of 9 mm diameter taken from the 7 days old stock culture grown starch nitrate agar medium. The flasks were incubated for 48 h in a rotatory incubator shaker at 30°C and 200 rpm and were used as inoculum for subsequent experiments.
Uricase assay: The principle of enzyme measurement was as follows: uricase can catalyse the oxidation of uric acid to form allantoin, carbon dioxide and hydrogen peroxide which is then analyzed by the oxidative coupling of 4-aminoantipyrine, phenol and peroxidase as chromogens. Uricase activity was measured by incubating 300 μL enzyme solution with a mixture of 400 μL sodium borate buffer (pH 8.5, 0.1 M) containing 2 mM uric acid, 150 μL 4-aminoantipyrine (30 mM), 100 μL phenol (1.5%), 50 μL peroxidase (15 U mL–1) at 37°C for 30 min (Suzuki, 1981). The reaction was stopped by addition of (200 μL of 0.1 M potassium cyanide solution. In the blank, the solution of potassium cyanide was added to the mixture before the addition of the crude enzyme. The absorbance was measured against the blank in a spectrophotometer at 540 nm. One unit of uricase enzyme is defined as the amount of enzyme that produces 1 μmol of H2O2 per minute under the standard assay conditions.
Morphology and cultural characteristics: Spore chain morphology and the spore surface ornamentation of strain NEAE-25 were examined on starch nitrate agar medium after 14 days at 30°C. The gold-coated dehydrated specimen can be examined at different magnifications with Jeol JSM-6360 LA Analytical scanning electron microscope operating at 20 Kv at the Central Laboratory, City for Scientific Research and Technological Applications, Alexandria, Egypt. Color of aerial mycelium, pigmentation of substrate mycelium and diffusible pigments production were observed on ISP media 2-7 as described by Shirling and Gottlieb (1966) and on starch-ammonium sulphate agar; all plates were incubated at 30°C for 14 days.
Physiological characteristics: Physiological characteristics were performed following the methods of Shirling and Gottlieb (1966). The ability of the organism to inhibit the growth of four bacterial strains (Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, or Klebsiella pneumoniae) and five fungal strains (Rhizoctonia solani, Alternaria solani, Bipolaris oryzae, Fusarium oxysporum and Fusarium solani) was determined. Some additional tests can be considered to be useful in completing the description of a strain or species, even if they are not very significant or indicative on their own, The ability of strain NEAE-25 to produce asparaginase (Gulati et al., 1997) and chitosanase activity (Choi et al., 2004) were tested.
Chemotaxonomy: The diagnostic whole-cell wall sugars were identified by the method described by Staneck and Roberts (1974).
16S rRNA sequencing: The preparation of genomic DNA of the strain was conducted in accordance with the methods described by Sambrook et al. (1989). The PCR amplification reaction was performed in a total volume of 100 μL which contained 1 μL DNA, 10 μL of 250 mM deoxyribonucleotide 5’-triphosphate (dNTP’s); 10 μL PCR buffer, 3.5 μL 25 mM MgCl2 and 0.5 μL Taq polymerase, 4 μL of 10 pmol (each) forward 16s rRNA primer 27f (5’-AGAGTTTGATCMTGCCTCAG-3’) and reverse 16s rRNA primer 1492 r (5’-TACGGYTACCTTGTTACGACTT-3’) and water was added up to 100 μL. The PCR-apparatus was programmed as follows: 5 min denaturation at 94°C, followed by 35 amplification cycles of 1 min at 94°C, 1 min of annealing at 55°C and 2 min of extension at 72°C, followed by a 10 min final extension at 72°C. The PCR reaction mixture was then analyzed via agarose gel electrophoresis and the remaining mixture was purified using QIA quick PCR purification reagents (Qiagen, USA). The purified PCR product of approximately 1400 bp was sequenced by using two primers, 518F; 5’-CCA GCA GCC GCG GTA ATA CG-3’ and 800R; 5’-TAC CAG GGT ATC TAA TCC-3’. Sequencing was performed by using Big Dye terminator cycle sequencing kit (Applied BioSystems, USA). Sequencing product was resolved on an Applied Biosystems model 3730XL automated DNA sequencing system (Applied BioSystems, USA) and deposited in the GenBank database under accession number HQ889312.
Sequence alignment and phylogenetic analysis: The partial 16S rRNA gene sequence of strain NEAE-25 was aligned with the corresponding 16S rRNA sequences of the type strains of representative members of the genus Streptomyces retrieved from the GenBank, EMBL, DDBJ and PDB databases by using BLAST program (www.ncbi.nlm.nih.gov/blast) (Altschul et al., 1997) and the software package MEGA4 version 2.1 (Tamura et al., 2007) was used for multiple alignment and phylogenetic analysis. The phylogenetic tree was constructed via the neighbor-joining algorithm (Saitou and Nei, 1987) based on the 16S rRNA gene sequences of strain NEAE-25 and related organisms.
Fermentation conditions: Fifty milliliter of fermentation medium were dispensed in 250 mL Erlenmeyer conical flasks, sterilized and inoculated with previously prepared inoculum. The inoculated flasks were incubated on a rotatory incubator shaker at 200 rpm and 30-35°C. After the specified incubation time for each set of experimental trials, the mycelium of the isolate was collected by centrifugation at 6000 g for 15 min. The cell free supernatant was used as a crude enzyme for further determinations.
Statistical experimental design: Stepwise optimization strategy including, evaluation of the most significant medium constituents and environmental factors affecting enzyme production using Plackett-Burman factorial design (Plackett and Burman, 1946) and elucidation of the optimal concentrations of the most significant independent variables by a central composite design.
Selection of significant variables using Plackett-Burman design: The Plackett-Burman statistical experimental design is a two factorial design, very useful for screening the critical physico-chemical factors that influence the enzyme production with respect to their main effects (Krishnan et al., 1998).
| Table 1: | Experimental independent variables at two levels used for the production of uricase by Streptomyces rochei strain NEAE-25 using Plackett-Burman design |
Table 1 shows the independent variables under investigations, where different carbon sources (uric acid and glycerol), nitrogen sources (KNO3, yeast extract), energy sources (K2HPO4), metals (NaCl, CaCl2, MgSO4.7H2O and FeSO4.7H2O) in addition to physical parameters like (incubation time, pH, temperature, inoculum size, inoculum age and medium volume) were tested. A total of 15 independent (assigned) and four unassigned variables (commonly referred as dummy variables) were screened in Plackett-Burman experimental design of 20 trials. Dummy variables (D1, D2, D3 and, D4) are used to estimate experimental errors in data analysis. Each variable is represented at two levels, high and low denoted by (+) and (-), respectively.
Plackett-Burman experimental design is based on the first order model:
| (1) |
where, Y is the response or dependent variable (uricase activity); it will always be the variable we aim to predict, β0 is the model intercept and βi is the linear coefficient and Xi is the level of the independent variable; it is the variable that will help us explain uricase activity. All trials were performed in duplicate and the average of uricase activities were treated as responses.
Central composite design: The levels and the interaction effects between various variables which influence the uricase production significantly were analyzed and optimized using Central Composite Design (CCD). The highest three independent variables which obtained from Plackett-Burman experiment with respect to their main effect namely; incubation time (X1), medium volume (X6) and uric acid (X7). In this study, the experimental plan consisted of 20 trials and the independent variables were studied at five different levels (-2, -1, 0, 1, 2). The central values (zero level) chosen for experimental design were: incubation time 5 days, medium volume 50 mL/250 mL flask and uric acid 6 g L–1.
The experimental results of CCD were fitted via the response surface regression procedure, using the following second order polynomial equation:
| (2) |
In which Y is the uricase activity, β0 is the regression coefficients, βi is the linear coefficient, βij is the interaction coefficients, βii is the quadratic coefficients and Xi is the coded levels of independent variables. However, in this study, the independent variables were coded as X1, X6 and X7. Thus, the second order polynomial equation can be presented as follows:
| (3) |
Statistical analysis: The experimental data obtained was subjected to multiple linear regressions using Microsoft Excel 2007. The p-values were used as a tool to check the significance of the interaction effects which in turn may indicate the patterns of the interactions among the variables (Montgomery, 1991). The statistical software package, STATISTICA software (Version 8.0, StatSoft Inc., Tulsa, USA) was used to plot the three-dimensional surface plots.
RESULTS
One hundred and thirty morphologically different actinomycetes strains were isolated and screened for their uricase activity using plate method (formation of clear zones around the colonies indicated the presence of uricase activity) (Fig. 1a). Out of these, 42% of the isolates exhibited urolytic activity during the preliminary screening experiment. Uricase producing isolates were categorized into 4 groups according to the width of inhibition zones; very strong (31-40 mm), strong (21-30 mm), moderate (11-20 mm) and weak (1-10 mm). The four groups were represented by 1, 16, 24 and 1% activity, respectively (Fig. 1b). Most promising isolate was selected and identified on the basis of morphological, cultural, physiological and chemotaxonomic properties, together with 16S rRNA sequence.
Morphology and cultural characteristics of the isolate NEAE-25: Morphology of strain NEAE-25 grown on yeast extract-malt extract agar (ISP medium 2) for 14 day revealed that strain NEAE-25 had the typical characteristics of the genus Streptomyces. Aerobic, gram-positive, mesophilic actinomycetes that develops abundant and well-developed substrate and aerial mycelium. It developed dark brown substrate mycelium, grey aerial mycelium on yeast extract-malt extract agar. Verticils are not present. The colour of the substrate mycelium was not sensitive to changes in pH.
| Fig. 1(a-b): | (a) Screening of actinomycete isolates for uricase activity using plate method (formation of clear zones around the colonies indicated the presence of uricase activity), (b) Plate screening of actinomycetes isolates for the uricase activity (Percentage indicates the frequency of actinomycetes isolates within specific category in relation to the total isolates) |
| Table 2: | Culture properties of the Streptomyces isolate NEAE-25 |
Strain NEAE-25 grew well on ISP medium 2, 3, 4, 5, 7 and starch-ammonium sulphate agar medium. It exhibited poor growth on ISP medium 6. Diffusible pigments are not produced on any medium tested (Table 2). A scanning electron micrograph of spore chains of strain NEAE-25 cultured on starch nitrate agar medium revealed that the organism produced spirales spore-chains (Fig. 2). Spirals are closed or opened, sometimes almost flexuous. Spore chains are moderately long up to 50, or often more than 50 spores per chain. Spore surface is smooth.
Physiological and chemotaxonomic characteristics of the isolate NEAE-25: The physiological and biochemical reactions of strain NEAE-25 are shown in Table 3.
Melanoid pigments not formed in peptone-yeast extract iron agar or tyrosine agar. As the sole carbon source, it utilizes D-glucose, D-galactose, D-xylose, rhamnose, L-arabinose, D-fructose, cellulose and D-mannose for growth. Only trace of growth on sucrose and raffinose as the carbon sources. It degrades cellulose, casein, gelatin, starch and uric acid but not chitosan and L-asparagine. Nitrate reduction, gelatin liquefaction, starch hydrolysis (Fig. 3), milk coagulation and peptonization were positive, whereas lecithinase hydrolysis is negative. Growth occurs in the presence of NaCl up to 5% (w/v). Hydrogen sulphide production was negative.
| Fig. 2(a-b): | Scanning electron micrographs showing the spore-chain morphology and spore-surface ornamentation of strain NEAE-25 grown on starch nitrate agar medium for 14 days at 30°C at magnification of, (a) 9000 X and (b) 18000 X. Spores in spiral chains and smooth surface of spores |
Strain NEAE-25 exhibited antifungal activity against Alternaria solani and Bipolaris oryzae but no antimicrobial activity against Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, Klebsiella, Rhizoctonia solani, Fusarium oxysporum or Fusarium solani. Chemotaxonomic tests showed that the whole-cell hydrolysates contained mainly mannose and arabinose.
Molecular phylogeny of the isolate NEAE-25: The almost-complete (1394 bp) 16S rRNA gene sequence of strain NEAE-25 was aligned with the sequences of the genus Streptomyces members retrieved from the GenBank databases by using BLAST (Altschul et al., 1997). The 16S rRNA gene sequence of strain NEAE-25 was deposited in the GenBank database under the accession number HQ889312. The sequence analysis revealed a close relationship to Streptomyces rochei strain AM 32 (GenBank accession no. JQ819732.1) with the maximum identity (99%).
| Fig. 3: | Plate assay showing zone of hydrolysis of starch by strain NEAE 25. All the starch in the medium near the microbe has been hydrolyzed by extracellular amylases |
| Table 3: | Phenotypic properties that separate strain Streptomyces NEAE–25 from related Streptomyces species. Data for reference species were taken from Bergey’s Manual® of Systematic Bacteriology-volume five, the actinobacteria (Goodfellow et al., 2012) and Wink (2012) electronic manual |
1Exhibited antifungal activity against Alternaria solani and Bipolaris oryzae. 2Produce amphomycin and cephamycin antibiotics that active against gram positive bacteria. Produce vineomacin that
has antibacterial and antitumor activities.3Streptomyces sp. NEAE- 25 in addition produces uricase and cellulase but not chitosanase and L-asparaginase and also utilizes maltose and ribose.
RF: Rectiflexibiles, RA: Retinaculiaperti, S: Spirales, +: Positive, -: Negative, ±: Doubtful, Blank cells: No data available |
|
| Fig. 4: | Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences, showing the relationships between strain NEAE-25 and related species of the genus Streptomyces. GenBank sequence accession numbers are indicated in parentheses after the strain names. Phylogenetic analyses were conducted in MEGA4 |
The phylogenetic tree (Fig. 4) showed that the isolate falls into one distinct clade with Streptomyces rochei strain AM 32 (GenBank accession no. JQ819732.1), Streptomyces rochei strain SM3 (GenBank accession no. JN128892.1), Streptomyces djakartensis strain RT-49 (GenBank accession no. HQ909758.1), Streptomyces mutabilis strain 13676E (GenBank accession no. EU741236.1), Streptomyces mutabilis strain HBUM173480 (GenBank accession no. EU841647.1), Streptomyces albogriseolus strain HBUM174033 (GenBank accession no. FJ486330.1) with which it shared 16S rRNA gene sequence similarity of 99.0%. It is clear that the strain NEAE-25 is closely similar to Streptomyces rochei.
Evaluation of the most significant factors affecting uricase activity: The experiment was conducted in 20 runs to study the effect of the selected variables on the production of uricase (Table 4). Fifteen different variables including medium component and physical parameters were chosen to perform Plackett-Burman experiment. The maximum uricase activity (43.58 U mL–1) was achieved in the run number 11, while the minimum uricase activity (6.77 U mL–1) was observed in the run number 18. The relationship between the independent variables and uricase production is determined by multiple-regression analysis. Statistical analysis of the uricase production was performed which is represented in Table 5. With respect to the main effect of each variables (Fig. 5), we can see that ten variables from the fifteen named incubation time, pH, temperature, inoculum size, inoculum age, medium volume, uric acid, yeast extract, CaCl2 and FeSO4.7H2O affect positively on uricase production, where the other five variables named (glycerol, KNO3, K2HPO4, NaCl and MgSO4.7H2O) affect negatively on uricase production. The significant variables with positive effect were fixed at high level. The variables which exerted a negative effect on uricase production (glycerol, KNO3, K2HPO4, NaCl and MgSO4.7H2O) were maintained at low level for further optimization by a central composite design. The model F value of 37.933 means that the model is significant.
| Table 4: | Twenty-trial Plackett-Burman experimental design for evaluation of fifteen independent variables at two levels with coded values along with the observed uricase activity |
X1-X15: Represent independent (assigned) variables, D1-D4: Dummy variables (unassigned), 1: High level of variables and -1: Low level of variables | |
| Table 5: | Statistical analysis of Plackett-Burman design showing coefficient values, t-test, p-values and confidence level for each variable affecting uricase production by Streptomyces rochei NEAE-25 |
t: Student’s test, p: Corresponding level of significance, Multiple R: 0.996503488, R Square: 0.993019202, Adjusted R Square: 0.96684121 | |
| Fig. 5: | The main effects of the fermentation medium constituents on uricase production by Streptomyces rochei NEAE-25 according to the Packett-Burman experimental results (The red color represent the most significant positive independent variables affecting enzyme production) |
| Fig. 6: | Pareto chart illustrates the order of significance of the variables affecting uricase production by Streptomyces rochei NEAE-25 (the blue color represent negative effects and the red color represent positive effects; Ranks (%) values ranging from 0.12 to 17.75) |
| Table 6: | Analysis of variance (ANOVA) for optimization of on uricase production by Streptomyces rochei NEAE-25 using Plackett-Burman design |
df: Degree of freedom, SS: Sum of squares, MS: Mean sum of squares, F: Fishers’s function significance, F: Corresponding level of significance | |
The values of Significance F<0.05 (0.0015) indicate that model terms are significant Table 6.
The Pareto chart illustrates the order of significance of the variables affecting uricase production in Plackett-Burman experimental design (Fig. 6). Among the 15 variables, incubation time showed the highest positive effect by 17.75%, NaCl showed the highest negative significance by 14.58%.
In a confirmatory experiment, to evaluate the accuracy of Plackett-Burman, a medium of the following composition: (g L–1: uric acid 5, glycerol 10, KNO3 1, yeast extract 1, K2HPO4 1, CaCl2 0.3, NaCl 1, MgSO4.7H2O 0.1 and FeSO4.7H2O 0.02), incubation time 5 days, pH 8, temperature 35°C, inoculum size 4%, v/v, inoculum age 48 h and medium volume 50 mL/250 mL flask, gives 43.58 U mL–1 which is higher than the result obtained before applying Plackett-Burman (16.1 U mL–1) by 2.7 times.
Optimization of medium composition using Central Composite Design (CCD): The CCD was employed to determine the optimal levels of the significant factors and also to study the interactions among these factors. The variables show highest main effects from Plackett-Burman experiment were selected to further investigation, the other variables in the study were maintained at a constant level which gave maximal yield in the Plackett-Burman experiments. The most significant positive independent variables affecting enzyme production named incubation time (X1), medium volume (X6) and uric acid (X7) were further investigated using CCD.
| Table 7: | Central composite design, with actual factor levels corresponding to coded factor levels, representing the response of uricase activity as influenced by incubation time, medium volume and uric acid |
| X1: Incubation time, X6: Medium volume, X7: Uric acid | |
Each factor in the design was studied at five different levels (-2, -1, 0, 1, 2). The results of experiments are presented along with predicted response (Table 7).
| Table 8: | Estimated regression coefficients, main effect, t-test, p-values and ANOVA for optimization of uricase production by Streptomyces rochei NEAE-25 using central composite design |
t: Student’s test, p: Corresponding level of significance, Multiple R: 0.98530, R Square: 0.97082, Adjusted R Square: 0.94456, df: Degree of freedom, SS: Sum of squares, MS: Mean sum of squares, F: Fishers’s function, Significance F: Corresponding level of significance | |
The results showed considerable variation in the uricase activity. Runs 6, 8, 12, 14, 15 and 18 showed a highest uricase activity (47.49 U mL–1). The minimum uricase activity (30.23 U mL–1) was observed in the run number 20. The uricase activity which is used was the average of a duplicate experiment made for all the cultures. The uricase activities in runs 6, 8, 12, 14, 15 and 18 were the mean of the six runs.
Multiple regression analysis and ANOVA: Multiple regression analysis was performed which is represented in Table 8.
Model verification: In order to determine the accuracy of the model and to verify the result, an experiment under the optimal conditions which obtained from CCD experiment was preformed and compared with the predicted data. The measured uricase activity obtained was 47.49 U mL–1, closed to the predicted one 47.57 U mL–1 revealing that a high degree of accuracy.
The predicted optimal levels of the process variables for uricase production by Streptomyces rochei strain NEAE-25 were incubation time (5 days), medium volume (50 mL/250 mL flask) and uric acid (6 g L–1).
DISCUSSION
On the basis of morphological, cultural properties above, together with the physiological and biochemical properties of strain NEAE-25 shown in Table 3, it is evident that strain NEAE-25 belongs to the genus Streptomyces. Strain NEAE-25 produced grey aerial mycelium and dark brown substrate mycelium on yeast extract-malt extract agar. The color of the substrate mycelium was not pH sensitive. The organism produced spirales spore chains. Spirals are closed or opened. Mature spore chains moderately long. Spore surface is smooth. Melanin or any diffusible pigments was not produced. A comparative study between strain NEAE-25 and its closest phylogenetic neighbours in morphological, cultural and physiological characteristics is summarized in Table 3. From the taxonomic features, the strain NEAE-25 matches with Streptomyces rochei in morphological, physiological and biochemical characters. Thus, it was given the suggested name Streptomyces rochei strain NEAE-25. The GenBank accession number of the sequence reported in this study is HQ889312.
Evaluation of the most significant factors affecting uricase activity: The R2 value is always between 0 and 1. When R2 is closer to the 1, this means that the model is strong and better to predict the response (Kaushik et al., 2006). In this study, the value of the determination coefficient (R2 = 0.9930) indicates that 99.30% of the variability in the uricase production was attributed to the studied independent variables and only 0.7% of the variations are not explained by these variables. In addition, the value of the adjusted determination coefficient (Adj. R2 = 0.9668) is also very high which indicates a high significance of the model (Akhnazarova and Kafarov, 1982). A higher value of the correlation coefficient (R = 0.9965) signifies an excellent correlation between the independent variables (Box et al., 1978), this indicated a good correlation between the experimental and predicted values.
Student’s t-test and p-values determine the significance of each coefficient (Table 5). Larger t-value and smaller p-value imply the more significant corresponding coefficient (Akhnazarova and Kafarov, 1982). Some investigators have found that confidence levels greater than 70% are acceptable (Stowe and Mayer, 1966). Thus, in the current experiment, variables evidencing p-values of less than 0.06 (confidence levels exceeding 94%) were considered to have significant effects on the uricase production. Incubation time, with a probability value of 0.0002, was determined to be the most significant factor, followed by NaCl (0.0004), uric acid (0.0010) and then medium volume (0.0018). Screened significant variables, incubation time (X1), medium volume (X6) and uric acid (X7) exerted positive effects whereas, NaCl exerted a negative effect on uricase production. The model F value of 37.933 means that the model is significant. The values of significance F<0.05 (0.0015) indicate that model terms are significant (Table 6). On the basis of the calculated t-values (Table 5), incubation time (X1), medium volume (X6) and uric acid (X7) were chosen for further optimization using central composite design, since these factors had the most positive effects on uricase production.
In this study, the best concentration of uric acid for maximum uricase production was 6 g L–1, its concentration higher than 6 g L–1 did not enhance the enzyme productivity. Uric acid was used as inducer; high concentrations of uric acid probably inhibit the production of uricase. The optimal incubation time for attaining a higher uricase yield by Streptomyces rochei NEAE-25 was 5 days. Abdel-Fattah and Abo-Hamed (2002) reported that uricase produced from A. flavus, Aspergillus terreus after 4 days incubation and from Trichoderma sp. after 6 days while Kon et al. (1976) reported that maximum uricase produced by Hyphomyces after 5 days incubation. To study the effect of aeration on uricase production, different volumes of medium were tried in 250 mL flasks. A 50 mL production medium in 250 mL flask gave maximum uricase production. Dissolved Oxygen (DO) is important parameter in uricase production. Increased levels of dissolved oxygen have lead to enhanced uricase production.
By neglecting the insignificant variables (p>0.06), the first order polynomial equation was derived representing uricase production as a function of the independent variables:
| (4) |
where, Y is the uricase activity and X1, X3, X5, X6, X7, X8, X10, X12, X13 and X15 are incubation time, temperature, inoculum age, medium volume, uric acid, glycerol, yeast extract, CaCl2, NaCl and FeSO4.7H2O, respectively. It can be seen from equation 4 that glycerol and NaCl exerted negative effect on uricase production by Streptomyces rochei NEAE-25, while other factors exerted positive effect.
Optimization of medium composition using Central Composite Design (CCD): Multiple regression analysis was performed which is represented in Table 8. R2-value of the regression model is higher than 0.9(0.9708) and this implies a very high correlation (Chen et al., 2009) and a very good fit between the experimental and predicted uricase production by Streptomyces rochei NEAE-25, the value of R is 0.9853. The value of the adjusted determination coefficient (Adj-R2 = 0.9445) also confirms the significance of the model.
ANOVA of the regression model is required to test the significance and adequacy of the model (Table 8). It is evident from the very low probability value (1.66E-06) and Fisher’s F-test (36.9705) that the model is highly significant. It can be seen from the t-values and p-values (degree of significance) listed in Table 8, that the linear coefficients of incubation time and quadratic effect of incubation time (X1), medium volume (X6) and uric acid (X7) are significant, meaning that they can act as limiting factors in the production rate of uricase and little variation in their values will alter the production. On the other hand, the p-values of the coefficient suggest that among the three variables studied, incubation time (X1) and medium volume (X6) showed maximum interaction between the two variables (0.037), indicating that 97.3% of the model affected by these variables. Furthermore, among the different interactions, interaction between X1, X7, interaction between X6, X7 and linear coefficient of X7 did not show significant effect on uricase production.
The second-order polynomial equation which defines uricase activity (Y) in terms of X1, X6 and X7 (the independent variables) was obtained using multiple regression analysis:
| (5) |
where, Y is the uricase activity, X1, X6 and X7 are the incubation time, medium volume and concentration of uric acid, respectively.
Description of the interaction between bioprocess variables and the response are illustrated in three-dimensional plots (Fig. 7a-c) when one of the variables is fixed at optimum value and the other two are allowed to vary. Figure 7a shows that lower and higher levels of incubation time (X1), medium volume (X6) support relatively low levels of uricase production. On the other hand, the maximum uricase production clearly situated close to the central point of incubation time (X1) and medium volume (X6). Figure 7b shows that higher levels of the uricase production were attained with central point of incubation time (X1) and uric acid (X7). Further increase in the concentration of uric acid lead to the decrease in the uricase production. Figure 7c represents the interaction between medium volume (X6) and uric acid (X7). It showed that the maximum uricase production clearly situated close to the central point of medium volume (X6) and uric acid (X7).
| Fig. 7(a-c): | Three-dimensional response surface and Contour plots showing the interactive effects of independent variables incubation time, medium volume and uric acid, on uricase production by Streptomyces rochei NEAE-25 |
CONCLUSION
The present study is to evaluate various parameters on uricase production by Streptomyces rochei NEAE-25 using response surface methodology and search optimal conditions to attain a higher uricase yield. As a result, a medium of the following formula is the optimum for producing an extracellular uricase in the culture filtrate of Streptomyces rochei NEAE-25: (g L–1: uric acid 6, glycerol 10, KNO3 1, yeast extract 1, K2HPO4 1, CaCl2 0.3, NaCl 1, MgSO4.7H2O 0.1 and FeSO4.7H2O 0.02), incubation time 5 days, pH 8, temperature 35°C, inoculum size 4%, v/v, inoculum age 48 h and medium volume 50 mL/250 mL flask. Significant improvement from 16.1-47.49 U mL–1 in the production of uricase.