Response surface method to optimize the low cost medium for protease production using anchovy meal from ascidian associated Bacillus sp . GA CAS 10

A protease producing Bacillus sp. GA CAS10 was isolated from ascidian Phallusia arabica, Tuticorin, Southeast coast of India. Response surface methodology was employed for the optimization of different nutritional and physical factors for the production of protease. Plackett-Burman method was applied to identify important factors (anchovy waste, KH2PO4, NaCl and temperature) influencing protease production. Further optimization was done by response surface methodology using central composite design. Under the optimized conditions by central composite design, the protease experimental yield (842.102 U/ml) closely matched the predicted yield by the statistical model (830.307 U/ml) with R = 99.94%. The time course of protease production was increased using the RSM optimized medium (856.29 U/ml) (anchovy waste 20.50 g/l, KH2PO4 3.06 g/l, NaCl 42.91 g/l, temperature 43.36°C, 42 h and pH 9) compared with the un-optimized basal medium (267.33 U/ml). The improvement of protease production by microbial conversion of anchovy waste suggested its potential utilization to generate high value added products using cheap carbon and nitrogen substrates.


INTRODUCTION
Proteases constitute 60 to 65% of the world's industrial enzyme market; most of which are alkaline proteases (Banik and Prakash, 2004).The use of alkaline proteases has increased remarkably in industries such as food, pharmaceutical, leather and textile which are the major consumers of these enzymes (Jellouli et al., 2009).
The anchovy fish catch accounts for a major portion of the world's overall fish catch, the majority being used for fish meal production and only a comparatively small amount being used for human consumption.Anchovy meal is known for its nutrient richness, containing 63 to 66% protein and 9.14% lipid, which may help promote excellent microbial growth and protease production (Esakkiraj et al., 2011;Kratzer et al., 1994;Bimbo, 1990;Turan et al., 2007).
Studies related to statistical optimization of medium constituents for protease production using anchovy meal by ascidian associated Bacillus sp. are meager and hence the present study was designed to optimize the medium and culture conditions to enhanced protease production by ascidian associated Bacillus sp.GA CAS10 using low-cost marine fish waste substrate by response surface methodology.

Microorganism
A protease producing bacterium GA CAS10 used in this study was isolated from an ascidian Phallusia arabica, obtained from Tuticorin, Southeast coast of India, following the method of Sathish Kumar et al. (2014).The strain GA CAS10 produced a clear zone when streaked on skim milk agar after 48 h, and was identified as Bacillus sp.based on the morphological and biochemical characteristics (Holt et al., 1994) and also confirmed through molecular characterization.The bacterial genomic DNA was extracted by phenol chloroform method (Marmur, 1961) and 16S rRNA gene was amplified by using forward primers 8F (5'-AGAGTTTGATCCTGGCTCAG-3') and reverse primer 1492R (5'-GGGCGGTGTGTACAAGGC -3').Polymerase chain reaction (PCR) was performed under the following conditions; 35 cycles consisting of initial denaturation at 95°C for 5 min, denaturation at 95°C for 30 S, annealing at 55°C for 30 s and followed by final extension of 5 min at 72°C.The 16S rRNA gene sequences were obtained by an automated DNA sequence (Megabace, GE) and homology was analyzed with sequences in the Gen Bank using CLUSTAL X software.The phylogenetic tree was constructed by the neighbor-joining method using software (MEGA 5.0) (Saitou and Nei, 1987).

One factor at a time experiments
Anchovy waste was purchased from the local fish market at Parangipettai, Tamil Nadu, India and powdered well.The strain GA CAS10 was cultured using Zobell marine broth with 2% NaCl.Then, 10% of enriched seed culture was inoculated into a 250 ml flask containing 50 ml basal medium (w/v) (glucose 0.5%, yeast extract 1.0%, potassium dihydrogen orthophosphate 0.1%, sodium chloride 3%, magnesium sulphate 0.3%).The culture was incubated in a shaker at 150 rpm for 48 h at 40°C.The cells were harvested by centrifugation at 10,000 rpm for 15 min and the supernatant was further used for protease assay.Initial screening of the most significant carbon and metal ions to maximize protease production were performed by one-variable-at-a-time approach.Carbon sources such as fructose, lactose, xylose, maltose, starch and mannitol were added individually in the basal medium at a concentration of 0.5 and 0.1% and various metal ions (calcium chloride, magnesium chloride, barium chloride, potassium chloride, zinc chloride and zinc sulphate) were tested for protease production.

Plackett -Burman design
Using Plackett-Burman design is an efficient way to screen the important medium components among a large number of process variables which is required for elevated protease production by screening 'n' variables in 'n + 1' experiment.Each factor was examined at two levels: -1 for a low level and +1 for a high level.The variables chosen for the present study were anchovy waste, fructose, KCl, KH 2 PO 4 , NaCl, temperature and pH (Table 1).Variables were evaluated in 12 experimental trials as shown in Table 2.The design was run in a single block and the order of the experiments was fully randomized.The design was developed by the Minitab package version 16.
Table 2. Plackett-Burman design matrix for seven variables with coded values along with the observed and predicted protease production.

Optimization by central composite design
The next level in the optimization of the medium required to determine the optimal levels of the significant variables in protease production.To this purpose, response surface methodology (RSM) was adopted for the augmentation of total protease production using a central composite design (CCD).The most significant variables were selected as follows: anchovy waste, KH 2 PO 4 , NaCl and temperature.A total of 31 experiments were formulated using the statistical software package 'Minitab 16.0'.The central values of all variable were coded as zero.The full experimental plan, with regard to their values and the corresponding experimental and predicted responses values (Y) are provided in Table 4.The data obtained from the RSM on protease production were subjected to analysis of variance (ANOVA).The experimental results of RSM were fitted with the response surface regression procedure using the following second order polynomial equation: Where, Y is the predicted response, Xi and Xj are independent factors, β0 is the intercept, βi is the linear coefficient, βii is the quadratic coefficient, and βij is the interaction coefficient.The statistical software package 'Minitab' (Version 16.0) was used to analyze the experimental data.

Time course of protease production
To study the relation between protease production and the growth profile of the bacterium, 50 ml of the optimized production media by response surface methodology was inoculated in 250 ml 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.

Enzyme assay
The protease activity was measured by incubating the reaction mixture containing 0.2 ml of diluted enzyme and 1.25 ml of 1.25% casein in 50 mM Tris-HCl buffer (pH 9) for 30 min at 37°C.The reaction was terminated by adding 5 ml of 0.19 M trichloroacetic acid.The reaction mixture was centrifuged and the soluble peptides in the supernatant were measured according to the method of Todd (1949) with tyrosine as reference.One unit of protease was defined as the amount of enzyme required to release 1 µg of tyrosine per min.

Microorganism
A protease producing strain GA CAS10 was isolated from the tissue of ascidian P. arabica, Tuticorin from Southeast coast of India.The morphological and biochemical characteristics of the strain revealed that the strain is a Gram-positive and endospore forming bacilli (rod) with catalase but without oxidase (Table 3).The phylogenetic analysis (Figure 1) and BLAST search confirmed the isolate as Bacillus sp.16S rRNA sequence of GA CAS10 has been deposited as Bacillus sp.GA CAS10 in the GenBank database (GenBank ID: JX627407.1).

Plackett-Burman design
A number of studies have been carried out on optimization of different physicochemical parameters of different organisms for maximum protease production using response surface methodology (Sathish Kumar et al., 2014).In general there is no defined medium designed for the production of alkaline protease from different microbial sources (Gupta et al., 2002).Based on the one-variable-at-a-time approach, protease production by Bacillus sp.GA CAS10 revealed fructose as optimum carbon source (267.33 U/ml) (Figure 2a) and potassium chloride as suitable source of metal ions (239.21U/ml)     (Figure 2b) for higher protease production.Similar results have been reported in previous studies where fructose have been found as suitable substrates and inducers for the production of proteases by Bacillus sp.I-312 (Joo and Chang, 2005) and Serratia proteamaculans AP-CMST (Esakkiraj et al., 2011).
Using Plackett-Burman design, a total of seven variables were analyzed with regard to their effects on protease production (Table 1).The designed matrix was selected to screen the significant variables for protease production and the corresponding responses are shown in Table 2.Among the seven variables screened, four components such as anchovy waste, KH 2 PO 4 , NaCl and temperature were selected for further response surface methodology analysis based on their positive effect towards protease production (Table 2); fructose, KCl, and pH showed negative effect towards protease production.Even though the protein content of anchovy was higher than that of lipid that may be more suitable as an inducer for protease production, previous study by Esakkiraj et al. (2011) showed anchovy meal powder as a suitable inducer for the production of proteases by S. proteamaculans AP-CMST.Factors having a confidence level greater than 95% and significant 'p' value (less than 0.05) were considered to have a significant effect on the protease production.The optimal levels of the four selected variables (anchovy waste, K 2 PHO 4 , NaCl and temperature) and their interactions were then examined by a central composite design.Present findings are in line with several reports showing the enhancement of protease production in the presence of fish meal as nitrogen sources by Bacillus mojavensis A21 (Haddar et al., 2010;Esakkiraj et al., 2009, Pseudomonas aeruginosa MN7 (Triki-Ellouz et al., 2003).

Central composite design
The overall price of industrial protease production is very high due to higher cost of the substrates used.Therefore, development of novel process to increase the production of proteases using low cost substrate is important.Hence, microbial protease producing industries are always looking for new and cheaper methods to enhance protease yield of enzyme and reduce the market price (Haddar et al., 2010;Annamalai et al., 2013).In the present study, a total of 31 experiments with different combinations of the four selected variables were performed (Table 4).The central composite design, employed to determine the optimum levels of the four screened factors and six levels, including five replicates at the centre point, was used for fitting a second-order response surface.The variance (ANOVA) analysis is presented in Table 5.The mathematical model relating to the protease production with the independent process variables, X 1 , X 2 , X 3 and X 4 is given in the second-order polynomial equation.Y= -15894.0 + 208.3X Where, Y is the predicted protease yield, X 1 is anchovy wastes, X 2 is K 2 PHO 4 , X 3 is NaCl and X 4 is temperature.The regression coefficients and the analysis of the variance (ANOVA) indicate the high significance of this model.For a good statistical model, the high F-value and non-significant lack of fit indicate that the model is a good fit and all of the factors should be positive and close to each other.Also, significant P-values (0.000) suggested  that the obtained experimental data was a good fit with the model and the fit of the model was also checked by determination of coefficient (R 2 ) with R 2 (multiple correlation coefficient) of 99.94%.The predicted R 2 and the adjusted R 2 was about 99.71 and 99.88%, respectively.The regression coefficients of all linear, quadratic terms and two cross products are significant at 1% level.The three dimensional response surface plots were presented in Figure 3 (graphical representations of the regression equation) from which the protease production for different concentration of the variable could be predicted.The responses are plotted on the Z-axis against two variables while other variable was maintained at level zero.The optimal values of anchovy waste, KH 2 PO 4 , NaCl and temperature were estimated as 20.50, 3.06, 42.91 (g/l) and 43.36°C, respectively, with a predicted protease production of 830.307U/ml.The confirmation experiment was conducted for predicted optimum conditions and the protease production was about 842.102 U/ml.The protease production from the experiment was near about the value predicted by the software which reveals high accuracy of the model.Similarly, maximum protease productions were obtained for Bacillus sp.RKY3 (939 U/ml) (Reddy et al., 2008) and B. mojavensis A21 (1830.60U/ml) (Haddar et al., 2010) under optimized culture conditions by response surface methodology.However, protease production by Bacillus sp.GA CAS10 is much more than protease production by B. subtilis A26 (269.36U/ml) (Agrebi et al., 2009), Bacillus sp.L21 (306.5 U/ml) (Tari et al., 2006), Bacillus sp.(410 U/ml) (Patel et al., 2005), B. mojavensis (558 U/ml) (Beg et al., 2003).

Time course of protease production and cell growth
The time course of protease production and the cell growth of Bacillus sp.GA CAS10 for the optimized (anchovy waste 20.50 g/l, KH 2 PO 4 3.06 g/l, NaCl 42.91 g/l, temperature 43.36°C and pH 9) conditions is shown in Figure 4.These results show that the enzyme production in relation with incubation time and cell growth revealed that both bacterial cell growth and protease production reached maximum (856.29 U/ml) at 42 h and started to decrease gradually after 48 h.This optimization strategy led to the enhancement of protease from unoptimized conditions (267.33 U/ml) to optimized conditions (856.29 U/ml).

Conclusion
The microbial protease production by utilizing anchovy waste not only solves ecological troubles but also enhances the economic value of the wastes.The optimized medium established in this work might result in a significant reduction in the cost of medium constituents and would thus offer advantages for large-scale fermentation.

Figure 1 .
Figure 1.Phylogenetic tree of isolate Bacillus sp.GA CAS10 and their closest NCBI (BLAST) strains based on the 16S rRNA gene sequences.

Figure 2a .
Figure 2a.Effect of different carbon sources on protease production.

Figure 2b .
Figure 2b.Effect of different metal ions on protease production.

Figure 3 .
Figure 3. Three dimensional response surface plot for protease production showing the interactive effects of the anchovy wastes and NaCl (a), anchovy wastes and temperature (b), KH 2 PO 4 and temperature (c), anchovy wastes and K 2 PHO 4 (d), K 2 PHO 4 and NaCl (e), NaCl and temperature (f).

Figure 4 .
Figure 4. Kinetics of growth (CFU/ml) and alkaline protease production in optimized medium.

Table 1 .
Experimental variables at different levels used for the production of alkaline protease by Bacillus sp.GA CAS10 using Plackett-Burman design.

Table 3 .
Morphological and biochemical characteristics of strain Bacillus sp.GA CAS10.

Table 4 .
Experimental conditions in variables of the central composite design and the corresponding experimental responses.

Table 5 .
Analysis of variance (ANOVA) for the quadratic model.