Optimization of Physical and Cultural Conditions of Fibrinolytic Enzyme from Bacillus altitudinis S-CSR 0020

The main aim of this work was optimization of physical conditions as well as medium components for the maximum production of fibrinolytic enzyme. A fibrinolytic enzyme producing organism was isolated from soil and identified as Bacillus altitudinis strain S-CSR 0020. Various physical parameters such as temperature, pH, incubation time and medium components such as inoculum size, substrate (nitrogen and carbon) concentrations were optimized. A cultivation medium was designed using optimized conditions by Box Behnken Design for mass production of fibrinolytic enzyme and specific activity of enzyme was analyzed. The maximum enzyme production seen at 50 °C temperature, 10.0 pH, 4% substrate concentration with 3 ml inoculum size and 96 h. of incubation time. Among the different carbon sources tested, fructose (8%) showed maximum enzyme activity of 325 U/ml with a specific activity of 812.5 U/ml. Box Behnken Design was used to optimize the percentage of best nitrogen sources (casein, peptone, soya bean and yeast extract) with an enzyme activity of 1089.8 U/ ml. Experiments were repeated with optimized condition manually and found with an enzyme activity of 1072.12 U/ml. Mass production of extracellular fibrinolytic enzyme was done with all these conditions and it showed an enzyme activity of 2070 U/ml with an initial activity of 400 U/ml without medium optimization. There was a fivefold increase in fibrinolytic activity after medium optimization that proved the reliability of optimization manually as well as using the Box Behnken design.

Cardiovascular diseases, due to thrombosis in the form of myocardial infarction and stroke are the major causes of death in worldwide 1 Para physiological disorders are the main causes of disturbance of dynamic balance between formation and degradation of fibrin 2 . Currently available thrombolytic agents include Alteplase (t-PA or Activase), Reteplase (r-PA or Retavaseurokinase (Abbokinase), prourokinase, Anisoylated Purified Streptokinase Activator Complex (APSAC) and streptokinase 2 are of human origin, which are very expensive. Due to serious limitations of fibrinolytic agents such as high cost, low specificity towards fibrin, gastrointestinal bleeding and resistance to allergic reactions 1 , there is much importance of fibrinolytic enzymes of microbial origin due to its wide range of applications in medicine, healthcare and pharmaceutical industry 3 . Fibrinolytic enzymes are derived mainly from plants, animals and microbial sources. Among various sources, due to advancement in molecular biology and fermentation technology, microbial fibrinolytic agents have much attention of research community 4 . There are fewer reports on fibrinolytic enzyme production optimization studies 5 . Enzyme production from microbes favors the economy of bulk production 6 . As compared to fungi, bacteria can use crude raw material as nutrients to grow faster and are easily flexible to genetic manipulations. The major cost determining factors for microbial enzyme production is the substrate used. To study the interaction between different variables, Response Surface Methodology (RSM) helps to design model in the significant factors evaluation and to select the optimum value of variables for maximum desirable response. It requires only a minimum number of experiments for a large number of factors. Successful application of RSM is used in optimization of various processes like bio fuel production, enzyme production including alkaline proteases and xylanases7 The aim of the present study is to optimize physical parameters such as temperature, pH and incubation time, cultural conditions such as substrate concentration, inoculum size and chemical components such as nitrogen sources and carbon sources for fibrinolytic enzyme production. Percentage of nitrogen sources were optimized using RSM for the maximum production of fibrinolytic enzyme.

Chemicals and reagents
All the chemicals used in the present study were of analytical grade and bought from Sigma Aldrich, Mumbai, Maharashtra.

Microorganism used and its maintenance
Novel fibrinolytic enzyme producing organism, Bacillus altitudinis strain S-CSR 0020 with Genbank accession number KT369312 was used for the study 8 . Culture was maintained on nutrient agar slants as glycerol stocks, and then stored at 4 °C.

Preparation of substrate
Clot was prepared by incubating buffalo blood (500 ml) at room temperature, which was collected from the slaughter house. The substrate was prepared by shredding the clot using the surgical blade, then placed in distilled water in a beaker which was kept on the magnetic stirrer for further shredding and RBC removal; then grinded into fine powder, sterilized using ethanol and dehydrated by acetone and stored at 4 °C in refrigerator 8 .

Enzyme assay and protein content
Fibrinolytic enzyme activity was estimated using 0.5% bovine fibrin in 0.1 M carbonate buffer (1 ml) with an equal volume of extracted enzyme solution and incubated at 37 °C for 30 min and the reaction was stopped by adding cold tri chloroacetic acid (TCA); centrifuged at 10000 r/min for 10 min and the supernatant collected was read at 660 nm against a reagent blank using UV-Vis (Shimadzu) spectrophotometer. Tyrosine served as the reference standard 9, 10 . One unit of fibrinolytic activity was defined as the amount of enzyme required to catalyze the release 1 µg of tyrosine per ml under the reaction conditions. Protein content estimated by Lowry's method 11 and specific activity also were determined.

Optimization of physical and other factors for maximum enzyme production
Temperature optimization was done by incubating 20 ml minimal media with 0.5% fibrin as substrate at different temperatures from 10 to 70 °C. The influence of pH on the enzyme production was determined using 0.2 M buffers at various pH values ranges from 4 to 12 and the effect of substrate concentrations checked with different concentrations ranges from 0.5 to 5%. Effect of incubation time and inoculum size were optimized by incubating with different time intervals (24 to 120 h.) and different inoculum sizes (1 to 5 ml) from already prepared McFarland's standard inoculum respectively. To study the effect of different carbon sources, media were prepared with 1% of different carbon sources such as glucose, lactose, fructose, maltose, sucrose, starch, glycerol, galactose, mannitol and malt extract in 20 ml minimal media with blood clot as substrate, the percentage of best carbon source also were optimized and the effect of different nitrogen sources by 1% of casein, peptone, soyabean meal, ammonium chloride, yeast extract, ammonium acetate, potassium nitrate, sodium nitrate, skim milk powder, and beef extract 12 . The enzyme activity and protein content were determined and thereby specific activity also calculated.

Statistical optimization of nitrogen source percentage using RSM
A four variable Box-Behnken design for randomized response surface methodology was used to study the combined effect of percentage of four nitrogen sources such as casein, peptone, yeast extract and soya bean on fibrinolytic enzyme production over three levels. The levels of the independent variables were selected based on preliminary experiments. The Box-Behnken design is suitable for the exploration of quadratic response surfaces and generates a second degree polynomial model, which in turn is used in optimizing a process using a small number of experimental runs. The design which was developed using Design Expert 10.0.6.0 resulted in 29 experimental runs which were randomized to maximize the effects of unexplained variability in the observed responses due to extraneous factors.

Mass production of enzyme
With all the physical and cultural conditions obtained through optimization studies (3 ml inoculum size, 4% substrate, 8% fructose, 3.99% casein, 3.32% yeast extract, 0.5% soya bean meal, 3.66% peptone, 10 pH, 50 °C temperature and incubation period of 96 h.), a final cultivation medium was designed to increase the fibrinolytic activity.

Optimization of physical and other factors for maximum enzyme production Effect of temperature on enzyme production
Physical conditions such as temperature, pH and incubation time should be optimized for maximum enzyme production. B. altitudinis S-CSR 0020 showed high enzyme production at 40-60 °C, which indicated a thermophilic organism. Enzyme activity increased progressively with increase in temperature from 10 °C reaching a maximum to 50 °C with an enzyme activity of 158 U/ml and protein content of 0.4 mg/ml, there by a specific activity of 395 U/mg ( Figure 1). Velocity of enzyme reaction determined by temperature and optimal temperature determines the maximum enzyme activity; in which rate of reaction is too fast without denaturation of enzyme [13]. Bacillus sphaericus MTCC 3672 had shown higher enzyme activity at 30 °C 4 . B. subtilis and mutant Bacillus cereus GD55 had produced fibrinolytic enzyme at 37 °C 14,15 .

Effect of pH on enzyme production
Bacillus altitudinis S-CSR 0020 showed an optimum enzyme production at pH 10.0, with an enzyme activity of 270 U/ml and the protein content was found to be 0.59 mg/ml with a specific activity of 457.63 U/mg ( Figure 2). Optimum pH for fibrinolytic enzyme production by B. altitudinis S-CSR 0020 was found to be 9.0-11.0, which indicated an alkaline organism. From the results it was understood that the organism isolated was highly thermo stable and pH tolerant. Change in pH can lead to change in the active site shape and due to denaturation enzyme activity loses at high or low pH values. B. subtilis showed an optimum pH at 9.0 12 . Mutant Bacillus cereus GD55 showed maximum activity at pH 8 15 . There were maximum fibrinolytic enzyme gene expression and very high biomass formation at optimum pH. Hence, all fermentation studies are performed at pH 8.0, but most of the Bacillus spp. grows best in a neutral or slightly basic pH environment. Thrombinase production has been studied at pH 7.4 from Bacillus sphaericus serotype H5a5b 16 and maximum fibrinolytic enzyme production from Bacillus sp. B24 obtained at pH 7 17 .

Effect of substrate concentration on enzyme production
For the enzyme production, the organism is active on 4% substrate (blood clot) with an enzyme activity of 470 U/ml, 0.7 mg/ml protein content and with a specific activity of 671 U/mg. Fibrinolytic activity increased with increase in the substrate concentration from 0.5% to 4% ( Figure  3).

Effect of inoculum size on enzyme production
The effect of inoculum size on fibrinolytic activity showed that enzyme activity increased progressively with increase in inoculum size from 1 ml reaching a maximum at 3 ml with an enzyme activity of 210 U/ml and protein content of 0.5 U/ mg. The specific activity was found as 420 mg/ml. Above 3 ml, there was a sudden decrease in the fibrinolytic activity ( Figure 4). Mutant Bacillus cereus GD55 showed maximum fibrinolytic enzyme production at 2 % inoculum size 15 .

Effect of incubation period on enzyme production
The influence of incubation period on fibrinolytic activity showed that enzyme activity increased progressively with increase in incubation period, reaching a maximum at 96 h. (4 d.). The enzyme activity at optimum incubation period was  found to be 690 U/mL, with a protein content of 0.85 mg/ml there by a specific activity of 811.76 U/mg ( Figure 5). Fibrinolytic enzyme production usually lies between 48 to 96 h 18 . In our results maximum enzyme production was seen at 96 h. Majority of extracellular enzyme produced during the log phase. Bacillus subtilis produced maximum enzyme production at 96 h 12 , but mutant Bacillus cereus GD55 and Bacillus sp. B24 showed maximum enzyme production after 3 days of incubation 15,17 .

Effect of carbon source on enzyme production
Among all supplementary carbon sources, fructose has been found to be the best carbon source for fibrinolytic enzyme production with an enzyme activity of 205 U/ml. The protein content was found to be 0.4 mg/ml and the specific activity was found to be 525 U/mg ( Figure 6). Mutant Bacillus cereus GD55 also showed maximum fibrinolytic enzyme production with fructose as a carbon souce 15 . Mannitol showed the best carbon source in the case of fibrinolytic enzyme production from Bacillus lichniformis B4 local isolate 13 .

Effect of fructose on enzyme production
Among the carbon sources, fructose showed maximum enzyme activity, so the influence of fructose concentration on fibrinolytic activity of the crude enzyme were checked and concluded that enzyme activity increased progressively with an increase in fructose concentration from 1% reaching a maximum at 8% was 325 U/ml. The protein content was 0.4 mg/ml and the specific activity was found to be 812.5 U/ml (Figure 7). An alkali-thermotolerant extracellular protease from a newly isolated Streptomyces sp. DP2, Fructose was found to be the best substrate for protease production, followed by maltose, lactose and wheat bran 6 .

Effect of nitrogen source on enzyme production
Among all supplementary nitrogen sources, casein has been found to be the best nitrogen source for fibrinolytic enzyme production with an enzyme activity of 525 U/ml with 0.55 mg/ml protein content and specific activity of 954 U/mg. Other nitrogen sources such as yeast extract, soya bean meal and peptone also showed increased fibrinolytic enzyme production ( Figure  8).Culture medium provides nutrients required for the organism to grow. It plays an important role for the enzyme production 13 . Due to high cost of commercial fibrin, the production cost can be minimized by utilizing cheaper substrates such as SMITHA & PRADEEP: STUDY OF FIBRINOLYTIC ENZYME FROM Bacillus altitudinis  blood clot as fibrin, carbon and nitrogen sources. Extracellular fibrinolytic enzyme production by Bacillus altitudinis is influenced greatly by cultural and environmental conditions and nitrogenous precursors in the medium . B. subtilis I-2 produced fibrinolytic protease using low-cost nitrogen sources such as cotton seed cake, soybean meal, malt extract, gelatin and beef extract. Medium components such as casein, peptone, yeast extract, soybean powder were reported as good nitrogen sources for protease enzyme production from Bacillus sp 1,19 . Among nitrogen sources tested for fibrinolytic enzyme production, four nitrogen sources (casein, peptone, soya bean and yeast extract) were found as most significant. Among 11 variables investigated for production of protease from B. mojavensis A2, four variables (hulled grain of wheat, NaCl, K 2 HPO 4 and KH 2 PO 4 ) were found as most significant 20 . In another study, Soybean meal supported maximum protease production (797.28 U/mL), followed by malt extract, cotton cake and beef extract 21 4 , peptone and soya peptone), the most promising one was found to be soy peptone 13 .

Optimization of different nitrogen sources using Box Behnken Design The results obtained from 29 experimental runs carried out according to the Box-Behnken
Design are summarized in Table 1. The significance of the fit of the second-order polynomial for enzyme activity was assessed by carrying out analysis of variance (ANOVA) for maximum enzyme production ( Table 2 and 3). Standard errors should be similar within type of coefficient. Smaller is better, the ideal VIF value is 1.0. VIFs above 10 are the causes for concern. VIFs above 100 are the causes for alarm, indicating coefficients are poorly estimated due to multicollinearity. Ideal Ri-squared is 0.0. High Ri-squared mean terms are correlated with each other, possibly leading to poor models. The Model F-value of 33.23 implies the model is significant. There is only a 0.01% chance that an F-value this large could occur due to noise. Values of "Prob > F" less than 0.0500 indicate model terms are significant. In this case A, B, C, D, AB, AC, CD, A 2 , B 2 , D 2 are significant model terms.  value of C.V suggests a high reliability of the experiment. The difference between the "Pred R-Squared" (0.8318) is in reasonable agreement with the "Adj R Squared"(0.9416). i.e., less than 0.2. Adeq Precision" measures the signal to noise ratio, represented by ratio 22.463 (ratio greater than 4 is desirable), this model can be used to navigate the design space. For given levels of factors, to make predictions, the equation in terms of actual factors can be used. From the 100 runs, 53 rd run with maximum enzyme activity was selected and  sources-casein, peptone, yeast extract and soya bean meal on fibrinolytic enzyme production. An increase in the concentration of casein with an accompanying increase in the concentration of yeast extract resulted in an increase in the fibrinolytic enzyme production with an enzyme activity of about 700 U/ml to a maximum value of 1049.4 U/ml at 3.99% casein and 3.56% yeast extract ( Figure 9). An increase in the concentration of casein with an accompanying decrease in the concentration of soya bean meal resulted in an increase in the fibrinolytic enzyme production from an enzyme activity of about 767.27 U/ml to a maximum value of 1089.8 U/ml at concentration of 3.99% casein and 0.5% soya bean meal ( Figure  10). An increase in the concentration of casein with an accompanying increase in the concentration of peptone resulted in an increase in the fibrinolytic enzyme production from 70.0 U/ml to a maximum value of 1042.55 U/ml at 3.99% casein and 3.53% peptone (Fig. 11). An increase in the concentration of yeast extract with a decrease in the concentration of soya bean meal resulted in an increase in the fibrinolytic enzyme production from 700 U/ml to a maximum value of 1037.37 U/ml at 3.16% yeast extract and 0.50% soya bean meal (Fig. 12). An increase in the concentration of yeast extract with an accompanying increase in the concentration of peptone resulted in an increae in the fibrinolytic enzyme production from 700 U/ml to a maximum value of 1004.66U/ml at 3.24% yeast extract and 3.54% peptone (Fig, 13). An increase in the concentration of peptone with an accompanying decrease in the concentration of soya bean meal resulted in an increase in the fibrinolytic enzyme production from 840.86 U/ml to a maximum value of 1033.47 U/ml at 3.89% peptone and 0.51% soya bean meal (Fig. 14).The R2 value showed a good fit to the model with the experimental data. The final optimized conditions obtained with RSM were 3.99% casein, 3.32% yeast extract, 0.5% soya bean meal and 3.66% peptone with a maximum enzyme activity of 1089.80 U/ml. Experiments were repeated with optimized condition manually and found with an enzyme activity of 1072.12 U/ml which is nearer to the model implies the accuracy.
With the aid of the Special features of RSM and 3D plots, the optimum value of the percentage combination of the four factors were validated. There is a better understanding between all possible attributes that empowers the use of RSM. Experiments were carried out in Erlenmeyer flask in triplicates under theoretically predicted conditions to validate the model 22 . In the present study, the percentage of four nitrogen sources on fibrinolytic enzyme were tested using Box-Behnken Design, which detected the optimal concentrations of individual components and also determines the interaction among four nitrogen sources. Significant interaction between Casein and yeast extract (AB), casein and soya bean (AC), casein and peptone (AD), Yeast extract and soya bean (BC), Yeast extract and peptone (BD) and soya bean and peptone (CD) revealed their importance for mass cultivation in the production medium for fibrinolytic enzyme production. Four variables tested with 0.5% and enzyme activity found to be casein-525 U/ml, peptone-475 U/ml, soya bean-400 U/ml and yeast extract 450 U/ml and from the optimized conditions the maximum enzyme activity obtained was 1089.8 U/ml. By regression analysis we found that the percentage interaction of different variables tested were significant and the model validated by comparing the observed and predicted values at the optimum enzyme activity. With the optimized conditions, wet lab experiments were carried out in triplicates and found to be 1072.12 U/ml, which confirmed the reliability of the model. RSM was used to optimize the media components for maximum production of metalloproteases from Xenorhabdus indica KB-3 23 . Statistical optimization of fibrinolytic enzyme production by Pseudoalteromonas sp. IND11 showed 3 fold increase in fibrinolytic enzyme 24 and also observed 4.0-fold increase in yield from Bacillus sp. strain AS-S20-I applying the Plackett-Burman and RSM experimental design 5 .
From a newly isolated marine bacterium Bacillus subtilis A26, 4.2-fold increased level of fibrinolytic production (269.36 U/ml) was observed by statistical optimization 25 . Optimization of subtilisin NAT production using inoculum densities, glucose concentration and soya bean concentration were done by RSM 26 . After optimization of all physical as well as medium components, a production medium designed and by mass cultivation, a fivefold increase in activity was observed with an enzyme activity of 2070 U/ml with an initial enzyme activity of 400 U/ml without medium optimization, which indicated the reliability of medium optimization.

Mass production of enzyme
With all the physical and cultural conditions obtained through optimization studies both manually as well as using statistical methods, a final cultivation medium was designed which increased the fibrinolytic activity to 2070 U/ml which was 5 times more than the initial enzyme activity of 400 U/ml. For the industrial applications, huge quantities of enzymes are required that can be produced by medium optimization with process economy and genetic manipulation 6 . Medium components optimization of extracellular fibrinolytic protease from Citrobacter braakii showed 5.5 fold increased level of fibrinolytic production 27

CONCLUSION
The present research is about media optimization by manual as well as statistical design and evaluation of a novel fibrinolytic enzyme from Bacillus altitudinis S-CSR 0020 through aerobic fermentation. Higher potency of a novel enzyme was evaluated by in-vitro blood clot degradation. Hence, an optimized medium established through present investigation will be useful for the development of an alternative to conventional fibrinolytic agents for cardiovascular disease treatments. Extensive purification, characterization and enzyme immobilization are on track. The optimized medium showed high fibrinolytic activities of 2070 U/ml, which were 5 -fold than that of the non-optimized medium.