Process optimization for amylase production of Bacillus subtilis M4 mutant strain

There are many factors that influence the character of bacterial metabolism and enzyme production.For the maximum production of the desired products, the media components and fermentation conditions should be optimized. In our investigation, we improved the amylase production of Bacillus subtilis M4 mutant strain by the combination of two optimization techniques. The cultural conditions (time period, temperature, pH, inoculum volume) and medium ingredients (various carbon, organic and inorganic nitrogen sources, chlorides, sulfates, phosphates, carbonates) were optimized by one factor at a time methodology (OFAT) and response surface methodology (RSM) to increase the amylase production. The optimum conditions for amylase production were found be the following: 35oC, pH range 7 and incubation time 72h, inoculum volume 8% (v/v). Optimum medium composition for amylase production was the following: starch 12.9 g, peptone 9.75 g, calcium carbonate 0.439 g, magnesium sulfate 0.464 g and potassium chloride 0.464 g per liter. When applied to our optimized medium in the fermentation process, the enzyme activity increased from 0.741 to 1.58 U/ml, which means a 2.1-fold increase compared to the original medium. KEYWORD: Amylase activity, Bacillus spp., Statistical optimization, OFAT, RSM


INTRODUCTION
Amylases are glycoside hydrolases enzymes, which break starch into glucose, maltose, maltotriose, and dextrin by hydrolyzing process of glycosidic bonds. These enzymes have been widely used many years.First enzyme produced industrially was an amylase from a fungal source in 1894 and used as a pharmaceuticalaid for the treatment of digestive disorders [1]. Amylases are the major class of industrial enzymes, which constitute approximately 25% of the enzyme market [2]. The biological organisms such as plants, animals, and microorganisms are major sources of enzymes. Microorganism produces amylase more beneficially than other sources. The production rate of microorganisms is high and can be engineered to obtain enzymes of good characteristics [3]. The Bacillus genus trends to dominate the enzyme industry, because of almost all microorganisms of this genus synthesis α-amylase. Amylases obtained from Bacillus licheniformis, Bacillus stearothermophilus, and Bacillus amyloliquefaciens are used in many industrial processes such as in food, fermentation, textiles and paper industries [4]. The composition and concentration of fermentation media significantly influence bacterial growth and extracellular amylase production. Optimization of cultural conditions is essential for the maximum production of bacterial strains. Almost all forms of microorganisms grow differently with specificity to different substrates in the fermentation medium.

Enzyme production
The basal media was inoculated with 10% (1x10 6 CFU/ml) of bacterial inoculum and incubated at 37°C for 24 h in shaking incubator with shaking of 110 rpm. When reached the fermentation period, the culture medium was centrifuged at 5000 rpm for 10 min and supernatant was used as a crude enzyme [8].

Amylase Assay
Amylase activity was determined by spectrophotometric method according to Fisher and Stein [9]. 1.0 ml of the crude enzyme was taken in a test tube and 1.0 ml of substrate (starch) was added. The test tube was incubated at 45°C in the water bath for 30 min. Then 2.0 ml dinitro-salicylic acid reagent was added in the tube and kept in boiling water bath for 5 min. After cooling at room temperature, the absorbance was read at 540 nm by spectrophotometer. One unit of amylase activity was measured as the amount of amylase required to liberate reducing sugar equivalent to one mmol of Dglucose per minute at 45°C [10].

Bacterial growth
The growth of bacteria was determined by the optical density of culture broth at 600 nm ina spectrophotometer.
Cells were isolated by centrifugation (5000 rpm for 5 min at 4ºC) of culture samples and washed two times with saline water (0.8% NaCl) [11].

Process Optimization for Amylase Production
One factor at a time methodology (OFAT)

Optimization of physicochemical parameters
The physicochemical parameters of the fermentation process, such as temperature, pH, inoculum volumeand incubation time were optimized. Enzyme activity was measured after incubation of bacterial strain at various temperatures ranging from 20˚C to 45˚C and pH from 3.0 to 10.0 and optimal temperature and pH were defined. The effect of inoculum volume on α-amylase activity was determined by inoculating the basal medium with different inoculum volumes ranging from 2% to 12% (v/v). To determine optimum incubation period, amylase activity and bacterial growth were observed during 120 h of incubation at the optimal temperature, pH and inoculum volume. Samples were taken every 24 h intervals [11].

Central composite rotatable design (CCRD)
In this experiment, we used the Central composite rotatable design method to find more accurate factor values to reach a good response. Five variables were selected from OFAT investigation results to evaluate their effects on amylase activity. We provided RSM analysis based on CCRD. Minitab version 18.1 program developed a five-level-five-variable CCRD with six replicates at the center point (13,14). In total 32 experiments were carried out in triplicate. The coded levels of the independent variables are prescribed in Table 1. Experimental data were analyzed by the response surface regression (RSREG) methodology and the following secondorder polynomial equation was calculated (1): whereY is the response variable (amylase activity), β k0, βki, βkii, and βkij are constant coefficients and xi the uncoded independentvariables [16]. MINITAB calculated the optimal values using equation (1).

Validation of amylase productionin optimized media
We compared cell growth and amylase production in optimized media with growth and amylase production in basal media to verify our statistical calculation results. Bacterial cell growth, amylase activity and pH in the fermentation media were determined every 24 h to monitor the changes during 120 h fermentation of B. subtilis M4 strain.

RESULTS AND DISCUSSION
To enhance amylase production, we applied two different optimization technique and optimized culture condition and medium composition. Incubation period: The Incubation period has an essential role in amylase activity. In our study, the incubation period for optimum production of amylase was 72h for B. subtilis M4. The enzyme activity reached a maximum of 0.799 U/ml (Fig 1), then decreased to 0.453 U/ml when the incubation time increased from 72 to 120 hours. Moreira et al.
reported that different species of Bacillus have shown a similar cultivation period of 72 h [17] for maximum amylase production [18]. A. oryzae produced the maximum α-Amylase at 72 h of incubation period [19]. B. amyloliquefaciens reached the optimum α-Amylase activity after fermentation for 72 hours [15]. In our experiment, the optical density of the B. subtilis M4 strain reached a maximum also after 72h of fermentation ( Fig. 1). It was noticed a positive correlation between growth and amylase activity for this strain that indicates the amylase production was growth associated with the B. subtilis M4 strain. Growth kinetics of the isolated Bacillus strain by Mishra and Behera, started lag phase right after inoculation. The stationary phase started from late 48 h which continued till 72 h and after that growth declined at 92 h [20]. OD 600 nm Amylase activity (U/ml)

Incubation period (h)
Amylase activity Bacterial growth Temperature: Enzyme activity monitored at various temperatures displayed that B. subtilis M4 reached maximum amylase production at 35°C (Fig 2). The optimal temperature of 35°C was showed for amylase production by B. subtilis CBTK 106, isolated from banana wastes [21]. According to Pokhrel et al., 35°C was found as the optimum temperature at which enzyme activity was found to be higher, of the bacterial isolate identified as Bacillus spp. [22]. Our results arein good agreement with this study.Behal et al., [24]investigated thermostable amylase producing Bacillus spp that revealed an optimum enzyme activity at pH 8.0 whereas in other species the optimum activity was at pH 7.0 [25]. Inoculum volume: In our experiment, the enzyme activity was increased as the inoculum volume increases and reached the optimum at 8% (Fig 4).
When the inoculum volume was further increased, the enzyme activity slowly decreased. It may be caused by the initial speedy growth of bacteria and the lack of nutrients in the medium. According to Tsurikova et al., the optimal inoculum volume for the production of amylase was found to be 8% [26]. Our experiment repeated the results regarding the negative effects of high inoculum volume on amylase production of bacteria [27]. The effect of carbon sources on amylase production was determined by substituting the carbon sources of the basal media and culturing the bacteria in optimum conditions. Starch was revealed as the most suitable carbon source for amylase production by B. subtilis M4 strain (Fig. 5).
Amylase is an inducible enzyme and is generally induced in the presence of starch or its hydrolytic product, maltose [28]. The utilization of soluble starch by Bacillus spp. was reported previously [29].
In the media containing lactose, the lowest amylase production was registered. Based on our experimental data, starch was supposed as the most suitable carbon source for B. subtilis M4 for amylase production. In the media containing peptone, the highest amylase production was recorded. Ammonium sulfate was revealed as the least suitable nitrogen source.
According to our study, the B. subtilis M4 strain produced 6-fold more amylase in the media with peptone with comparing with ammonium sulfate (Fig. 6). Tryptone, peptone, and casein were registered as suitable sources for the production of amylase [30].

Chlorides:
We have found that calcium chloride affects the highest amylase production and potassium chloride is the second suitable chloride for B.subtilis M4 (Fig 7). The addition of salts of some metal ions resulted in good growth of microorganisms and so better enzyme production (most α-amylases are recognized as metalloenzymes). Ca 2+ ions are revealed being present in the majority of these enzymes. The addition of CaCl2 to the culture media enhanced enzyme production [31]. Sulfates: In our study, calcium sulfate was registered as the best sulfate source for amylase production (Fig  8). The next one was found to be magnesium sulfate. Mg 2+ had a great role and enzyme production was decreased to 50% when Mg 2+ was eliminated from the medium. Na + and Mg 2+ jointly stimulated enzyme production by Bacillus spp. CRP strain [32].  9). Phosphate has a significant regulatory role in the synthesis of primary and secondary metabolites in microorganisms [33] and affects the growth of the organism and amylase production. Above 0.2 M phosphate levels significantly increased enzyme production and conidiation in A. oryzae [34]. In this experiment, calcium carbonate greatly increased the production of amylase (Fig. 9), so we defined it to be the best calcium source for B. subtilis M4 strain for amylase production.

Central composite design (CCD)
We have determined the effect of the selected 5 variables on amylase activity by the response surface methodology. CCRD matrix with experimental and predicted data are described in Amylase activity (U/ml)  The calculated response surface for the most important factors is demonstrated by CCRD in a 3dimensional graph (Fig. 10). 3-dimensional graph of starch and peptone (A), calcium carbonate and magnesium sulfate (B), starch and potassium chloride (C) against amylase activity demonstrate the outcomes of the statistical and mathematical analyses.
The calculated response surface for the most important factors is demonstrated by CCRD in a 3dimensional graph (Fig. 10). 3-dimensional graph of starch and peptone (A), calcium carbonate and magnesium sulfate (B), starch and potassium chloride (C) against amylase activity demonstrate the outcomes of the statistical and mathematical analyses.
According our study the optimum concentrations of selected factors were as following (Fig. 11): starch -0.42 (12.9 g/l), peptone -0.10 (9.75 g/l), calcium carbonate 0.30 (0.439 g/l), magnesium sulfate -0.18 (0.464 g/l), potassium chloride -0.18 (0.464 g/l), respectively. At this optimized condition, our model predicted 1.563 U/ml of amylase activity. The culture broth pH during the cultivation was between 6.5-7.5. In the optimized medium, the enzyme activity increased from 0.741 to 1.58 U/ml, which means a 2.1 fold. According to Deljou and Arezi, in the result of medium optimization, amylase production by B. licheniformisAZ2 increased by 2.4 fold [11]. Another study reports that for optimization of the fermentation medium components and environmental factors was used OFAT approach and Plackett-Burman design. As a result of this work amylase production by Bacillus lichineformis AH214, was enhanced 2.0 fold compared to the original medium [35].  A B C Figure. 10. Three-dimensional graph for amylase production: A unction of starch and peptone(A), calcium carbonate and magnesium sulfate (B), starch and potassium chloride(C) Figure 11. The optimum concentrations of selected factors, founded by solving the secondary order polynomial equation (2) Comparison between basal and optimized media Fig.11 describes changes of amylase production (A), cell density (B), and pH (C) during the incubation period of B. subtilis M4 in the optimized and basal medium. Cells grew more and reached the highest level at 72 h of incubation, then the growth reduced slightly. The bacterial growth kept stable after 48 h of cultivation. The culture broth pH during the cultivation was between 6.5-7.5. In the optimized medium, the enzyme activity increased from 0.741 to 1.58 U/ml, which means a 2.1 fold. According to Deljou and Arezi, in the result of medium optimization, amylase production by B. licheniformisAZ2 increased by 2.4 fold [11]. Another study reports that for optimization of the fermentation medium components and environmental factors was used OFAT approach and Plackett-Burman design. As a result of this work amylase production by Bacillus lichineformis AH214, was enhanced 2.0 fold compared to the original medium [35].   Figure.12.Change in amylase production (A), cell density(B), and pH (C) during fermentation of B.subtilis M4. Optimized medium (-----)and basalmedium (-----). Each data states the mean of triple independent assays (the standard errors were less than 5% of the means).

CONCLUSION
In this study, we aimed to increase the amylase production by the B. subtilis M4 mutant strain. For this purpose, the cultural conditions and medium ingredients were optimized by one factor at a time approach and response surface methodology. The optimal culture conditions for amylase production were the following: temperature 35ºC, pH value 7, incubation time 72 h, inoculum volume 8% (v/v). Optimal fermentation medium for amylase production contained starch 12.9, peptone 9.75, calcium carbonate 0.439, magnesium sulfate 0.464 and potassium chloride 0.464 g/l. In the optimized fermentation media, the enzyme activity reached 1.58 U/ml, which means a 2.1 fold compared to the basal medium. To be applied commercially, further study is needed for the improvement of this enzyme production.

ACKNOWLEDGMENTS
This study was a part of the Ph.D. work and we deeply appreciate all supports from the School of Animal Science and Biotechnology, Mongolian University of Life Sciences for assisting this research paper.