Optimisation of Media and Cultivation Conditions for Alkaline Protease Production by Alkaliphilic Bacillus halodurans

MATERIALS AND METHODS

Microorganism and Culture Maintenance
The microorganism used in this study was isolated from indigenous soil samples from Wadi El-Natrun, an Egyptian Soda Lake and were screened using a skim milk agar plate and later in alkaline broth (Ibrahim and El-diwany, 2007). The isolate was identified as Bacillus halodurans according to morphological and biochemical tests and 16S rDNA sequence analysis (Ibrahim and El-diwany, 2007).

Stock cultures of the isolate were stored in 30% glycerol at-70°C. Prior to each experiment, the bacterium was sub-cultured from the frozen stocks onto Horikoshii agar medium (pH 10.5) containing 10 glucose, 5 peptone, 5 yeast extract, 1 KH₂PO₄, 0.2 MgSO₄•7H₂O, 10 Na₂CO₃ and 15 g g L^-1 agar (Horikoshi, 1999b). Alkaline broth was used as the basal medium for preliminary studies of bacterial growth and protease production.

Production of Alkaline Proteases
A loop of culture from an agar plate was inoculated into a 50 mL glass tube containing 5 mL of alkaline protease production medium and incubated overnight at 180 rpm and 50°C. This culture was then inoculated into a 500 mL capacity Erlenmeyer flask containing 95 mL of the same medium and incubated at 50°C for 48 h. Cells and insoluble materials were removed by centrifugation at 10,000 g for 10 min at 4°C and the cell-free supernatant was filtered through a 0.45 μm pore-size membrane filter and was used as the source of the crude alkaline protease enzyme. Five different media were used for enzyme production: (1) Horikoshii medium, (2) Luria-Bertani broth (LB) plus 10 g L^-1 Na₂CO₃, (3) tryptic soy broth (TSB) plus 10 g L^-1 Na₂CO₃, (4) nutrient broth (NB) plus 10 g L^-1 Na₂CO₃ and (5) a modification of a medium previously described by Beg et al. (2003) containing 5 casamino acids, 10 glucose, 1 KH₂PO₄, 3 K₂HPO₄, 2 Na₂SO₄, 0.1 MgSO₄•7H₂O and 10 g L^-1 Na₂CO₃.

Enzyme Assay
Proteolytic activity was assayed by a modification of the method of Kunitz (1947). Samples containing 400 μL of 0.5% (w/v) casein in 50 mM Tris-HCl buffer, pH 10, with 100 μL of enzyme sample were incubated in a water bath at 50°C for 20 min. The enzyme reaction was terminated by addition of 500 μL of 10% (w/v) trichloroacetic acid and incubation at room temperature for 10 min.

The reaction mixture was centrifuged at 10,000 g for 10 min at 4°C and the absorbance was measured against a blank (non-incubated sample) at 280 nm. One unit of protease was defined as the amount of enzyme that releases the equivalent of 1 ìmol of tyrosine per minute under the defined assay conditions. Standard curve of tyrosine was constructed using 10, 20, 30, 40, 50, 60, 70, 80 and 100 mg mL^-1 tyrosine in 50 mM glycine-NaOH buffer, pH 10.

Protein Determination
Protein concentrations were determined according to the method described by Bradford (1976). One millilitre of Bradford reagent was added to 50 μL of sample and the absorbance was measured after 5 min at 595 nm. Different concentrations of Bovine Serum Albumin (BSA) were used as a protein standard, including 10, 20, 40, 60, 80 and 100 μg mL^-1 in distilled water. One milliliter of Bradford reagent was added to 50 μL BSA standard and after 5 min the absorbance was measured at 595 nm.

Optimization of Alkaline Protease Production
The alkaline protease production optimization studies of Bacillus halodurans included different media and conditions, e.g., five different media with different carbon and nitrogen sources, including cheap nutrient sources with different concentrations and metal ions. The effect of different cultivation conditions, including incubation temperatures (25-55°C), aeration conditions (0-300 rpm), incubation times and inoculum sizes, were investigated. All experiments were done in triplicate.

RESULTS AND DISCUSSION

Optimization of Alkaline Protease Production
The microorganism used in this study was isolated from indigenous soil samples of Wadi El-Natrun, an Egyptian soda lake and were screened using a skim milk agar plate and later in alkaline broth (Ibrahim and El-diwany, 2007). The organism was identified as Bacillus halodurans according to morphological and biochemical tests and 16S rDNA sequence analysis (Ibrahim and El-diwany, 2007). The crude alkaline protease showed reasonable activity at a temperature range of 65 to 75°C with maximum activity at 70°C. The crude enzyme also had a relatively wide pH range of activity between pH 8 to 11, with maximum enzyme activity at pH 10 in 50 mM Tris-HCl buffer, indicating the enzyme is a thermo-alkaline protease (Ibrahim and El-diwany, 2007).

The time course of alkaline protease production showed that maximum enzyme production was seen after 48 h of incubation (data not shown). Five different alkaline media were tested for maximum alkaline protease production by Bacillus halodurans. The results presented in Fig. 1 indicated that Medium I was the best medium for alkaline protease production by Bacillus halodurans (70.2 U mg^-1 protein). Therefore, Medium I was selected for further analysis.

Fig. 1:	Effects of different media on alkaline protease production by Bacillus halodurans at pH 10.5 and 50°C after 48 h of incubation

Table 1:	Effects of different carbon sources on alkaline protease production by Bacillus halodurans at pH 10.5 and 50°C after 72 h of incubation

Fig. 2:	Effects of different lactose concentrations on alkaline protease production by Bacillus halodurans at pH 10.5 and 50°C after 72 h of incubation

Effects of Carbon Sources
There are several reports that different carbon sources have different influences on extracellular enzyme production by different strains (Chi and Zhao, 2003; Beg et al., 2003; Kanekar et al., 2002). Therefore, the effect of different carbon sources on alkaline protease production by B. halodurans was investigated. The results presented in (Table 1) show that lactose was the best carbon source for protease production with a specific activity of 135.5 U mg^-1 protein, which indicated that B. halodurans was able to produce β-galactosidase to hydrolyze lactose and consume glucose and galactose as carbon sources. This result is similar to that reported for alkaline protease production by Salinvibrio sp., where lactose was the best carbon source for enzyme production (Amoozegara et al., 2007). However, increased alkaline protease production was also reported by several other studies that used different sugars, such as starch, sucrose, fructose and maltose (Genckal and Tarib, 2006; Reddy et al., 2008), indicating that the best carbon source for enzyme production is different according to different microorganisms. While glucose showed the highest cell growth (115.16 mg/50 mL), it showed low enzyme production, indicating that alkaline protease production was catabolically repressed by glucose (Mehta et al., 2006; Kaur et al., 2001). The enzyme specific activity increased with increasing lactose concentrations (Fig. 2) with a maximum enzyme production at 1.5% lactose (224.66 U mg^-1 protein). Further increases in the lactose concentration resulted in reductions of enzyme production, which was mostly due to catabolic repression of enzyme production by the accumulation of glucose produced after lactose hydrolysis (Mehta et al., 2006). Optimizing the carbon source resulted in an increase in enzyme production of about three fold.

Effects of Different Nitrogen Sources on Alkaline Protease Production
In microorganisms, nitrogen (both organic and inorganic forms) is metabolized to produce amino acids, nucleic acids, proteins and cell wall components. Alkaline protease production depends heavily on the availability of nitrogen sources in the medium, which has regulatory effects on enzyme synthesis (Patel et al., 2005).

Table 2:	Effects of different nitrogen sources on alkaline protease production by Bacillus halodurans at pH 10.5 and 50°C 48 h of incubation

Fig. 3:	Effect of different soybean concentration on alkaline protease production by Bacillus halodurans at pH 10.5 and 50°C after 48 h incubation

The effects of the nitrogen source on alkaline protease production by Bacillus halodurans, including different inorganic, organic and cheap nitrogen sources, were examined. As shown in Table 2, the inorganic nitrogen sources urea, ammonium chloride and ammonium sulfate showed the lowest alkaline protease productions of 32.2 protein, 66.22 protein and 67.56 U mg^-1 protein, respectively. Interestingly, soybean, which is a cheap nitrogen source, showed the highest enzyme production of the nitrogen sources used (382.65 U mg^-1 protein). Soybean was shown to stimulate protease production in the marine bacterium Teredinobacter turnirae (Elibola and Moreira, 2005), while protease production in haloalkaliphilic Bacillus sp. was enhanced by a combination of peptone and yeast extract (Patel et al., 2005). However, the inorganic nitrogen source sodium nitrate was shown to stimulate alkaline protease production in another microorganism (Chi et al., 2007), indicating that the best nitrogen source for enzyme production is different according to different microorganisms. Alkaline protease production by Bacillus halodurans was increased by increasing soybean concentrations and reached a maximum level at 6 g L^-1 (835.6 U mg^-1 protein) and decreased with further increasing soybean concentrations (Fig. 3). A similar effect of soybean was reported in Teredinobacter turnirae (Elibola and Moreira, 2005). Optimization of the nitrogen source resulted in a further increase in alkaline protease production of about 3.5 fold.

Effects of Different Metals on Alkaline Protease Production by B. halodurans
The effects of different metal ions on the production of alkaline protease by Bacillus halodurans were investigated. Among different metals examined, Mg, Mn and Ca showed the highest enzyme productions at concentrations of 5 mM with specific activities of 893.73, 905.70 and 918.54 U mg^-1 protein, respectively (Table 3).

Table 3:	Effects of different metal concentrations on alkaline protease production by Bacillus halodurans at pH 10.5 and 50°C after 48 h of incubation

Fig. 4:	Effects of different concentrations of a trace element mixture (Ca, Mn and Mg) on alkaline protease production by Bacillus halodurans at pH 10.5 and 50°C after 48 h of incubation

Zn, Cu and Co strongly inhibited enzyme production. Incorporation of Ca, Mn and Mg together in the growth medium showed higher enzyme production than using them individually with maximum production at 5 mM of the trace element mixture (1381 U mg^-1 protein) (Fig. 4).

Effects of Different Incubation Periods on Growth and Alkaline Protease Production
It is known that temperature is one of the most critical parameters that has to be controlled in bioprocesses (Chi and Zhao, 2003). The effect of the incubation temperature on alkaline protease production by Bacillus halodurans was investigated at temperatures ranging from 25-55°C.

Fig. 5:	Effects of different incubation temperatures on alkaline protease production by Bacillus halodurans at pH 10.5 and after 48 h of incubation

Fig. 6:	Effects of shaking speed on the production of alkaline protease by Bacillus halodurans at an incubation temperature of 37°C

The results revealed that maximal cell growth was seen at 50°C (Fig. 5); however, maximum enzyme production was found at 37°C (3416.14 U mg^-1 protein) with about a 2.5-fold increase in the specific enzyme activity. Other researchers have reported that there is an inverse relationship between alkaline protease production and cell growth (Genckal and Tarib, 2006). It has been reported that the optimum temperature for alkaline protease production by Aureobasidum pullulans was much lower than the optimum growth temperature (Chi et al., 2007).

Effects of Agitation Rate
Aeration is another important parameter that affects enzyme production in bacteria. It has been reported that enzymes are also susceptible to mechanical force, which may disturb the elaborate shape of complex molecules to such a degree that denaturation occurs (Banerjee et al., 1999; Gupta et al., 2002a; Joo et al., 2002). Alkaline protease production by Bacillus halodurans was investigated at different agitation speeds. The results represented in (Fig. 6) revealed that enzyme production increases with increasing agitation speed. The maximum protease production was observed at 200 rpm (3416 U mg^-1 protein). However, enzyme production was reduced by further increases in the agitation rate. A similar profile was determined for the effect of agitation speed on alkaline protease production by Teredinobacter turnirae (Elibola and Moreira, 2005). The effect of inoculum size and age was also investigated, but they showed no significant effects on alkaline protease production by Bacillus halodurans (data not shown).

CONCLUSION

An alkaliphilic bacterium isolated from an alkaline Egyptian soda lake and identified as Bacillus halodurans showed thermo-alkaline protease production. The media and cultivation conditions were investigated to optimise alkaline protease production. The goal of this study was to enhance the enzyme production which was achieved through optimization of nutrients and cultivation conditions.

The specific enzyme activity was increased by about 48.8 fold using different nutrient sources and cultivation conditions. The maximum specific enzyme activity was obtained in a medium containing 15 g lactose, 6 g soybean and 5 mM of a mixture of Mg, Mn and Ca after fermentation at 37°C and 200 rpm.