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Chung-Soon Chang, Department of Biochemistry, College of Medicine, Inha University, 7-241 Shinheung-Dong 3 Ga, Chung-Ku, Inchon 400-103, Korea (e-mail: firstname.lastname@example.org).
Aims: An investigation was carried out on an oxidative and SDS-stable alkaline protease secreted by Bacillus clausii of industrial significance.
Methods and Results: Maximum enzyme activity was produced when the bacterium was grown in the medium containing (g l−1): soyabean meal, 15; wheat flour, 10; liquid maltose, 25; K2HPO4, 4; Na2HPO4, 1; MgSO4·7H2O, 0·1; Na2CO3, 6. The enzyme has an optimum pH of around 11 and optimum temperature of 60°C. The alkaline protease showed extreme stability towards SDS and oxidizing agents, which retained its activity above 75 and 110% on treatment for 72 h with 5% SDS and 10% H2O2, respectively. Inhibition profile exhibited by phenylmethylsulphonyl fluoride suggested that the protease from B. clausii belongs to the family of serine proteases.
Conclusions:Bacillus clausii produced high levels of an extracellular protease having high stability towards SDS and H2O2.
Significance and Impact of the Study: The alkaline protease from B. clausii I-52 is significant for an industrial perspective because of its ability to function in broad pH and temperature ranges in addition to its tolerance and stability in presence of an anionic surfactant, like SDS and oxidants like peroxides and perborates. The enzymatic properties of the protease also suggest its suitable application as additive in detergent formulations.
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It is well known that proteases constitute one of the most important groups of industrial enzymes, as it accounts for at least a quarter of the total global enzyme production (Layman 1986). In recent years, the use of alkaline proteases in a variety of industrial processes like detergents, food, leather and silk has increased remarkably (Kembhavi et al. 1993; Gessesse 1997; Kumar and Takagi 1999; Kumar and Tiwari 1999). Although a wide range of micro-organisms are known to date to produce proteases, a large proportion of the commercially available alkaline proteases are derived from Bacillus strains because of its ability to secrete large amounts of alkaline proteases having significant proteolytic activity and stability at considerably high pH and temperatures (Jacobs 1995; Ito et al. 1998; Kumar et al. 1998; Yang et al. 2000). Further, these enzymes are compatible and stable to various detergent components and active at washing temperatures and pH values. Among them, a facultative alkalophile Bacillus clausii is known to produce a commercially important alkaline serine protease, Savinase® (Novozyme, Novo Nordisk, Bagsvaerd, Denmark), which finds use as detergent additive to remove protein-containing spots from laundry (Christiansen and Nielsen 2002).
In general, most of the alkaline proteases applied for industrial purposes face some limitations. First, many of the available alkaline proteases exhibited low activity and stability towards anionic surfactants like SDS and oxidants like hydrogen peroxide, which have been the common ingredients in modern bleach-based detergent formulations. Secondly, around 30–40% of the production cost of industrial enzymes is estimated to be accounted for the cost of the growth medium. Considering these facts, we attempted to isolate and screen new marine alkalophilic micro-organisms from the tidal mud flat in Inchon, Korean West Sea (Yellow Sea), and use some low-cost and easy available medium ingredients for cost-effective alkaline protease. We recently isolated an alkalophilic strain of B. clausii I-52 from the heavily polluted tidal mud flats of Yellow Sea, which produced a strong alkaline proteolytic enzyme having industrial potential. In this paper, we report an oxidant and SDS-stable protease from this alkalophilic isolate and the optimization of the protease production parameters which finds its potential use as detergent additive and for other industrial applications.
Materials and methods
Micro-organism and enzyme production
Protease-producing isolates were isolated from the heavily polluted tidal mud flat in Inchon, Korean West Sea, and screened using a skimmed milk agar plate in tryptic soya broth (TSB). Among the isolates, B. clausii I-52 which exhibited a prominent clear zone was selected for the study of protease production. The isolate was maintained on TSB agar plate and stored at 4°C. The basal culture medium for the protease production contained (g l−1): K2HPO4, 4; Na2HPO4, 1; MgSO4·7H2O, 0·1; Na2CO3, 6. Sodium carbonate solution was sterilized separately, and then added to the medium. The medium (100 ml) was inoculated with 1 ml of a 24-h-old seed culture in 500 ml baffled flasks, and incubated at 37°C with shaking at 250 rev min−1 for 48 h. The cell-free supernatant was recovered by centrifugation (8000 g, 4°C, 20 min), and used for determining the protease activity.
Alkaline protease activity was determined using casein as a substrate at a concentration of 0·5% in 0·1 mol l−1 glycine-NaOH buffer (pH 11·0) (Kumar et al. 1999). One unit of enzyme activity is defined as the amount of the enzyme resulting in the release of 1 μg of tyrosine per minute at 60°C under the standard assay conditions.
Partial purification of the enzyme
Partially purified alkaline protease was used to investigate some enzymatic properties. The cell-free supernatant was harvested by centrifugation at 8000 g for 20 min, and adsorbed to Diaion HP 20 (5%, w/v; Mitsubishi Chemical, Tokyo, Japan) according to the method reported previously (Joo et al. 2001). The resin was recovered by suction filtration and then eluted with buffer A (0·1 mol l−1 sodium phosphate buffer, pH 7·5) containing 25% acetone. The solution eluted from the resin was mixed with one volume of buffer A containing 2 mol l−1 (NH4)2SO4 and then applied on a Phenyl-Sepharose column (2·5 × 10 cm), which had been equilibrated with buffer A containing 1 mol l−1 (NH4)2SO4. The column was washed with the same buffer until the optical density of the effluent at 280 nm almost reached zero, and then eluted with buffer A. The flow rate was 100 ml h−1 and 5 ml fractions were collected. Fractions with high protease activity were pooled and concentrated using Centriprep PM10 (Millipore, Bedford, MA, USA) and stored as aliquots at −70°C for further use.
Partial characterization of the enzyme activity
Protease activity was measured by the standard assay method in the following buffer systems: 0·1 mol l−1 citric acid (pH 3·0–3·5), 0·1 mol l−1 sodium acetate (pH 4·0–5·5), 0·1 mol l−1 sodium phosphate (pH 6·0–7·5), 0·1 mol l−1 Tris–HCl (pH 8·0–9·0), 0·1 mol l−1 glycine-NaOH (pH 9·5–11), 0·1 mol l−1 sodium phosphate (pH 11·5–12·0) and 0·1 mol l−1 sodium carbonate (pH 12·5–13·0), respectively. To check the pH stability, 10 μl of the enzyme solution was mixed with 190 μl of the buffer solutions mentioned above and aliquots of the mixture were taken to measure the protease activity under standard assay conditions after incubation for 72 h. To evaluate the heat stability of the protease, the partially purified protease was incubated at various temperatures ranging from 30 to 70°C for 30 and 60 min, respectively. To examine the effect of surfactants and oxidizing agents on the enzyme activity, several agents were added to the enzyme solution at the indicated concentrations, allowed to stand for 72 h at room temperature and the remaining activities were measured.
The isolate is a Gram-positive, motile and rod-shaped bacterium having a size of 1·7 × 3·1–3·5 μm, and can ferment maltose and sucrose. It was also positive for the formation of acetoin and hydrolysis of starch and casein. Based on the 16S rRNA sequence data, the isolate I-52 was identified as B. clausii as it showed high homology with B. clausii DSM 8716T (99·29% similarity within the determined sequence of 708 nucleotides). The isolate has been deposited in the Korean Collection for the Type Cultures, Taejon, Korea, under the accession no. KCTC 10277 BP.
Effect of nitrogen and carbon sources on the protease production
Among the organic nitrogen sources used, soyabean meal had the significant effect on the production of the extracellular protease, and high level of the production was achieved when the cells were grown in a medium containing 1·5% (w/v) soyabean meal (6780 U ml−1). However, casein (680 U ml−1) and gelatin (830 U ml−1) exhibited poor effect on the protease production in B. clausii I-52, as the yields were less than approx. 10 and 12% in comparison with soyabean meal, respectively (Table 1). In order to investigate the effect of sodium carbonate on the optimal production of the protease, sodium carbonate solution at a concentration ranging from 0·2 to 1% (w/v) was sterilized separately and then added to a basal medium containing 1·5% (w/v) soyabean meal. As shown in Table 2, an increased protease production (6560 U ml−1) was achieved at a concentration of 0·6% (w/v) sodium carbonate (initial pH, 10·45). Among the various starches tested (Table 3), the addition of 1% (w/v) wheat flour was observed to be the effective concentration for protease production (11 830 U ml−1). In addition to wheat flour, we also examined the effect of other carbon sources on the protease yield. As shown in Table 4, it was observed that significant improvement in protease yield was obtained with supplementation with 2·5% (v/v) liquid maltose (24 270 U ml−1). However, glucose (9580 U ml−1) and lactose (6060 U ml−1) showed decrease in protease yields, and reduced approx. 18 and 48%, respectively (Table 4). Following the optimization, the highest yield (24 270 U ml−1) and specific yield (28 220 U mg−1) were achieved in B. clausii I-52 (Table 4).
Table 1. Effect of the nitrogen sources on the production of the extracellular protease. Cells were grown in the basal medium containing 0·6% sodium carbonate and supplemented with each nitrogen source at 37°C for 48 h
Enzyme activity (U ml−1, ×10−2)
Specific activity (U mg−1, ×10−2)
Cotton seed flour
Corn steep solids
Table 2. Effect of sodium carbonate on the protease production. Cells were grown in the basal medium containing 1·5% soyabean meal and sodium carbonate at 37°C for 48 h
Sodium carbonate (%)
Enzyme activity (U ml−1, ×10−2)
Table 3. Effect of the different types of starch on the production of the extracellular protease. Cells were grown in the basal medium containing 1·5% soyabean meal, 0·6% sodium carbonate and supplemented with each starches at 37°C for 48 h
Enzyme activity (U ml−1, ×10−2)
Specific activity (U mg−1, ×10−2)
Table 4. Effect of the carbon sources on the production of the protease. Cells were grown in the basal medium containing 1·5% soyabean meal, 1% wheat flour and 0·6% sodium carbonate (control) and supplemented with each carbon sources at 37°C for 48 h
Enzyme activity (U ml−1, ×102)
Specific activity (U mg−1, ×10−2)
Optimal pH and temperature of the enzyme
Optimal pH for protease activity was determined to be around pH 11·0 (Fig. 1a). The maximum protease activity recorded was between 60 and 65°C under the standard reaction conditions, while the activity decreased rapidly above 70°C (Fig. 1b). When the enzyme was incubated with different buffers for 72 h, the protease was very stable over a broad pH range from 5 to 12, which retained more than 90% of its activity even after incubation for 72 h, however, more than 80% of the maximum activity was lost below pH 4·5 or above pH 12·5 (Fig. 2a). By analyzing the thermal stability, the protease was found to be stable up to 55°C for 1 h incubation, but lost approx. 45% of its activity at 60°C even after 30 min incubation (Fig. 2b).
Effect of surfactants and oxidizing agents on the enzyme activity
The partially purified protease produced by B. clausii I-52 was stable not only towards the non-ionic surfactants like Triton-X-100 and Tween-20 but also towards strong anionic surfactant like SDS. Especially, it showed high stability against SDS and retained approx. 73% of its activity even after treatment with 5% SDS for 72 h. Further, the enzyme exhibited an enhanced activity on treatment for 72 h with 2·5% sodium perborate and 5% hydrogen peroxide (Table 5).
Table 5. Effect of oxidizing agents and surfactants on protease activity from B. clausii I-52. The protease was preincubated with oxidizing agents and surfactants for 72 h period at room temperature and the remaining activity was measured using the standard protease assay. Residual activity was determined as percentage of control with no additions
Remaining activity (%)
Bacillus-derived alkaline proteases are the major industrial workhorses and the recent trend towards the use of alkaline proteases from these sources in different process applications like detergents, tanning, food, waste treatment and peptide synthesis has increased remarkably becausae of their increased production capacities, high catalytic activity and high degree of substrate specificity (Kumar et al. 1998; Kumar and Takagi 1999). In this paper, we reported a new strain of B. clausii, which produced high levels of an extracellular alkaline protease with optimal pH of 11 and temperature of around 60°C. The protease activity was strongly inhibited by phenylmethylsulphonyl fluoride confirming it as a serine protease (Table 6). We identified that soyabean meal was an effective medium ingredient for the protease production by B. clausii I-52 among the organic nitrogen sources tested (Table 1). With respect to the nitrogen sources, soyabean meal (Glycine max) is one of the potentially useful cost-effective medium substrate because of its easy availability and low-cost as it is produced as a by-product during oil extraction (Gattinger et al. 1990). However, the addition of casein and gelatin showed no or little effect on the protease production in B. clausii I-52. This result was somewhat different from some other Bacillus species. It was earlier reported that the addition of casein substantially improved the protease production in B. licheniformis MIR29 (Ferrero et al. 1996) and Bacillus sp. (Puri et al. 2002). Protease production was increased approx. 30% by the addition of 1% (w/v) casein in B. horikoshii isolated from the haemolymph of a unique Korean polychaeta, Periserrula leucophryna (Joo et al. 2002). The protease yield was greatly enhanced approx. 1·75-fold by the addition of 1% (w/v) wheat flour (Table 3) and, especially, 3·58-fold by the addition of 2·5% (v/v) liquid maltose (Table 4) to a culture medium when compared with a basal medium containing 1·5% (w/v) soyabean meal. However, lactose exhibited a negative effect on the protease production. Contrary to this result, Mabrouk et al. (1999) reported the enhancement of protease production in B. licheniformis ATCC 21415 by the addition of lactose, but a lowered yield was observed with the addition of maltose to the culture medium. Based on the optimization studies, we achieved a yield of 24 270 U ml−1 with specific activity of 28 220 U mg−1 protein when cultivated for 48 h at 37°C in a medium containing (g l−1): soyabean meal, 15; wheat flour, 10; liquid maltose, 25; K2HPO4, 4; Na2HPO4, 1; MgSO4·7H2O, 0·1; Na2CO3, 6 (Table 4). Despite the use of low-cost medium ingredients, the proteases must also exhibit a strong stability against surfactants and oxidants, which have been the common ingredients in modern bleach-based detergent formulations. The protease from B. clausii showed stability and compatibility towards strong anionic surfactants like SDS and oxidizing agents such as H2O2 and sodium perborate. Kobayashi et al. (1995) reported that an alkaline protease from Bacillus sp. KSM-K16 retained approx. 75% activity on treatment with 5% SDS for 4 h. Earlier reports on the stability of alkaline proteases towards oxidants had indicated that an alkaline protease from Bacillus sp. RGR-14 showed 40% loss in enzyme activity with 1% H2O2 (Oberoi et al. 2001), while a subtilisin-like protease from Bacillus sp. KSM-KP43 lost little or no enzyme activity on treatment with 10% H2O2 for 30 min (Saeki et al. 2002). However, there is little published literature available concerning the stability studies of protease towards both SDS and hydrogen peroxide. Gupta et al. (1999) reported that the protease from Bacillus sp. SB5 retained about 60 and 95% of its activity on treatment for 1 h with 1% SDS and 5% H2O2, respectively, while the Bacillus sp. JB-99 protease retained 75 and >95% of its activity on treatment for 1 h with 0·5% SDS and 5% H2O2, respectively (Johnvesly and Naik 2001). Comparing these results, the B. clausii protease exhibited a significant compatibility and stability towards both surfactants and oxidizing agents, which retained its activity of 73 and 116% after incubation for 72 h with 5% SDS and 5% H2O2 (Table 5). As the protease produced by B. clausii I-52 was more stable over a wide range of pH and temperatures and also towards both surfactants and oxidants, it is envisaged that the isolate can be a potential source of alkaline protease for use as additive in industrial applications like detergent industry.
Table 6. Effect of various inhibitors on the enzyme activity of the partially purified extracellular protease from B. clausii I-52