Production of protease from a new alkalophilic Bacillus sp. I-312 grown on soybean meal: optimization and some properties

Process Biochemistry 40 (2005) 1263–1270 www.elsevier.com/locate/procbio

Production of protease from a new alkalophilic Bacillus sp. I-312 grown on soybean meal: optimization and some properties Han-Seung Joo, Chung-Soon Chang* Department of Biochemistry, College of Medicine, Inha University, 7-241 Shinheung-Dong 3 Ga, Chung-Ku, Inchon 400-103, Korea Received 4 August 2003; accepted 16 May 2004

Abstract An oxidative and SDS-stable alkaline protease having industrial significance and produced by an alkalophilic Bacillus sp. I-312, which was isolated from the heavily polluted tidal mud flat and sea water near Songdo in Inchon, Korean West Sea (Yellow Sea). Maximum activity (42,520 U ml 1) was obtained when the bacterium was grown in a medium containing (g l 1): soybean meal, 15; wheat flour, 10; fructose, 5; K2HPO4, 4; Na2HPO4, 1; CaCl2, 0.05; Na2CO3, 8 at 32 8C for 48 h incubation period with agitation of 250 rpm. The optimum pH and temperature of the enzyme were around 11 and 60 8C, respectively. The alkaline protease also showed extreme stability towards SDS and oxidizing agents, which retained its activity 87.8 and 87.5% activity on treatment with 5% SDS and 5% H2O2 for 72 h, respectively. The alkaline serine protease secreted by Bacillus sp. I-312 is industrially important from the perspectives of its abilities to function in alkaline pH and to show stability in broad pH ranges in addition to its stability towards SDS and hydrogen peroxide, suggesting that it is a potential candidate as an additive in detergent formulations. # 2004 Elsevier Ltd. All rights reserved. Keywords: Bacillus sp.; Alkaline protease; Optimization; Detergent additive

1. Introduction Proteases represent one of the most important groups of industrial enzymes and account for at least a quarter of the total global enzyme production [1]. Recently, the use of alkaline proteases has increased significantly in various industrial processes such as detergent and feed additives, dehairing, decomposition of gelatin on X-ray films and peptide synthesis. Among these, use as laundry detergent additivs is one of the various industrial applications for alkaline proteases [2–6]. Although a number of microorganisms produced proteases, Bacillus strains are recognized as important sources of commercial alkaline proteases because of their ability to secrete large amounts enzymes with high Abbreviations: EDTA, ethylenediaminetetraacetic acid; LBTI, limabean trypsin inhibitor; PMSF, phenylmethylsulfonyl fluoride; SBM, soybean meal; SBTI, soybean trypsin inhibitor; SDS, sodium dodecyl; sulphate; TCA, trichloroacetic acid; TLCK, N-a-tosyl-L-lysine chloromethyl ketone; TPCK, N-a-tosyl-L-phenylalanine chloromethyl ketone * Corresponding author. Tel.: +82 32 890 0931; fax: +82 32 884 6726. E-mail address: [email protected] (C.-S. Chang). 0032-9592/$ – see front matter # 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2004.05.010

activity [7–14]. In addition, these enzymes also must be compatible and stable to various detergent components and active at working temperatures and pH values. Thus, the important parameters of these proteases for use as detergent additives are their stability against pH, temperature, detergents and even in bleaching agents. However, many of the alkaline proteases applied to industrial purposes face some limitations. Firstly, exhibited low activity and stability towards anionic surfactants and oxidants, which have been the common ingredients in modern bleach-based detergent formulations. Secondly, around 30–40% of the production cost of the industrial enzymes was estimated to account for the cost of the growth medium. It is well established that extracellular protease production in microorganisms is greatly influenced by media components, especially carbon and nitrogen sources, metal ions, and physical factors such as pH, temperature, dissolved oxygen and incubation time [15–19]. There are various commercially available detergent proteases such as Alcalase1, Esperase1 and Savinase1 (Novozymes Biotech Inc., Denmark), and Purafect1 and Properase1 (Genencor Int.,

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USA). All these enzymes are stable in the presence of the various components of detergents and are active at washing temperatures and pH values and are derived from microorganisms [20–22]. Considering these facts, we attempted to screen and isolate new marine alkalophilic microorganisms having significant protease activity from the heavily polluted tidal mud flat and sea water near Songdo in Inchon, Korean West Sea (Yellow Sea). In this paper, we report an oxidant and SDS-stable protease from an alkalophilic isolate Bacillus sp. I-312 and the optimization of the protease production parameters for its potential use as a detergent additive and for other industrial applications.

2. Materials and methods 2.1. Microorganism and enzyme production Protease-producing isolates were isolated from the heavily polluted tidal mud flat and sea water near Songdo in Inchon, Korean West Sea and screened using a skim milk agar plate in tryptic soy broth (TSB). The isolate was maintained on TSB agar plate and stored at 4 8C. The basal culture medium for the protease production contained (g l 1): K2HPO4, 4; Na2HPO4, 1; CaCl2, 0.05; Na2CO3, 6. Sodium carbonate solution was sterilized separately, and then added to the medium. The medium (100 ml) in 500 ml baffled flasks was inoculated with 1 ml of a 24 h-old seed culture, and incubated at 37 8C with shaking at 250 rpm for 48 h. The cell-free supernatant was recovered by centrifugation (8000  g, 4 8C, 20 min), and used for determining the protease activity and protein concentration. 2.2. Protease assay Alkaline protease activity was determined using casein as a substrate at a concentration of 0.5% in 0.1 M glycine– NaOH buffer, pH 11.0 [23]. One unit of enzyme activity is defined as the amount of the enzyme resulting in the release of 1 mg of tyrosine per min at 60 8C under the standard assay conditions. 2.3. Partial purification of protease Partially purified alkaline protease was used to investigate some enzym properties. The cell-free supernatant was harvested by centrifugation at 8000  g for 20 min, and adsorbed to Diaion HP 20 (5%, w/v) according to a method reported previously [24]. The resin was recovered by suction filtration and then eluted with buffer A (0.1 M sodium phosphate buffer, pH 7.5) containing 25% (v/v) acetone. The eluent was mixed with one volume of buffer A containing 2 M (NH4)2SO4 and then applied to a Phenyl-Sepharose column (2.5 cm  10 cm), which had been equilibrated with buffer A containing 1 M (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 (Amicon) and stored as aliquots at 70 8C for further use. 2.4. Partial characterization of enzyme activity Protease activity was measured using the standard assay method in the following buffer systems: 0.1 M citric acid (pH 3.0–3.5); 0.1 M sodium acetate (pH 4.0–5.5); 0.1 M sodium phosphate (pH 6.0–7.5); 0.1 M Tris–HCl (pH 8.0– 9.0); 0.1 M glycine–NaOH (pH 9.5–11); 0.1 M sodium phosphate (pH 11.5–12.0); and 0.1 M sodium carbonate (pH 12.5–13.0), respectively. Ten microlitres of the enzyme solution was mixed with 190 ml of the each buffer solution and then the protease activity was measured under standard assay conditions after incubation for 72 h to check the pH stability. Partially purified protease was incubated at various temperatures ranging from 35 to 80 8C for 30 and 60 min, respectively, and then the residual activity was assyaed to evaluate heat stability. To examine the effect of surfactants and oxidizing agents on 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 measured.

3. Results 3.1. Microorganism Several bacterial strains secreting alkaline proteases were screened from the heavily polluted tidal mud flat and sea water near Songdo in Inchon, Korean West Sea (Yellow Sea). Of these, isolate I-312 exhibited a large zone of hydrolysis and was selected for the optimization of production conditions and protease characterization. The isolate was a Gram-positive, motile, rod-shaped bacterium having a size of 1.2–1.5 mm  3.1–3.5 mm, and strictly aerobic. The isolate was positive for the utilization of L-arabinose, ribose, fructose, glucose and maltose. However, it was negative for galactose, xylose, inositol, xylitol and lactose. Based on the partial 16S rRNA sequence data, isolate I-312 was identified as belonging to the genus Bacillus. 3.2. Effect of nitrogen sources on the protease production Among the organic nitrogen sources tested, SBM exhibited a prominent effect on the production ability and higher protease yield was achieved using a basal medium supplying 1.5% (w/v) SBM (10,520 U ml 1). In addition, isolate I-312 produced protease when grown in a basal medium supplemented with 1% (w/v) cotton seed flour (5820 U ml 1), corn steep solid (6290 U ml 1) and feather meal (4770 U ml 1),

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Table 1 Effect of the nitrogen sources on the protease production N sources

Concentration (%)

Enzyme activity (U ml 1,  10 2)

Specific activity (U mg 1,  10 2)

None

0

12.8

40.0

Soybean meal

0.5 1.0 1.5 2 2.5

54.4 90.1 105.2 85.3 36.9

164.8 200.2 228.7 158.0 72.4

Casein Gelatin Cotton seed flour Peptone Corn steep solids Feather meal

1.0 1.0 1.0 1.0 1.0 1.0

2.3 5.3 58.2 4.8 62.9 47.7

10.0 21.2 132.3 20.0 118.7 86.7

Cells were grown in the basal medium containing 0.6% sodium carbonate and supplemented with each nitrogen source at 37 8C for 48 h.

respectively. However, casein (230 U ml 1), gelatin (530 U ml 1) and Bacto-peptone (480 U ml 1) caused a significant reduction in the protease yields (Table 1). This result was somewhat different from some other Bacillus species and it was reported earlier that the addition of casein substantially improved protease production in B. licheniformis MIR29, B. mojavensis and B. horikoshii 104 [11,12,25].

3.3. Effect of sodium carbonate on the protease production To investigate the effect of sodium carbonate on the protease production, sodium carbonate solution was sterilized separately and then added to a basal medium containing 1.5% SBM at a concentration ranging from 0.6 to 1.0% (w/v). An optimal protease production (11,470 U ml 1) was observed at a concentration of 0.8% (w/v) sodium carbonate (initial medium pH, 11.04) (Table 2).

3.4. Effect of carbon sources on the protease production The addition of starches such as corn starch, potato starch and wheat flour also had an effect on protease production. Wheat flour was the effective substrate for protease production among the starches examined and the yield was enhanced 2.4-fold by the addition of 1% wheat flour (28,630 U ml 1) when compared with a basal medium without wheat flour (12,120 U ml 1) (Table 3). In addition to starch, we also investigated the effect of a few rapidly metabolizable carbon sources like citrate, glucose, fructose and disaccharides were investigated. Protease yield was increased approximately 30% by supplying 0.5% (w/v) fructose (36,810 U ml 1) to the basal medium containing 1.5% SBM and 1% wheat flour when compared with the control (29,030 U ml 1) (Table 4). However, lactose (26,130 U ml 1) produced a small decrease in protease yields of approximately 10% (Table 4). This was similar

Table 2 Effect of sodium carbonate on the protease production. Cell was grown in the basal medium containing 1.5% SBM and sodium carbonate at 37 8C for 48 h Concentration (%)

Enzyme activity (U ml 1,  10 2)

Specific activity (U mg 1,  10-2)

Medium pH

0.6 0.7 0.8 0.9 1.0

107.9 100.1 114.7 105.7 101.8

199.4 222.4 221.0 222.1 191.7

10.59 10.84 11.04 11.32 11.65

Table 3 Effect of the different type of starches on the protease production Starch

Concentration (%)

Enzyme activity (U ml 1,  10 2)

Specific activity (U mg 1,  10 2)

None Potato starch Corn starch Wheat flour

– 1.0 1.0 0.2 0.4 0.6 0.8 1.0

121.2 126.3 136.2 121.0 178.4 212.0 270.2 286.3

220.3 221.6 247.6 220.0 297.3 341.9 415.8 421.0

Cell was grown in the basal medium containing 1.5% SBM, 0.8% sodium carbonate and supplemented with each starches at 37 8C for 48 h.

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Table 4 Effect of the rapidly metabolizable carbon sources on the protease production Carbon sources

Concentration (%)

Enzyme activity (U ml 1,  10 2)

Specific activity (U mg-1,  10 2)

None Sodium citrate Sodium succinate Glucose Lactose Sucrose Liquid maltose Fructose

– 0.5 0.5 0.5 0.5 0.5 0.5 0.25 0.5 0.75

290.3 332.7 305.8 327.5 261.3 311.1 297.3 305.5 368.1 325.7 298.1

414.7 443.6 418.9 430.9 378.7 420.4 396.4 407.3 500.8 446.2 405.6

Cell was grown in the basal medium containing 1.5% SBM, 1% wheat flour and 0.8% sodium carbonate and supplemented with each carbon sources at 37 8C for 48 h.

Table 5 Effect of the growing temperature on the protease production Temperature (8C)

Enzyme activity (U ml 1,  10-2)

Specific activity (U mg 1,  10 2)

30 32 35 37 40 42

217.3 425.2 393.8 372.9 227.1 100.4

265.3 548.0 487.9 480.6 281.4 141.9

Cell was grown in the basal medium containing 1.5% SBM, 1% wheat flour, 0.5% fructose and 0.8% sodium carbonate and incubated with each indicated temperature for 48 h.

to results observed in B. horikoshii 104, Bacillus sp. 103 and Bacillus clausii I-52 as reported previously [11,13,14]. In contrast to this result, Mabrouk and co-workers reported that lactose was the preferred carbon source in B. licheniformis ATCC 21415 and B. brevis MTCC B0016, respectively [26,27]. According to optimization experiments, the optimum production medium formulated contained (g l 1): soybean meal, 15; wheat flour, 10; fructose, 5; K2HPO4, 4; Na2HPO4, 1; CaCl2, 0.05; Na2CO3, 8. 3.5. Effect of growth temperature on protease production Growth temperature is another critical parameter that needs to be controlled. The optimum growth temperature for protease production by Bacillus sp. I-312 was found to be 32 8C rather than 37 8C, and the highest yield (42,520 U ml 1) with specific activity of 54,800 U mg protein-1 was achieved when the cell was cultivated using optimized medium at 32 8C for 48 h with shaking at 250 rpm (Table 5). 3.6. pH and thermal stability of the protease The optimal pH for protease activity was determined to be around pH 11.0, and enzyme activity decreased rapidly above pH 12.0 (Fig. 1A). This distinctive property of high pH optimum is a common feature among all alkaline

Fig. 1. (A) Optimum pH of the protease activity. (B) Optimum temperature of the protease.

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Fig. 2. (A) Effect of pH on the stability of the protease. The partially purified protease was incubated in various buffers with different pH(s) ranging from 3 to 13 for 72 h; (B) heat stability of the protease. The partially purified protease was incubated at various temperatures ranging from 30 to 70 8C for 30 and 60 min, respectively.

proteases [13,14,28,29] and the commercially available proteases from Bacillus species [22,30]. The alkaline protease was very stable over a broad pH range from 4.5 to 12 after incubation for 72 h, indicating its potential for practical use in industrial purposes which require stability over wide pH ranges (Fig. 2A). The optimum temperature of the protease was between 60 and 65 8C, while activity decreased rapidly above 70 8C (Fig. 1B). In Bacillus sp. SSR1 the optimal temperature was 40 8C, while in B. pumilus, the enzyme activity is at a maximum at 50–55 8C, and between 60 and 75 8C in Bacillus sp. SB5, Bacillus sp. JB99, Bacillus sp. KSM-KP43, B. clausii I-52 and Bacillus sp. PS179 [14,21,28,29,31–33]. The protease from Bacillus sp. I-312 was stable up to 55 8C after incubation for 1 h, but lost its activity rapidly above 65 8C (Fig. 2B). When the enzyme was stored at 37 8C, its activity was reduced by 50 and 80% within 2 and 8 weeks, respectively. The stability of the

enzyme was increased on adding glycerol or propylene glycol at a concentration of 10% (v/v), and retained its activity of 80 and 95%, respectively, after incubation for 12 weeks at 37 8C, indicating the efficient applicability of propylene glycol as a stabilizer for long-term storage of the protease preparations (Fig. 3). This result was similar to earlier reports that addition of polyhydric alcohols such as glycerol, mannitol and polyethylene glycol caused an increase in the thermal stability of alkaline proteases [12,34]. 3.7. Enzyme stability towards the surfactants and oxidizing agents The partially purified protease from Bacillus sp. I-312 was stable not only towards the nonionic surfactants like Triton X-100 and Tween 20 but also the strong anionic

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Fig. 3. Stability of the protease from Bacillus sp. I-312 in the presence of polyhydric alcohols. The stabilizers were mixed with the protease at a concentration of 10% (v/v), and stored at 37 8C. The relative activity was measured at regular time intervals under standard assay conditions.

None

–

100.0

TritonX-100

1 5

121.4 108.3

Tween 20

1 5

129.7 113.6

SDS

1 5

110.5 87.8

nic surfactants like SDS and oxidants like hydrogen peroxide. An alkaline protease from Bacillus sp. KSM-K16 retained approximately 75% activity on treatment with 5% SDS for 4 h, while B. pumilus alkaline protease lost 22% activity on treatment with 0.1% SDS for 1 h [32,35]. We also previously reported the SDS-stable alkaline proteases have been reported from some Bacillus species. An alkaline protease from Bacillus sp. 103 retained 93.4% activity after incubation with 1% SDS for 72 h, whereas B. clausii I-52 protease lost 27% activity on treatment with 5% SDS for 72 h [13,14]. In the case of H2O2-resistant proteases, an alkaline protease from Bacillus sp. RGR-14 showed 40% loss in enzyme activity with 1% H2O2, while a subtilisin-like protease from Bacillus sp. KSM-KP43 lost little or no enzyme activity on treatment with 10% H2O2 for 30 min [19,33]. Alkaline proteases from Bacillus sp. 103 and B. clausii I-52 retained 91 and 114% of the enzyme activity by treating with 1% H2O2 for 72 h, respectively [13,14]. When compared with other SDS and H2O2–resistant proteases described above, Bacillus sp. I-312 alkaline protease showed significant stability towards both SDS and H2O2 suggesting it is a potential candidate for use in industrial applications.

H2O2

1 5

115.0 87.5

3.8. Inhibitor studies

Sodium perborate

1 2.5

112.9 101.5

surfactant, SDS. In particular, it showed high stability against SDS and hydrogen peroxide, and lost no enzyme activity on treatment with 1% SDS and 1% H2O2 for 72 h, and retained approximately 87.8 and 87.5% activity even after incubation with 5% SDS and 5% H2O2 for 72 h, respectively. Further, the enzyme activity was not decreased on treatment for 72 h with 2.5% sodium perborate (Table 6). Although few reports has been published on SDS and H2O2stable alkaline proteases, many of the available alkaline proteases exhibited low activity and stability towards anioTable 6 Effect of oxidizing agents and surfactants on protease activity from Bacillus sp. I-312 Surfactants/oxidizing agents Concentration (%) Remaining activity (%)

The protease was preincubated with oxidizing agents and surfactants for 72 h at room temperature and the remaining activity was measured according to the standard assay method. Residual activity was determined as percentage of control with no additions.

The effect of natural and synthetic inhibitors on partially purified protease was also investigated. Enzyme activity was strongly inhibited only by PMSF, and inhibited approximately 95% in the presence of 0.5 mM PMSF, while inhibitors like TLCK, a trypsin selective reagent and TPCK,

H.-S. Joo, C.-S. Chang / Process Biochemistry 40 (2005) 1263–1270 Table 7 Effect of various inhibitors on enzyme activity of the partially purified extracellular protease from Bacillus sp. I-312 Inhibitor

Concentration

Remaining activity (%)

None SBTI Benzamidine Bestatin Leupeptin

– 50 ug/ml 1 mM 50 ug/ml 50 ug/ml

100 96.9 105.4 102.4 94.1

PMSF

0.5 mM 1 mM 1 mM 0.5 mM 0.5 mM

4.7 2.0 103.1 95.2 96.1

EDTA TLCK TPCK

a chymotrypsin alkylating agent did not inhibit enzyme activity (Table 7). This inhibition profile suggests that the extracellular protease from Bacillus sp. I-312 belongs to a family of serine protease. Further, the alkaline protease from Bacillus sp. I-312 was not affected in the presence of 1 mM EDTA suggesting that metal cofactors are not required for enzyme activity. This property of the enzyme was very useful for application as detergent additive since chelating agents which function as water softeners and are involved in the removal of stains are components of the detergent and then specifically bind divalent cations [12]. 4. Conclusion Alkaline proteases are employed primarily as cleaning additives and account for 20% of the world market [27,36], and Bacillus-derived proteases considered as major industrial workhorses because of their high production capacities and activity [7–14]. Bacillus sp. I-312 in a new strain, which produces high levels (42,520 U ml 1) of an extracellular alkaline protease with optimum pH of 11 and optimum temperature of around 60 8C. Based on the optimization studies, SBM was ascertained to be as the most effective substrate for protease production, and this was useful because SBM is a cheap and easily available medium substrate, and produced as a by-product during oil extraction [37]. In conclusion, it is envisaged that the isolate can be a potential source of alkaline protease for use as additive in industrial applications such as the detergent industry because Bacillus sp. I-312 produced an alkaline protease at a prominent level using cost-effective medium ingredients. This enzyme was not only functionally stable over a wide range of pH and temperatures but also compatible to both surfactants and oxidants. Acknowledgments This work was supported by a research grant from the MOMAF and in part for the specialized research project in bioengineering from Inha University in 2003.

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