Biotechnology Letters 26: 1421–1424, 2004. © 2004 Kluwer Academic Publishers. Printed in the Netherlands.
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Purification and characterisation of an alkaline protease used in tannery industry from Bacillus licheniformis Xue-Ming Tang1,2,∗ , F.M. Lakay2 , Wei Shen1 , Wei-Lan Shao1 , Hui-Ying Fang1 , B.A. Prior2 , Zheng-Xiang Wang1 & Jian Zhuge1 1 The
Key Laboratory of Industrial Biotechnology, Ministry of Education, Research Center of Industrial Microbiology, Southern Yangtze University, Wuxi 214036, P.R. China 2 Department of Microbiology, University of Stellenbosch, Private Bag X1, Matieland 7602, South Africa ∗ Author for correspondence and current address: Department of Microbiology, University of Stellenbosch, Private Bag X1, Matieland 7602, South Africa (Fax: +27 218085846; E-mail:
[email protected]) Received 29 April 2004; Revisions requested 17 May 2004; Revisions received 6 July 2004; Accepted 6 July 2004
Key words: alkaline protease, Bacillus licheniformis, characterisation, purification
Abstract An extracellular alkaline protease produced by Bacillus licheniformis AP-1 was purified 76-fold, yielding a single 28 kDa band on SDS-PAGE. It was optimally active at pH 11 and at 60 ◦ C (assayed over 10 min). The protease was completely inhibited by phenylmethylsulfonyl fluoride and diodopropyl fluorophosphate, with little increase upon Ca2+ and Mg2+ addition.
Introduction Environmental pollution during leather processing is an industrial problem in some countries, such as China and India, where leather is a major export commodity. Hazardous chemicals such as lime, sodium sulphide, solvents, etc., arise mainly from the pre-tanning stages of leather processing. Therefore, to overcome these hazards, cleaner and more eco-friendly technologies for leather processing and effluent treatment need to be established. Enzymes offer viable alternatives in pre-tanning operations, such as soaking, dehairing, bating, degreasing and offal treatment. Some bacterial alkaline proteases possessing elastolytic and keratinolytic activity offer an effective bio-treatment of leather, especially the dehairing and bating of skins and hides. The alkaline conditions enable the swelling of hair roots and subsequent attack of proteases on the hair follicle protein aid in the easy removal of hair. Despite the strong alkaline conditions, this process is safer and more suitable than traditional methods using sodium sulphide treatment (Kumar & Takagi 1999, Thangam & Rajkumar 2002).
Species of Bacillus are widely used in the industrial large-scale production of enzymes, including proteases. Of particular industrial importance are proteases with activity at alkaline pH and high temperatures. Earlier studies with the alkaline protease from Bacillus licheniformis AP-1 used as a dehairing agent, not only indicated elastolytic and keratinolytic activities, but also low hydrolytic collagen activity (Zhang 1998). In this study we report the purification and characterisation of the extracellular alkaline protease from B. licheniformis AP-1.
Materials and methods Bacterial strains and culture conditions Bacillus licheniformis AP-1, from Prof Xinyun Huo (China Enzyme Association), was grown at 36 ◦ C for 40 h on medium consisting of (g l−1 ): soy bean cake meal, 50; corn flour, 50; bran, 25; KH2 PO4 , 0.3; Na2 HPO4 , 0.4; and Na2 CO3 , 1. Cultivation with 50 ml medium in 250 ml conical flasks with shaking at 220 rpm.
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Fig. 1. SDS-PAGE of alkaline protease (according to Laemmli 1970). (1) Protein molecular weight marker; (2) ammonium sulfate fraction, 20 µg; (3) DEAE-Sephadex A50 fraction, 10 µg; (4) CM-Sephadex C50 fraction, 1.5 µg; (5) Sephadex G75 fraction, 0.5 µg.
Fig. 2. Optimum pH (a) and temperature (b) profile of purified enzyme. Sp. act. of enzyme = 159 381 U mg−1 .
Enzyme assay Protease activity was determined using casein/trichloroacetic acid (TCA)-Lowry assay according to the method of An et al. (1994). Purified enzyme (1%, w/v) was added to the reaction mixture, containing 1% (w/v) casein and 625 µl reaction buffer (0.1 M citric acid/0.2 M Na2 HPO4 , pH 2–7.5; 0.1 M NaH2 PO4 /0.05 M Na2 B4 O7 , pH 8–10.5; and 0.1 M Na2 B4 O7 /NaOH buffer, pH 11–14). The mixture was incubated at various pH and temperatures for 10 min. The reaction was terminated by addition of 200 µl 50% (w/v) TCA. Unhydrolysed proteins were precipitated at 4 ◦ C for 15 min, followed by centrifugation at 9500 × g for 10 min. The oligopeptide content in the supernatant was determined by the Lowry assay. Activity was expressed as tyrosine equivalents in TCA-supernatant. One unit of activity was defined as that releasing 1 nmol tyrosine per min (nmol Tyr min−1 ). A blank was run in the same manner, except the enzyme was added after the addition of 50% (w/v) TCA. All experiments were done in duplicate.
Fig. 3. Enzyme stability over pH (a) and temperature (b) range. Temperature legend: ♦ 45 ◦ C; 50 ◦ C; + 55 ◦ C; 60 ◦ C; 65 ◦ C. Sp. act. of enzyme = 159 381 U mg−1 .
SDS-PAGE and molecular weight determination The purified product gave only one band upon SDSPAGE (Figure 1), with an apparent size of 28 kDa. pH and temperature profile
Results and discussion Enzyme purification The protease was purified approx. 76-fold, with a recovery of ca. 20% protein (see Table 1).
The purified enzyme was active over a wide pH range (4–13), with an optimal activity at pH 11 (Figure 2a). It was stable over the pH range 7–11.5, with the highest stability at pH 11.5 (Figure 3a). At pH 11 it showed activity over the temperature range of 30– 70 ◦ C, with optimal activity at 60 ◦ C (Figure 2b).
1423 Table 1. Summary of enzyme purification. Purification stepa
Total protein (U mg−1 )
Specific activity (U mg−1 )
Enzyme recovery (%)
Purification (fold)
Supernatantb Ammonium sulfate fractionationc DEAE Sephadex A50d CM-Sephadex C50e Sephadex G75f
1080 349 63 7.6 2.9
2092 4602 20481 6040 159381
100 71 58 31 21
1 2.2 10 44 76
a All fractions, except the supernatant were subjected to dialysis in 20 m M phosphate buffer (pH 6) and concentration by ultrafiltration (MWCO 10000 Da, Millipore) at 4 ◦ C. b Obtained by centrifugation of culture broth at 5000 × g for 15 min. c Supernatant was precipitated with saturated ammonium sulfate. d Concentrated fraction was separated on DEAE Sephadex A50 column, pre-equilibrated with 50 M phosphate
buffer (pH 6), against a linear NaCl gradient of 0–500 m M. e Concentrated Sephadex A50 fraction was separated on CM-Sephadex C50 column, pre-equilibrated with
20 m M phosphate buffer (pH 6), against 1 M NaCl. f Concentrated CM-Sephadex C50 fraction was separated on Sephadex G75 column, pre-equilibrated with 20 m M phosphate buffer (pH 6), against 150 m M NaCl.
Enzyme stability was tested from 45 to 65 ◦ C, revealing a half-life of 2 h at 60 ◦ C and 15 min at 65 ◦ C (Figure 3b). The enzyme was completely inactivated after 80 min at 65 ◦ C, whereas only ca. 15% of enzyme stability was lost over 2 h when incubated at either 45, 50, or 55 ◦ C. Therefore, these results indicate an alkaline protease with high alkaline- and heat-stability. Effects of cations and chemical reagents The effect of various metal ions and inhibitors on the activity of purified protease are summarised in Table 2. Both Ca2+ and Mg2+ slightly enhanced enzyme activity. These cations probably protect the enzyme against thermal denaturation and therefore maintain the active conformation of the enzyme at high temperatures (Donaghy & McKay 1993). However, all other tested additives had a negative effect on the enzymatic activity, especially Hg2+ and Ag+ . In addition, the alkaline protease activity was completely inhibited by PMSF and diiodopropyl fluorophosphate (DFP), suggesting that this protease is a serine protease. Although many serine proteases have been reported from microbial origins, there were few reports about the purification and characterisation of proteases that can be used for leather dehairing and bating processes. Previously, an extracellular alkaline serine protease from a Bacillus pumilus strain, demonstrated good dehairing function and had maximum activity at pH 10 and 55 ◦ C (Huang et al. 2003). Although alkaline serine proteases from B. stearothermophilus F1 and Bacillus sp. KSM-K16 exhibit highest activ-
Table 2. Effect of metal ions and chemical reagents on enzyme activity. Reagenta
Relative activityb (%)
Control Ca2+ Mg2+ Pb2+ , Ba2+ , Mn2+ , Urea, SDS Cu2+ , Fe2+ , Ni2+ , Zn2+ , EDTA Ag+ Hg2+ PMSF, DFP
100 108 104 90–96 70–88 36 21 0
a All reagents were used at 1 m M . b All experiments were done in triplicate, yielding con-
sistent reproducibility. Values are given as the average of such obtained data. Purified enzyme used at 159 381 U mg−1 .
ity towards casein, these enzymes could not hydrolyse fibrous proteins such as collagen, elastin or keratin (Thangam & Rajkumar 2002). The alkaline protease used in tannery industry from B. licheniformis AP-1 has significant elastolytic and keratinolytic activity at pH 9–12 and 55 ◦ C (data not shown). In this present study, this protease had good stability at high alkaline pH values and broad heat stability, thereby permitting its biotechnological application potential to be exploited in many industries as an eco-friendly product.
1424 Acknowledgement The authors wish to thank RCIM (Research Center of Industrial Microbiology) in Southern Yangtze University for financial support.
References An H, Seymour TA, Wu J, Morrissey MT (1994) Assay systems and characterization of Pacific whiting (Merluccius productus) protease. J . Food Sci. 59: 1013–1017. Chakrabarti SK, Matsumura N, Ranu RS (2000) Purification and characterization of an extracellular alkaline serine protease from Aspergillus terreus (IJIRA 6.2). Curr. Microbiol. 40: 239–244.
Donaghy JA, McKay AM (1993) Production and properties of an alkaline protease by Aureobasidium pullulans. J. Appl. Bacteriol. 74: 662–666. Huang Q, Peng Y, Li X, Wang HF, Zhang YZ (2003) Purification and characterization of an extracellular alkaline serine protease with dehairing function from Bacillus pumilus. Current Microbiol. 46: 169–173. Kumar GC, Takagi H (1999) Microbial alkaline protease: from a bioindustrial viewpoint. Biotech. Adv. 17: 561–594. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685. Thangam EB, Rajkumar GS (2002) Purification and characterization of alkaline protease from Alcaligenes faecalis. Biotechnol. Appl. Biochem. 35: 149–154. Zhang SZ (1998) Enzyme industry. In: Wu TS, ed. Protease, Vol. 17. Beijing: Chinese Science Press, pp. 431–446.
