Purification and characterization of recombinant Bacillus subtilis 168 catalase using a basic polypeptide from ribosomal protein L2

Biochemical Engineering Journal 72 (2013) 83–89

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Regular article

Purification and characterization of recombinant Bacillus subtilis 168 catalase using a basic polypeptide from ribosomal protein L2 Junhuan Li a,b,1 , Yang Zhang a,b,1 , Haiying Chen a,b , Yuntao Liu a,b , Yanjun Yang a,b,∗ a b

The State Key Laboratory of Food Science and Technology, Wuxi 214122, China School of Food Science and Technology, Jiangnan University, Wuxi 214122, China

a r t i c l e

i n f o

Article history: Received 24 October 2012 Received in revised form 16 December 2012 Accepted 5 January 2013 Available online 11 January 2013 Keywords: Bioseparations Chromatography Downstream processing Enzyme activity Enzyme production Ribosomal protein L2

a b s t r a c t An efficient purification system for purifying recombinant Bacillus subtilis 168 catalase (KatA) expressed in Escherichia coli was developed. The basic region containing 252–273 amino acids derived from E. coli ribosomal protein L2 was used as an affinity tag while the small ubiquitin-like modifier (SUMO) was introduced as one specific protease cleavage site between the target protein and the purification tags. L2 (252–273)–SUMO fusion protein purification method can be effectively applied to purify the recombinant catalase using cation exchange resin. This purification procedure was used to purify the KatA and achieved a purification fold of 30.5, a specific activity of 48,227.2 U/mg and an activity recovery of 74.5%. The enzyme showed a Soret peak at 407 nm. The enzyme kept its activity between pH 5 and 10 and between 30 ◦ C and 60 ◦ C, with the highest activity at pH 8.0 and 37 ◦ C. The enzyme displayed an apparent Km of 39.08 mM for hydrogen peroxide. These results agree well with the previous reports about B. subtilis catalase. L2 (252–273)–SUMO fusion protein purification technique provides a novel and effective fusion expression system for the production of recombinant proteins. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Catalase [hydrogen-peroxide: hydrogen-peroxide oxidoreductase (EC1.11.1.6)], which catalyzes the dismutation of hydrogen peroxide into oxygen and water, has been widely used in biological and clinical assays [1]. The catalase produced in vegetative cells of Bacillus subtilis had been purified and characterized [2]. The enzyme kept its activity well over a broad pH range of 5–11 and had relatively excellent thermal stability. Although the recombinant catalase from B. subtilis had been successfully expressed in B. subtilis [3,4], it was difficult to purify it from the host cells or the culture medium directly. The complicated purification steps limit the large-scale preparation of highly purified catalase. In previous reports, purification protocols for catalase (Table 1) usually consist of one step affinity chromatography, including Cu(II)-chelating Sepharose Fast Flow [5] and iminodiacetic acidCu(II)-chelating membrane [6], one step self-cleaving elastin-like polypeptide tag purification [7] and two or more other steps, like salting-out, gel-filtration and ion-exchange chromatography [2,4,8,9]. However, catalase purified by immobilized metalchelated affinity chromatography needed the subsequent step to

∗ Corresponding author at: The State Key Laboratory of Food Science and Technology, Wuxi 214122, China. Tel.: +86 510 85329080; fax: +86 510 85329080. E-mail address: [email protected] (Y. Yang). 1 Joint first authors. 1369-703X/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bej.2013.01.002

remove imidazole or heavy metal ions. Moreover, the inefficiency of traditional multi-step purification techniques also made the above protocol time consuming and had relatively low productivity. Since the application of catalase as therapeutic agents for human diseases has increased, it is of great significance to develop more effective and less costly affinity methods for the purification of high purity of catalase [10]. Introducing a purification tag may benefit the purification of recombinant proteins. On the other hand, adding a tag has also been reported to negatively affect the target protein resulting in e.g. (i) inhibition of enzyme activity [11]. Importantly, removal of the tag needs to be considered when designing an affinity purification method for the production of a recombinant protein that is intended for human use to enable production of a tag-free protein. For most of high purity of proteins, the downstream purification steps usually contribute to the bulk of the overall production costs. Cation exchange chromatography is a well-established unit operation in the downstream processing of poly lysine tagged protein [12] and without the use of the expensive immobilized metal-chelated affinity resin and toxic chemicals (such as heavy ions and imidazole) [13]. Kweon et al. reported that approximately 95% of Escherichia coli intracellular proteins had pI values below pH 7.4, with only 5% at basic pH [14]. This result indicates that more than 95% of intracellular proteins were negatively charged at basic pH and could be easily removed from the positively charged protein species using a cation exchanger. The cationic binding module Zbasic2 can be used to purify and immobilize recombinant protein at neutral pH

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Table 1 Comparison of purification methods for catalase. Adsorbents/affinity tags

Activity recovery (%)

Specific activity (U/mg)

Source

Ref.

Cu(II)-chelating sepharose fast flow IDA-Cu(II)-chelating membrane Self-cleaving elastin-like polypeptide tags Ammonium sulfate fractionation; Sepharose 4B IDA-Zn(II) Ammonium sulfate fractionation; anion exchange column; hydroxyapatite column Ammonium sulfate fractionation; Q-Sepharose column; hydroxyapatite column Ammonium sulfate fractionation; DEAE-Sephadex A-50; Bio-Gel A-1.5 m; Bio-Gel HTP

89 67.7 29.6 39.7 69 67 32

– 2660 703.3 25,704 53,400 34,600 172,037

Penicillium chrysogenum Bovine liver Escherichia coli Sprouted black gram Human Bacillus subtilis Bacillus subtilis

[5] [6] [7] [8] [9] [4] [2]

value and clearly shows that most proteins cannot be adsorbed on cation exchangers supports at neutral pH value [15,16]. Thus, it is reasonable to choose a cationic tag as a purification tag. In our previous reports, we have successfully applied the poly lysine tag into the purification of the small ubiquitin-like modifier (SUMO) protease [17] and was also applied the basic polypeptide L2 (203–273) from E. coli ribosomal protein L2 into the purification of enhanced green fluorescence protein (EGFP) [18]. The basic polypeptide L2 (203–273) is the diatomite binding domain and functions as an affinity tag. In our preliminary study, the core basic polypeptide L2 (252–273) from L2 (203–273) was found to have more effective purification performance than that of L2 (203–273) (unpublished data). In this paper, a novel affinity purification method which combined the core basic polypeptide L2 (252–273) from E. coli ribosomal protein L2 with SUMO tag removal method was established. This affinity purification method uses industrial grade cation exchange resin as the affinity matrix which is relatively cheap. The classical ion exchange explanation is based on the isoelectric point of protein and opposite charge of the support. However, there are many reports that show that an enzyme has been immobilized on PEI and dextran sulfate coated support at the same pH value [19], an enzyme has been immobilized on mixed ionic supports not having change [20]. The above studies show that to fix a protein to a support via ionic exchange, the protein need to establish several interactions with the support. The ionic exchange between target protein and support depends on the distribution of protein surface amino/carboxyl groups. Pessela et al. reported that large proteins (multimeric enzyme, beta-galactosidase and bovine liver catalase) can be strongly adsorbed onto conventional anion exchangers since their large external surface (large surface amino groups) permits a great area of interaction with the support [21]. In this study, the small basic polypeptide L2 (252–273) was selected as the purification tag, because this small basic polypeptide may form less ionic bonds with cation exchangers and make the desorption of the fusion protein become easy. We attempted to weaken the electricstatic attraction between the large surface carboxyl groups of KatA and cation exchangers, and strengthen the electricstatic attraction between L2 (252–273) tag (high surface probability) and support by optimizing the purification conditions. Herein we reported the application of L2 (252–273)–SUMO fusion purification technique in the purification of recombinant B. subtilis 168 catalase (KatA) expressed in E. coli and verified the feasibility of L2 (252–273) basic tag to purify a multimeric enzyme. In addition, the physicochemical properties of the purified catalase were also characterized. 2. Materials and methods

previously preserved in our laboratory. E. coli BL21 (DE3) and pET28a (+) were purchased from Novagen (Novagan, Madison, WI, USA). Bacteria were cultured in auto-induction medium ZYM5052 [22]. The poly lysine tagged SUMO protease was prepared in our laboratory according to the description [17].

2.2. Plasmid construction For the plasmid pET28a–L2 (252–273)–SUMO–KatA construction coding for the L2 (252–273)–SUMO–KatA protein based on the commercial pET28a (+), L2 (252–273), SUMO, and KatA DNA fragments were amplified by PCR amplification with the primers of L2252 for (5 -CGTGCCATGGCAAAAACCAAAGGTAAGAAGACCCG-3 ) and L2rev (5 -CAAGGATCCATTTGCTACGGCGACGTACGA-3 ) from E. coli BL21 (DE3) genomic DNA, SUMO for (5 -CGCGGATCCGAATGTCGGACTCAGAAGTCAA-3 ) and SUMOrev (5 -CCCAAGCTTGCACCACCAATCTGTTCTCTGT-3 ) from S. cerevisiae genome, and KatA for (5 -CATGAAGCTT GATGAGTTCAAATAAACTGACAACTA-3 ) and KatArev (5 -GATCTCGAG TTAAGAATCTTTTTTAATCGGC-3 ) from B. subtilis 168 genome, the PCR products were respectively digested with NcoI/BamHI, BamHI/HindIII and HindIII/XhoI and ligated into the pET-28a(+) in sequence, yielding pET28a–L2 (252–273)–SUMO–KatA.

2.3. Adsorption studies of L2 (252–273)–SUMO–KatA on Amberlite Cobalamion Plasmid pET28a–L2 (252–273)–SUMO–KatA was transformed into E. coli strain BL21 (DE3). The recombinant cells were incubated overnight at 37 ◦ C, and then inoculated into 50 mL of ZYM-5052 medium with 50 ␮g/mL kanamycin sulfate. The cells were cultured at 37 ◦ C for 3 h and 20 ◦ C for another 13 h. Cell pellets were harvested by centrifugation, and then were suspended in 4 mL of phosphate buffer (40 mM, pH 7.0) and disrupted by sonication. The cell lysate supernatant containing L2 (252–273)–SUMO–KatA was mixed with Amberlite Cobalamion and shaken in a thermostated shaker (150 rpm) at room temperature for 75 min to reach equilibrium. Samples were withdrawn at suitable time intervals and subjected to determine the catalase activity of unbound target protein. The amounts of L2 (252–273)–SUMO–KatA adsorbed onto the Amberlite Cobalamion were calculated using the following equation: q=

V (Co − Ce ) m

(1)

2.1. Materials, bacterial strains, plasmids and media All chemicals were reagent grade, purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, PR China), unless otherwise noted. Industrial grade weak acid cation exchange resin (Amberlite Cobalamion) was purchased from Rohm and Haas (Philadelphia, PA, USA). B. subtilis 168 and Saccharomyces cerevisiae were

where q is the amount of L2 (252–273)–SUMO–KatA adsorbed onto the Amberlite Cobalamion (U/g); Co and Ce are the catalase activity of L2 (252–273)–SUMO–KatA in the initial solution and the supernatant phase after adsorption, respectively (AU/mL); V is the volume of cell lysate supernatant (mL); m is the mass of the Amberlite Cobalamion (g).

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the catalase activity monitored with the spectrophotometer (SHIMADZU UV-2450). The initial absorbance was approximately 0.5. The decrease in absorbance was monitored continuously for at least 60 s. The molar absorptivity for hydrogen peroxide at 240 nm was assumed to be 43.6 M−1 cm−1 and one unit of catalase activity was defined as the amount that degrades 1 ␮mol H2 O2 per min at 30 ◦ C. 2.6. Biochemical characteristics of recombinant catalase

Fig. 1. Recombinant protein purification flow chart using L2 (252–273)–SUMO fusion technology.

To determine the pH dependency of catalase activity, recombinant catalase was diluted with corresponding buffer pH range from 3.0 to 10.0. To determine the effect of temperature on catalase activity, samples were incubated at temperatures from 30 ◦ C to 60 ◦ C at the pH 7.0. The highest activity assayed was considered as the reference value (100%). Kinetic parameters were determined by incubating the enzyme with different amounts of substrate. Catalase was incubated with H2 O2 (2.5–50 mM) in phosphate buffer pH 7.0, at 30 ◦ C. The value of the Michaelis constant was calculated from a Lineweaver–Burk curve. 3. Results and discussion

2.4. Purification of recombinant catalase The L2 (252–273)–SUMO fusion purification method was shown in Fig. 1. The first purification protocol involves the purification of fusion protein L2 (252–273)–SUMO–KatA, the cleavage of L2 (252–273)–SUMO fusion tag from fusion protein and the adsorption of L2 (252–273)–SUMO fusion tag, the second purification protocol involves the adsorption of L2 (252–273)–SUMO–KatA on affinity matrix, the cleaning of the affinity matrix and the in situ cleavage of the L2 (252–273)–SUMO fusion tag adsorbed on the affinity matrix. The target protein can be purified using either of the above protocols. Briefly, for the first protocol, the cell lysate supernatant from 50 mL culture (5.42 mL) was incubated with cation exchange resin (1.1 g Amberlite Cobalamion) at room temperature for 75 min. The cation exchange resin was collected and eluted with 20 mM phosphate buffer (pH 8.0) containing 200 mM, 400 mM, 600 mM NaCl. The eluted fraction using 20 mM phosphate buffer (pH 8.0) containing 600 mM NaCl was diluted and mixed with the poly lysine tagged SUMO protease (the weight ratio of lysine-tagged SUMO protease to the fusion protein was approximately 1:400). After 3h incubation at room temperature, the mixture was mixed with the above resin to remove contaminants and L2 (252–273)–SUMO tag. After 75-min incubation, the released KatA existed in the supernatant. For the second protocol, the cation exchange resin adsorbed with the fusion protein (average 5.5 mL cell lysate/1.1 g Amberlite Cobalamion, average activity 28,465.4 U/mL cell lysate, n = 3) was collected and washed three times with 20 mM phosphate buffer (pH 8.0) containing 150 mM NaCl. It was further suspended in 20 mM phosphate buffer (pH 8.0) and finally mixed with poly lysine tagged SUMO protease. After 3-h incubation at room temperature, the supernatant containing the released KatA was collected. The protein concentration was determined by the Bradford protein assay, using bovine serum albumin as the standard. The purification steps were monitored by SDS-PAGE stained with Coomassie Brilliant Blue.

3.1. Effect of pH on the adsorption of L2 (252–273)–SUMO–KatA and KatA from the cell lysate supernatant The theoretical isoelectric point (pI) value of L2 (252–273) is 12.29 (computed by DNAstar software). The L2 (252–273) tag has the 6 lysine and 5 arginine. Since the L2 (252–273) basic polypeptide has several basic amino acids that resembles the poly lysine tag, L2 (252–273) could also be used as a purification tag when cation exchange resin was chosen as affinity matrix. The dissociation property of weak acid cation exchange resins are similar to weak organic acids, which are weakly dissociated. As the degree of the dissociation of a weak acid resin is strongly influenced by the solution pH, the resin capacity depends partly on the solution pH. As shown in Fig. 2, the optimal pH was 7.0 for the adsorption of L2 (252–273)–SUMO–KatA (theoretical pI value 7.04, computed by DNAstar software) on Amberlite Cobalamion. This result may be ascribed to the complete dissociation of the carboxylic acid (COOH) in Amberlite Cobalamion at pH 7.0. The basic amino acids in L2 (252–273) tag are also completely dissociated at pH 7.0. When the pH value of the buffer solution is lower than 7.0, the degree of dissociation of the carboxylic acid decrease, and so does the adsorption capacity of L2 (252–273)–SUMO–KatA on Amberlite

2.5. Catalase activity assay Catalase activity of the supernatant was assayed as described by Beers et al. [23]. Briefly, catalase-catalyzed decomposition of H2 O2 was monitored by the decrease in the absorbance at 240 nm. The H2 O2 concentrations should be adjusted immediately before

Fig. 2. Effect of the pH on the relative adsorption capacity of L2 (252–273)–SUMO–KatA and KatA on Amberlite Cobalamion (1 mL cell lysate/0.2 g Amberlite Cobalamion, initial catalase activity: 28142.2 U/mL; temperature: 25 ◦ C; ionic strength: 0 M).

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Cobalamion. When the pH of the buffer solution is greater than 7.0, the fusion protein L2 (252–273)–SUMO–KatA will possess less positive charge. Therefore, the electrostatic interaction between the fusion protein and the cation exchange resin is weakened, this reason may lead to the lower adsorption capacity of the fusion protein on the cation exchange resin at pH 8.0. The purified KatA was added into the crude proteins derived of the same host harboring pET28a. The effect of pH on the adsorption of KatA on Amberlite Cobalamion was also studied. It can be seen from Fig. 2 that a small portion of KatA can be adsorbed by Amberlite Cobalamion at pH 7.0. This phenomenon may be ascribed to ionic bonds between the surface amino groups of the multimeric nature of KatA and cation exchangers. But this electrostatic attraction can be eliminated by the electrostatic repulsion between the surface carboxylic groups of KatA and cation exchangers at pH 8.0. The tagfree KatA only can be effectively adsorbed by Amberlite Cobalamion at the buffer pH 5.0 and 6.0 (this pH is lower than the isoelectric point of KatA). The above results indicate that the L2 (252–273) tag can significantly improve the adsorption of KatA on Amberlite Cobalamion. Although the adsorption capacity of L2 (252–273)–SUMO–KatA differs with the buffer pH, L2 (252–273)–SUMO tagged KatA can be effectively adsorbed by Amberlite Cobalamion at the different buffer pH (from pH 5.0 to pH 8.0). Fuentes et al. reported that the adsorption of proteins on supports bearing almost neutral charge is not driven by the existence of opposite charges between the adsorbent and the biomacromolecule but just by the possibility of forming a high number of enzyme-support ionic bonds [20]. Although L2 (252–273)–SUMO–KatA bears almost neutral charge at buffer pH 7.0, this fusion protein still has the excellent adsorption performance. These results suggest that the adsorption of proteins on cation exchanger is not only driven by the existence of opposite charges between the adsorbent and the fusion protein, but also by the possibility of forming a number of L2 (252–273) tag-support ionic bonds. According to the above results, we can draw the following tentative conclusion: L2 (252–273) basic tag will be hopefully used as a general purification tag as Zbasic2 tag which can be used as fusion partner to different target proteins [15]. 3.2. Adsorption kinetic property In order to ascertain the time required to reach adsorption equilibrium of L2 (252–273)–SUMO–KatA on Amberlite Cobalamion, binding experiments were performed according to Section 2. The adsorption kinetic property was studied and the result is

Fig. 3. Adsorption kinetic curve of L2 (252–273)–SUMO–KatA on Amberlite Cobalamion (1 mL cell lysate/0.2 g Amberlite Cobalamion, initial catalase activity: 28142.2 U/mL; pH 7.0; temperature: 25 ◦ C; ionic strength: 0 M).

shown in Fig. 3. Fig. 3 shows that it took 45 min for 90% of the L2 (252–273)–SUMO–KatA adsorbed onto the Amberlite Cobalamion, the L2 (252–273)–SUMO–KatA had obviously lower binding rate on Amberlite Cobalamion than that of the poly lysine tagged SUMO protease [17]. This result may be due to the relative low isoelectric point of L2 (252–273)–SUMO–KatA. The relatively weak electrostatic interaction between the L2 (252–273)–SUMO–KatA and the Amberlite Cobalamion leads to the lower binding rate and longer time to achieve the adsorption equilibrium (around 75 min). 3.3. Adsorption isotherm The adsorption model was studied using 0.2 g of Amberlite Cobalamion (H+ form) and different initial volume of the cell lysate supernatant containing L2 (252–273)–SUMO–KatA. The adsorption behavior of L2 (252–273)–SUMO–KatA on Amberlite Cobalamion can be simulated with the Langmuir model of which the equation can be written as: Ce 1 Ce = + qe qm qm k

(2)

where Ce (U/mL) and qe (U/g adsorbent) are the catalase activities of the unbound fusion protein and the adsorbed fusion protein on adsorbent at equilibrium, respectively; qm is the maximum adsorption capacity (U/g adsorbent) and k is the adsorption constant (mL/U). The experimental data was analyzed by the linear regression method according to Eq. (2). Fig. 4 was obtained from the linear fit and could be expressed as follows: Ce /qe = 0.00000913Ce + 0.01854, qm = 110,000 U/g adsorbent, k = 0.000492 mL/U, R2 = 0.993. The high R2 indicated the Langmuir model fitted well with the adsorption behavior of L2 (252–273)–SUMO–KatA onto Amberlite Cobalamion. 3.4. Purification of recombinant catalase The purification procedure of catalase using cation exchange resin was summarized in Fig. 5. It can be seen from Fig. 5, L2 (252–273)–SUMO–KatA cannot be eluted from Amberlite Cobalamion until the phosphate buffer containing 600 mM NaCl was used. The eluate of 200 mM, 400 mM NaCl stepwise elution did not show any catalase activity. The second protein purification protocol is obviously easier than the first one because of the fewer operation steps. Since cation exchange resin (AMBERLITETM COBALAMION) exhibited excellent mechanical stability, the second purification

Fig. 4. Adsorption isotherm of L2 (252–273)–SUMO–KatA on Amberlite Cobalamion (pH 7.0; temperature: 25 ◦ C; ionic strength: 0 M).

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the first purification step will dramatically simplify the subsequent purification steps. Since the L2 (252–273) (pI, 12.29, computed by DNAstar software) was a basic polypeptide, L2 (252–273) mediated affinity purification technique should be mainly applied in the purification of acid proteins (pI < 7) as the L2 (203–273) tag reported previously [18]. L2 (252–273) tag resembles the cationic binding module Zbasic2 [15,16]. L2 (252–273) tag may be used as the Zbasic2 tag described by Bolivar as a method not only to purify, but also to immobilize and even stabilize the multimeric structure of the enzyme. 3.5. Effect of pH and temperature on the catalase activity

Fig. 5. SDS-PAGE analysis of the purification of the recombinant catalase. SDS-PAGE analysis (12%) of the purification of recombinant KatA from the fusion protein by Amberlite Cobalamion. Lanes 1 and 8, soluble cell lysate; lanes 2 and 9, non-retained fraction of Amberlite Cobalamion; lanes 3–5, the eluate of 200 mM, 400 mM and 600 mM NaCl stepwise elution; lane 6, pooled fractions of the cleaved product of L2 (252–273)–SUMO–KatA; lane 7, pooled fractions after the incubation of the cleaved product with resin; lane 10, the released KatA from L2 (252–273)–SUMO–KatA adsorbed by Amberlite Cobalamion.

protocol was selected to purify the recombinant catalase (KatA). Though the purification protocol, the purity of the purified KatA was greater than 95%. It can be seen from Table 2 that a purification fold of 30.5, a high specific activity of 48,227.2 U/mg and a high activity recovery of 74.5% was obtained with the recombinant KatA. The lost catalase activity remained in the unbinding portion, this result can be ascribed to the inadequate cation exchange resin when we tested our second purification protocol. From Table 1 we can see that our catalase purification recovery was higher than most of the other catalase purification methods. Although the specific activity of the recovered catalase in our study was lower than the catalase directly purified from the B. subtilis 168 [18], the catalase yield of the former (2.42 mg purified catalase from 99.17 mg total protein) was significantly higher than that of the latter (3.3 mg purified catalase from 5980 mg total protein). The purified recombinant catalase using the optimal culture condition and purification procedure in this study had a higher specific activity than that of the recombinant B. subtilis 168 catalase expressed in the B. subtilis 168 [20]. If necessary, L2 (252–273) tagged catalase could also be immobilized on the cation exchangers for reversible use. Using cation exchangers as affinity purification or immobilization support may be more feasible at industrial scale due to its inexpensive cost. The catalase purified or immobilized by metal-chelate affinity sorbents may be not suitable for the direct applications in the food industry in the removal of hydrogen peroxide from food products after cold pasteurization due to the heavy metal leakage. In this study, the KatA was inserted adjacent to the constructed L2 (252–273)–SUMO tag, so that the KatA can be adsorbed by a cation exchange resin and then released from the fusion tag by the robust cleavage of poly lysine tagged SUMO protease. With the application of recombinant catalase in clinical assays and cancer therapy, there will be a great demand for the high-purity catalase. The traditional chromatography separation process involving flexible steps leads to the high production cost of the high purity proteins. Using the L2 (252–273)–SUMO fusion purification method as

The optimum pH of catalase activity was selected in different pH buffer solutions range from 3.0 to 10.0 at 30 ◦ C under the standard assay conditions. In order to determine the effect of temperature on enzymatic activity, samples were incubated at the temperature range from 30 ◦ C to 60 ◦ C at pH 7.0. The catalase exhibited a relatively high activity over a broad pH range of 6–10, with the maximum activity at pH 8 (data not shown). There was a dip in the purified KatA activity at pH 9.0, this phenomenon had also appeared at the catalase–peroxidase from Oceanobacillus oncorhynchi subsp. Incaldaniensis which had a dip in catalase activity at pH 9.0 with glycine–NaOH as a buffer [24], catalase from B. subtilis 168 which had a dip in catalase activity at pH 8.0 [2], and rice plant catalaseA from recombinant E. coli, which had a dip in catalase activity at pH 9.0 [25]. The heme binding pocket in catalase from B. subtilis 168 (Genebank accession no. CAB04807.1) includes His54, Ser93, Asn127, Phe132, Phe140, Arg333, Tyr337. The pH profile of the catalase could be due to the pH-induced structural changes. Carpena et al. reported that the pH profile of the catalase activity can be correlated with the distribution of Arg conformations of which the side chain conformation changes from one predominant conformation at low pH to a second at high pH [26]. The side chains of Arg, His and Tyr in the heme pocket may create a charged microenvironment which will discharge when the buffer pH is around 9.0, this reason may result in the decreased interaction between heme and heme binding pocket and the relatively low activity of the purified KatA at pH 9.0. Further studies needed to be conducted to determine why there was a dip in catalase activity at pH 9.0. The purified KatA exhibited a temperature optima at the tested temperatures from 37 ◦ C to 42 ◦ C. Incubation at 60 ◦ C for 1 h resulted in the inactivation of 74% of the purified KatA. 3.6. Km and spectroscopic properties In our preliminary test, high concentration of H2 O2 (more than 50 mM) resulted in a decrease of catalase activity. Therefore, the Km value for the purified KatA was determined at the low concentration of H2 O2 (less than 50 mM). The equation, as determined from a Lineweaver–Burk plot was expressed as follows: Y = 2.49685X + 0.06389, R2 = 0.994. The apparent Km for hydrogen peroxide was 39.08 mM, which was very similar to the result of 40.1 mM reported by Loewen et al. [2]. The absorption spectrum of the native state of recombinant catalase (Fig. 6) shows a Soret peak at 407 nm and smaller peaks at 504, 539 and 628 nm. This absorbance spectrum agrees well with the result of Loewen et al. [2]. This result indicates that the B. subtilis catalase expressed in recombinant E. coli still also has heme like the catalase

Table 2 Purification of recombinant catalase from E. coli (mean ± SE, n = 3). Step

Total protein (mg)

Total activity (U)

Specific activity (U/mg)

Cell lysate Amberlite Cobalamion

99.17 ± 5.23 2.42 ± 0.06

156,559.7 ± 11,002.8 116,709.9 ± 2676.4

1578.8 ± 110.9 48,227.2 ± 1,105.9

Yield (%) 100 74.5

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Fig. 6. Absorption spectra of the native states of the purified KatA.

purified from B. subtilis. The absorbance ratio (A406 /A280 ) ratio of the enzyme was 0.82, which meant 0.8 iron atoms per subunit. However, the A406 /A280 ratio of homogeneous B. subtilis catalase purified by Loewen et al. was 1.0 [2]. The reduction in specific activity of recombinant KatA could be ascribed to the reduction of heme content. The recombinant expression of heme containing proteins in E. coli usually resulted in the incomplete heme incorporation due to the limitation of endogenous heme pool and the decrease specific activity of heme containing proteins [27–30]. Preparations of catalase from Streptomyces sp. [31], Mycobacterium smegmatis [32], alkalophilic Bacillus sp. [33] and other resources had also been reported with less than the expected ratio of one heme per subunit. Although the specific activity of the purified KatA was lower than that of catalase from B. subtilis [2,4] due to the incomplete heme incorporation, this problem can be solved by optimizing the culture media as described by Kery et al. [29]. 4. Conclusion In this study, we reported a rapid and effective protein expression and purification approach. Our study indicated the expression host of E. coli could be effectively used to produce recombinant B. subtilis catalase. L2 (252–273)–SUMO fusion protein purification method can help avoid the complex purification process of recombinant proteins and provide a potential costeffective affinity purification method for pharmaceutical proteins. L2 (252–273)–SUMO fusion tag have also been successfully applied into the purification of recombinant EGFP in our laboratory (unpublished data). Thus, this affinity purification technique shows some generality in the purification of recombinant protein. Acknowledgment This work was supported by grants from the Chinese National High Technology Research and Development Program 863 (2013AA102207). References [1] H.-S. Ko, H. Fujiwara, Y. Yokoyama, N. Ohno, S. Amachi, H. Shinoyama, T. Fujii, Inducible production of alcohol oxidase and catalase in a pectin medium by Thermoascus aurantiacus IFO 31693, J. Biosci. Bioeng. 99 (2005) 290–292. [2] P.C. Loewen, J. Switala, Purification and characterization of catalase-1 from Bacillus subtilis, Biochem. Cell Biol. 65 (1987) 939–947.

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