- Email: [email protected]
Site-directed mutagenesis of methionine residues for improving the oxidative stability of α-amylase from Thermotoga maritima
Journal of Bioscience and Bioengineering VOL. 116 No. 4, 449e451, 2013 www.elsevier.com/locate/jbiosc
NOTE
Site-directed mutagenesis of methionine residues for improving the oxidative stability of a-amylase from Thermotoga maritima Handan Ozturk, Selin Ece, Ersin Gundeger, and Serap Evran* Department of Biochemistry, Faculty of Science, Ege University, Izmir 35100, Turkey Received 17 March 2013; accepted 9 April 2013 Available online 21 May 2013
The oxidative stability of a-amylase (AmyC) from Thermotoga maritima was improved by mutating the methionine residues at positions 43 and 44, 55, and 62 to oxidative-resistant alanine residues. The most resistant M55A variant showed 50% residual activity in the presence of 100 mM H2O2, whereas the wild-type enzyme was inactive. Ó 2013, The Society for Biotechnology, Japan. All rights reserved. [Key words: a-Amylase; Thermotoga maritima; Oxidative stability; Protein engineering; Site-directed mutagenesis of methionine]
a-Amylases (EC 3.2.1.1) are amylolytic enzymes that hydrolyze a-1,4 glycosidic bonds of starch, and they are widely used in starch, food, textile, paper and detergent industries (1). For their use in laundry detergent formulations, a-amylases with optimum pH values higher than 8.0 are required (2). Oxidative stability is an important issue, since detergents contain oxidizing agents. Methionine is one of the residues that is susceptible to oxidation, and its side chain is substituted with a larger and more polar methionine sulphoxide. As a result of this chemical modification, enzyme activity and stability can be greatly disrupted (3). The intracellular a-amylase from Thermotoga maritima (AmyC) has been shown to be active in the alkaline pH range and at 60e90 C (4). Due to its alkaline optimum pH and high thermal stability properties, AmyC is a potential candidate for use in detergent industry. AmyC monomer contains twenty methionine residues, which can limit its effective use in oxidizing environments. Glu185 and Asp349 have been assigned as catalytic residues (4,5), and we attempted to improve the oxidative stability by replacing four of the methionine residues in the vicinity of the active site. The solved crystal structure (5) enabled us to identify Met43, Met44, Met55 and Met62 residues (Fig. 1A). Genomic DNA of T. maritima, which was a kind gift from Prof. Dr. Konstantinos Vorgias (Athens University, Greece) was used as the template for polymerase chain reaction (PCR) amplification of amyC gene using the primers 50 -GGAATTCCATATGAGAGGAAAAATACTGAT ATTTCTGC-30 and 50 -CGCGGATCCAATCACATCCCTCG-30 (NdeI and BamHI restriction sites are underlined, respectively). The PCR conditions were as follows: 95 C, 3 min; 95 C, 45 s; 68 C, 45 s; 72 C, 1 min and 30 s; 72 C, 10 min. The 2e4 steps were repeated 30 times. The amplified fragment was then cloned into pET21a(þ) vector (Merck, Darmstadt, Germany), which allowed introducing a 6xHistag at the C-terminal of AmyC. The selected methionines at positions 43 and 44, 55 and 62 were mutated to alanine residues by the * Corresponding author. Tel.: þ90 232 3112304; fax: þ90 232 3115485. E-mail address: [email protected] (S. Evran).
megaprimer method. (6). The primers used for site-directed mutagenesis are listed in Table 1. After verification of the gene sequences, plasmids with the cloned gene fragments were transformed into Escherichia coli Rosetta competent cells (Merck, Darmstadt, Germany) for heterologous expression. 2 L of LB medium supplemented with 150 mg/mL ampicillin was inoculated with 20 mL of the overnight culture of E. coli Rosetta cells transformed with the pET21a(þ)/ amyC. After an OD600 of 0.4e0.6 was reached, the temperature was decreased to 30 C, and protein expression was induced by 0.4 mM isopropyl-b-D-thiogalactopyranoside (IPTG) overnight. The cells were harvested by centrifugation (20 min, 4000 rpm, 4 C), and resuspended in 50 mM potassium phosphate buffer containing 500 mM NaCl and 5 mM imidazole (pH 7.5). The cells were then sonicated at 40% power for 5 min using a Vibra-cell VCX-500 (Sonics, Newtown, CT, USA) sonicator. The insoluble fraction was removed by centrifugation (30 min, 13,000 rpm, 4 C), and the soluble extract was applied to a HisTrapFF crude column (5 ml; GE Healthcare, Uppsala, Sweden). The His-tagged AmyC enzymes were purified by Ni-chelate affinity chromatography. Protein concentrations were quantified by Bradford method using bovine serum albumin (BSA) as the protein standard (7). a-Amylase activity was assayed by measuring the amount of reducing sugar released during hydrolysis of 1% soluble starch in Tris buffer (250 mM, pH 8.5) at 70 C for 30 min. The amount of reducing sugar was measured by dinitrosalicylic acid (DNS) method (8). One unit of enzyme activity was defined as the amount of enzyme that released 1 mmol of maltose under the assay conditions. The specific activity of enzyme was calculated by the ratio of enzyme activity (U) to protein concentration (mg) in the assay mixture. The specific activity of wild-type AmyC was taken as 100%, and it was compared with that of each mutant AmyC enzyme. The methionine to alanine exchanges resulted in reduced specific activities compared to the wild-type enzyme. As shown in Fig. 1B, M43A þ M44A, M55A and M62A variants had 82%, 77% and 44% relative activities, respectively. Compared to Met55 and Met62, the residues Met43 and Met44 were less severely affected by alanine
1389-1723/$ e see front matter Ó 2013, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2013.04.018
OZTURK ET AL.
J. BIOSCI. BIOENG.,
A M44
M43
D349 M55
E185
M62
B Relative activity (%)
100
80 60 40 20 0 Wild-type
M43A+M44A
M55A
M62A
FIG. 1. Location of methionine mutations in the structure of AmyC. (A) The figure was generated by Swiss-Pdb Viewer (14) using the published structure (PDB: 2B5D) (5). The catalytic residues E185 and D349 are indicated by red color. The effect of mutations on enzymatic activity was investigated. (B) The specific activity of the wild-type AmyC was taken as 100%, and it was compared with that of each mutant AmyC enzyme. The data represent the average values of three measurements. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
exchanges. This could be attributed to that Met43 and Met44 are located on the surface of the enzyme, and they are more distant from the active site (Fig. 1A). However, it should not be excluded that M43A þ M44A variant exhibited 82% activity, which indicated a change in the hydrophobicity resulting from introducing alanine residues at these positions. Met317Ser exchange, which is nearby the enzyme surface and away from the catalytic residues, has not resulted in a significant decrease in the activity of the alkaline amylase from Alkalimonas amylolytica (9). The reduced specific activity of M55A and M62A mutants confirmed the previous findings that the methionine mutations that are close to the active site severely affects the catalytic activity (9,10). Oxidative stability was tested by incubating 0.9 mg of wild-type or mutant AmyC enzymes at different concentrations of H2O2 (1e100 mM) in Tris buffer (250 mM, pH 8.5) at 30 C for 30 min. Following incubation, catalase (SigmaeAldrich, St. Louis, MO, USA) with a final concentration of 1904 U/ml was added in order to quench the remaining H2O2. The residual activity was measured using 0.5 ml of the sample under standard assay conditions. The relative activity (%) was calculated by taking the activity in the absence of H2O2 as 100%. Wild-type AmyC was susceptible to oxidation at increasing concentrations of H2O2, with a complete loss of activity in the presence of 50 mM H2O2. The mutants M43A þ M44A and M55A retained 39% and 50% of the initial TABLE 1. The primers used for site-directed mutagenesis. Primer M43AþM44A M55A M62A
Sequence (50 / 30 )a TCGAACGCCGCTAAAAGC ATCGACGCGGTCAACCTG AGCATCTCCGCCAGCGGAG
a The nucleotide exchanges for methionine mutations are underlined.
activity at 100 mM H2O2, respectively (Fig. 2). The mutant M62A had no residual activity after treatment with 100 mM H2O2, but it was more resistant to oxidation than the wild-type at 50 mM H2O2. Methionine residues have been replaced by non-oxidizable residues such as leucine, alanine or serine for improved oxidative stability of several enzymes (9e12). These previous studies suggest that the methionine residues, which are solvent-accessible (10,11) or at the catalytic domain (9,12) are the primary targets for oxidation. Our results indicated that the M55A substitution nearby the active site enabled a higher oxidative resistance than that of the solvent-accessible M43A þ M44A. We assumed that M55A substitution was important for avoiding steric hindrance, which might have resulted from the formation of methionine sulfoxide nearby the active site. However, M62A substitution resulted in only a slight improvement in the oxidative stability (Fig. 2). Moreover, a remarkable decrease in the specific activity was observed (Fig. 1B). It has been shown that replacement of methionine residues nearby the active site increased the sensitivity to H2O2, which has been attributed to the antioxidant role of these residues (13). Met62 might have a similar role, and the remarkable decrease in specific activity upon M62A substitution suggests a structural or catalytic role for this residue. Due to their potential use in detergents, aamylase enzymes from Bacillus sp. TS-23 and A. amylolytica have been engineered for improved oxidative stability at 500 mM H2O2 (9,11). Introducing a single methionine substitution has been shown to increase the oxidative stability of Geobacillus stearothermophilus sp. US100 at 1.8 M H2O2 (12). We achieved a significant improvement in oxidative stability at 100 mM H2O2, with the most oxidative resistant M55A retaining 50% of its activity. It could be concluded that oxidative stability is greatly influenced by the total number methionine residues, as well as their location in enzyme structure. The effect of methionine mutation on pH and thermal stability was evaluated for the most oxidative resistant M55A mutant. Wild-type AmyC and M55A mutant were incubated in Tris buffer (100 mM, pH 8.5) at 30 C for 4 h, and then the residual activities were measured under standard assay conditions. Wildtype enzyme and M55A mutant retained 93% and 88% of their initial activities, respectively. The results indicated that the methionine substitution had only a slight effect on pH stability. To investigate the thermal stability, both enzymes were incubated at 70 C for 4 h. Following incubation, the remaining activities were measured under standard assay conditions. Both wild-type and M55A mutant enzyme had 88% residual activity. Compared to M43A þ M44A and M62A mutants, M55A mutant exhibited the most improved oxidative stability properties, albeit with a 23% decrease in specific activity. Moreover, it retained the pH and thermal stability properties. The engineered AmyC could be a potential candidate for industrial applications, particularly in the
100
Relative activity (%)
450
80 60 40 20 0
0
20
40
60
80
100
[H2O2] (mM) FIG. 2. Inactivation of wild-type (filled circles), M55A mutant (filled squares), M43A þ M44A mutant (filled triangles), and M62A mutant (open triangles) enzymes by different concentrations of H2O2. The relative activity (%) was compared with the activity without the addition of H2O2.
VOL. 116, 2013 presence of oxidizing agents. Here, we provided the first protein engineering attempt for AmyC from T. maritima. References 1. Prakash, O. and Jaiswal, N.: Alpha-amylase: an ideal representative of thermostable enzymes, Appl. Biochem. Biotechnol., 160, 2401e2414 (2010). 2. Fujinami, S. and Fujisawa, M.: Industrial applications of alkaliphiles and their enzymesepast, present and future, Environ. Technol., 31, 845e856 (2010). 3. Kim, Y. H., Berry, A. H., Spencer, D. S., and Stites, W. E.: Comparing the effect on protein stability of methionine oxidation versus mutagenesis: steps toward engineering oxidative resistance in proteins, Protein Eng., 14, 343e347 (2001). 4. Ballschmiter, M., Futterer, O., and Liebl, W.: Identification and characterization of a novel intracellular alkaline alpha-amylase from the hyperthermophilic bacterium Thermotoga maritima MSB8, Appl. Environ. Microbiol., 72, 2206e2211 (2006). 5. Dickmanns, A., Ballschmiter, M., Liebl, W., and Ficner, R.: Structure of the novel alpha-amylase AmyC from Thermotoga maritima, Acta Crystallogr. D Biol. Crystallogr., 62, 262e270 (2006). 6. Sarkar, G. and Sommer, S. S.: The “megaprimer” method of site-directed mutagenesis, Biotechniques, 8, 404e407 (1990). 7. Bradford, M. M.: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem., 72, 248e254 (1976).
NOTE
451
8. Miller, G. L.: Use of dinitrosalicylic acid reagent for determination of reducing sugar, Anal. Chem., 31, 426e428 (1959). 9. Yang, H., Liu, L., Li, J., Du, G., and Chen, J.: Structure-based replacement of methionine residues at the catalytic domains with serine significantly improves the oxidative stability of alkaline amylase from alkaliphilic Alkalimonas amylolytica, Biotechnol. Prog., 28, 1271e1277 (2012). 10. Roger Chien, H. C., Hsu, C. L., Hu, H. Y., Wang, W. C., and Hsu, W. H.: Enhancing oxidative resistance of Agrobacterium radiobacter N-carbamoyl Damino acid amidohydrolase by engineering solvent-accessible methionine residues, Biochem. Biophys. Res. Commun., 297, 282e287 (2002). 11. Lin, L. L., Lo, H. F., Chiang, W. Y., Hu, H. Y., Hsu, W. H., and Chang, C. T.: Replacement of methionine 208 in a truncated Bacillus sp. TS-23 alpha-amylase with oxidation-resistant leucine enhances its resistance to hydrogen peroxide, Curr. Microbiol., 46, 211e216 (2003). 12. Khemakhem, B., Ali, M. B., Aghajari, N., Juy, M., Haser, R., and Bejar, S.: Engineering of the alpha-amylase from Geobacillus stearothermophilus US100 for detergent incorporation, Biotechnol. Bioeng., 102, 380e389 (2009). 13. Kuo, L. Y., Hwang, G. Y., Yang, S. L., Hua, Y. W., Chen, W., and Lin, L. L.: Inactivation of Bacillus stearothermophilus leucine aminopeptidase II by hydrogen peroxide and site-directed mutagenesis of methionine residues on the enzyme, Protein J., 23, 295e302 (2004). 14. Guex, N. and Peitsch, M. C.: SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling, Electrophoresis, 18, 2714e2723 (1997).
NOTE
Site-directed mutagenesis of methionine residues for improving the oxidative stability of a-amylase from Thermotoga maritima Handan Ozturk, Selin Ece, Ersin Gundeger, and Serap Evran* Department of Biochemistry, Faculty of Science, Ege University, Izmir 35100, Turkey Received 17 March 2013; accepted 9 April 2013 Available online 21 May 2013
The oxidative stability of a-amylase (AmyC) from Thermotoga maritima was improved by mutating the methionine residues at positions 43 and 44, 55, and 62 to oxidative-resistant alanine residues. The most resistant M55A variant showed 50% residual activity in the presence of 100 mM H2O2, whereas the wild-type enzyme was inactive. Ó 2013, The Society for Biotechnology, Japan. All rights reserved. [Key words: a-Amylase; Thermotoga maritima; Oxidative stability; Protein engineering; Site-directed mutagenesis of methionine]
a-Amylases (EC 3.2.1.1) are amylolytic enzymes that hydrolyze a-1,4 glycosidic bonds of starch, and they are widely used in starch, food, textile, paper and detergent industries (1). For their use in laundry detergent formulations, a-amylases with optimum pH values higher than 8.0 are required (2). Oxidative stability is an important issue, since detergents contain oxidizing agents. Methionine is one of the residues that is susceptible to oxidation, and its side chain is substituted with a larger and more polar methionine sulphoxide. As a result of this chemical modification, enzyme activity and stability can be greatly disrupted (3). The intracellular a-amylase from Thermotoga maritima (AmyC) has been shown to be active in the alkaline pH range and at 60e90 C (4). Due to its alkaline optimum pH and high thermal stability properties, AmyC is a potential candidate for use in detergent industry. AmyC monomer contains twenty methionine residues, which can limit its effective use in oxidizing environments. Glu185 and Asp349 have been assigned as catalytic residues (4,5), and we attempted to improve the oxidative stability by replacing four of the methionine residues in the vicinity of the active site. The solved crystal structure (5) enabled us to identify Met43, Met44, Met55 and Met62 residues (Fig. 1A). Genomic DNA of T. maritima, which was a kind gift from Prof. Dr. Konstantinos Vorgias (Athens University, Greece) was used as the template for polymerase chain reaction (PCR) amplification of amyC gene using the primers 50 -GGAATTCCATATGAGAGGAAAAATACTGAT ATTTCTGC-30 and 50 -CGCGGATCCAATCACATCCCTCG-30 (NdeI and BamHI restriction sites are underlined, respectively). The PCR conditions were as follows: 95 C, 3 min; 95 C, 45 s; 68 C, 45 s; 72 C, 1 min and 30 s; 72 C, 10 min. The 2e4 steps were repeated 30 times. The amplified fragment was then cloned into pET21a(þ) vector (Merck, Darmstadt, Germany), which allowed introducing a 6xHistag at the C-terminal of AmyC. The selected methionines at positions 43 and 44, 55 and 62 were mutated to alanine residues by the * Corresponding author. Tel.: þ90 232 3112304; fax: þ90 232 3115485. E-mail address: [email protected] (S. Evran).
megaprimer method. (6). The primers used for site-directed mutagenesis are listed in Table 1. After verification of the gene sequences, plasmids with the cloned gene fragments were transformed into Escherichia coli Rosetta competent cells (Merck, Darmstadt, Germany) for heterologous expression. 2 L of LB medium supplemented with 150 mg/mL ampicillin was inoculated with 20 mL of the overnight culture of E. coli Rosetta cells transformed with the pET21a(þ)/ amyC. After an OD600 of 0.4e0.6 was reached, the temperature was decreased to 30 C, and protein expression was induced by 0.4 mM isopropyl-b-D-thiogalactopyranoside (IPTG) overnight. The cells were harvested by centrifugation (20 min, 4000 rpm, 4 C), and resuspended in 50 mM potassium phosphate buffer containing 500 mM NaCl and 5 mM imidazole (pH 7.5). The cells were then sonicated at 40% power for 5 min using a Vibra-cell VCX-500 (Sonics, Newtown, CT, USA) sonicator. The insoluble fraction was removed by centrifugation (30 min, 13,000 rpm, 4 C), and the soluble extract was applied to a HisTrapFF crude column (5 ml; GE Healthcare, Uppsala, Sweden). The His-tagged AmyC enzymes were purified by Ni-chelate affinity chromatography. Protein concentrations were quantified by Bradford method using bovine serum albumin (BSA) as the protein standard (7). a-Amylase activity was assayed by measuring the amount of reducing sugar released during hydrolysis of 1% soluble starch in Tris buffer (250 mM, pH 8.5) at 70 C for 30 min. The amount of reducing sugar was measured by dinitrosalicylic acid (DNS) method (8). One unit of enzyme activity was defined as the amount of enzyme that released 1 mmol of maltose under the assay conditions. The specific activity of enzyme was calculated by the ratio of enzyme activity (U) to protein concentration (mg) in the assay mixture. The specific activity of wild-type AmyC was taken as 100%, and it was compared with that of each mutant AmyC enzyme. The methionine to alanine exchanges resulted in reduced specific activities compared to the wild-type enzyme. As shown in Fig. 1B, M43A þ M44A, M55A and M62A variants had 82%, 77% and 44% relative activities, respectively. Compared to Met55 and Met62, the residues Met43 and Met44 were less severely affected by alanine
1389-1723/$ e see front matter Ó 2013, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2013.04.018
OZTURK ET AL.
J. BIOSCI. BIOENG.,
A M44
M43
D349 M55
E185
M62
B Relative activity (%)
100
80 60 40 20 0 Wild-type
M43A+M44A
M55A
M62A
FIG. 1. Location of methionine mutations in the structure of AmyC. (A) The figure was generated by Swiss-Pdb Viewer (14) using the published structure (PDB: 2B5D) (5). The catalytic residues E185 and D349 are indicated by red color. The effect of mutations on enzymatic activity was investigated. (B) The specific activity of the wild-type AmyC was taken as 100%, and it was compared with that of each mutant AmyC enzyme. The data represent the average values of three measurements. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
exchanges. This could be attributed to that Met43 and Met44 are located on the surface of the enzyme, and they are more distant from the active site (Fig. 1A). However, it should not be excluded that M43A þ M44A variant exhibited 82% activity, which indicated a change in the hydrophobicity resulting from introducing alanine residues at these positions. Met317Ser exchange, which is nearby the enzyme surface and away from the catalytic residues, has not resulted in a significant decrease in the activity of the alkaline amylase from Alkalimonas amylolytica (9). The reduced specific activity of M55A and M62A mutants confirmed the previous findings that the methionine mutations that are close to the active site severely affects the catalytic activity (9,10). Oxidative stability was tested by incubating 0.9 mg of wild-type or mutant AmyC enzymes at different concentrations of H2O2 (1e100 mM) in Tris buffer (250 mM, pH 8.5) at 30 C for 30 min. Following incubation, catalase (SigmaeAldrich, St. Louis, MO, USA) with a final concentration of 1904 U/ml was added in order to quench the remaining H2O2. The residual activity was measured using 0.5 ml of the sample under standard assay conditions. The relative activity (%) was calculated by taking the activity in the absence of H2O2 as 100%. Wild-type AmyC was susceptible to oxidation at increasing concentrations of H2O2, with a complete loss of activity in the presence of 50 mM H2O2. The mutants M43A þ M44A and M55A retained 39% and 50% of the initial TABLE 1. The primers used for site-directed mutagenesis. Primer M43AþM44A M55A M62A
Sequence (50 / 30 )a TCGAACGCCGCTAAAAGC ATCGACGCGGTCAACCTG AGCATCTCCGCCAGCGGAG
a The nucleotide exchanges for methionine mutations are underlined.
activity at 100 mM H2O2, respectively (Fig. 2). The mutant M62A had no residual activity after treatment with 100 mM H2O2, but it was more resistant to oxidation than the wild-type at 50 mM H2O2. Methionine residues have been replaced by non-oxidizable residues such as leucine, alanine or serine for improved oxidative stability of several enzymes (9e12). These previous studies suggest that the methionine residues, which are solvent-accessible (10,11) or at the catalytic domain (9,12) are the primary targets for oxidation. Our results indicated that the M55A substitution nearby the active site enabled a higher oxidative resistance than that of the solvent-accessible M43A þ M44A. We assumed that M55A substitution was important for avoiding steric hindrance, which might have resulted from the formation of methionine sulfoxide nearby the active site. However, M62A substitution resulted in only a slight improvement in the oxidative stability (Fig. 2). Moreover, a remarkable decrease in the specific activity was observed (Fig. 1B). It has been shown that replacement of methionine residues nearby the active site increased the sensitivity to H2O2, which has been attributed to the antioxidant role of these residues (13). Met62 might have a similar role, and the remarkable decrease in specific activity upon M62A substitution suggests a structural or catalytic role for this residue. Due to their potential use in detergents, aamylase enzymes from Bacillus sp. TS-23 and A. amylolytica have been engineered for improved oxidative stability at 500 mM H2O2 (9,11). Introducing a single methionine substitution has been shown to increase the oxidative stability of Geobacillus stearothermophilus sp. US100 at 1.8 M H2O2 (12). We achieved a significant improvement in oxidative stability at 100 mM H2O2, with the most oxidative resistant M55A retaining 50% of its activity. It could be concluded that oxidative stability is greatly influenced by the total number methionine residues, as well as their location in enzyme structure. The effect of methionine mutation on pH and thermal stability was evaluated for the most oxidative resistant M55A mutant. Wild-type AmyC and M55A mutant were incubated in Tris buffer (100 mM, pH 8.5) at 30 C for 4 h, and then the residual activities were measured under standard assay conditions. Wildtype enzyme and M55A mutant retained 93% and 88% of their initial activities, respectively. The results indicated that the methionine substitution had only a slight effect on pH stability. To investigate the thermal stability, both enzymes were incubated at 70 C for 4 h. Following incubation, the remaining activities were measured under standard assay conditions. Both wild-type and M55A mutant enzyme had 88% residual activity. Compared to M43A þ M44A and M62A mutants, M55A mutant exhibited the most improved oxidative stability properties, albeit with a 23% decrease in specific activity. Moreover, it retained the pH and thermal stability properties. The engineered AmyC could be a potential candidate for industrial applications, particularly in the
100
Relative activity (%)
450
80 60 40 20 0
0
20
40
60
80
100
[H2O2] (mM) FIG. 2. Inactivation of wild-type (filled circles), M55A mutant (filled squares), M43A þ M44A mutant (filled triangles), and M62A mutant (open triangles) enzymes by different concentrations of H2O2. The relative activity (%) was compared with the activity without the addition of H2O2.
VOL. 116, 2013 presence of oxidizing agents. Here, we provided the first protein engineering attempt for AmyC from T. maritima. References 1. Prakash, O. and Jaiswal, N.: Alpha-amylase: an ideal representative of thermostable enzymes, Appl. Biochem. Biotechnol., 160, 2401e2414 (2010). 2. Fujinami, S. and Fujisawa, M.: Industrial applications of alkaliphiles and their enzymesepast, present and future, Environ. Technol., 31, 845e856 (2010). 3. Kim, Y. H., Berry, A. H., Spencer, D. S., and Stites, W. E.: Comparing the effect on protein stability of methionine oxidation versus mutagenesis: steps toward engineering oxidative resistance in proteins, Protein Eng., 14, 343e347 (2001). 4. Ballschmiter, M., Futterer, O., and Liebl, W.: Identification and characterization of a novel intracellular alkaline alpha-amylase from the hyperthermophilic bacterium Thermotoga maritima MSB8, Appl. Environ. Microbiol., 72, 2206e2211 (2006). 5. Dickmanns, A., Ballschmiter, M., Liebl, W., and Ficner, R.: Structure of the novel alpha-amylase AmyC from Thermotoga maritima, Acta Crystallogr. D Biol. Crystallogr., 62, 262e270 (2006). 6. Sarkar, G. and Sommer, S. S.: The “megaprimer” method of site-directed mutagenesis, Biotechniques, 8, 404e407 (1990). 7. Bradford, M. M.: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem., 72, 248e254 (1976).
NOTE
451
8. Miller, G. L.: Use of dinitrosalicylic acid reagent for determination of reducing sugar, Anal. Chem., 31, 426e428 (1959). 9. Yang, H., Liu, L., Li, J., Du, G., and Chen, J.: Structure-based replacement of methionine residues at the catalytic domains with serine significantly improves the oxidative stability of alkaline amylase from alkaliphilic Alkalimonas amylolytica, Biotechnol. Prog., 28, 1271e1277 (2012). 10. Roger Chien, H. C., Hsu, C. L., Hu, H. Y., Wang, W. C., and Hsu, W. H.: Enhancing oxidative resistance of Agrobacterium radiobacter N-carbamoyl Damino acid amidohydrolase by engineering solvent-accessible methionine residues, Biochem. Biophys. Res. Commun., 297, 282e287 (2002). 11. Lin, L. L., Lo, H. F., Chiang, W. Y., Hu, H. Y., Hsu, W. H., and Chang, C. T.: Replacement of methionine 208 in a truncated Bacillus sp. TS-23 alpha-amylase with oxidation-resistant leucine enhances its resistance to hydrogen peroxide, Curr. Microbiol., 46, 211e216 (2003). 12. Khemakhem, B., Ali, M. B., Aghajari, N., Juy, M., Haser, R., and Bejar, S.: Engineering of the alpha-amylase from Geobacillus stearothermophilus US100 for detergent incorporation, Biotechnol. Bioeng., 102, 380e389 (2009). 13. Kuo, L. Y., Hwang, G. Y., Yang, S. L., Hua, Y. W., Chen, W., and Lin, L. L.: Inactivation of Bacillus stearothermophilus leucine aminopeptidase II by hydrogen peroxide and site-directed mutagenesis of methionine residues on the enzyme, Protein J., 23, 295e302 (2004). 14. Guex, N. and Peitsch, M. C.: SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling, Electrophoresis, 18, 2714e2723 (1997).