Structure-based engineering of methionine residues in the catalytic cores of alkaline amylase from

35 Pages·2012·0.67 MB·English

AEM Accepts, published online ahead of print on 3 August 2012
Appl. Environ. Microbiol. doi:10.1128/AEM.01307-12
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
1 Structure-based engineering of methionine residues in the catalytic cores of alkaline
2 amylase from Alkalimonas amylolytica for improved oxidative stability
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4 Haiquan Yanga, b, Long Liua, b*, Mingxing Wanga, b, Jianghua Lia, b, Nam Sun Wangc, Guocheng Dua, b,
5 Jian Chend* D
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7 Running Title: Protein engineering of alkaline amylase
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9 a Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi h
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10 214122, China //
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11 b School of Biotechnology, Jiangnan University, Wuxi 214122, China .
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12 cDepartment of Chemical & Biomolecular Engineering, University of Maryland, College Park, MD,
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13 20742, USA /

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14 d National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi A
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15 214122, China il 3
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18 Corresponding authors: Long Liu, Tel.: [phone], Fax: [phone], E-mail: e
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19 [email protected]; Jian Chen, Tel.: [phone], Fax: [phone], E-mail:
20 [email protected]
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22
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23 ABSTRACT
24 This work aims to improve the oxidative stability of alkaline amylase from Alkalimonas amylolytica
25 through structure-based site-directed mutagenesis. Based on an analysis of the tertiary structure, five
26 methionines (Met 145, Met 214, Met 229, Met 247, and Met 317) were selected as the mutation sites
27 and individually replaced with leucine. In the presence of 500 mM HO at 35°C for 5 h, the wild-type D
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28 enzyme and the mutants M145L, M214L, M229L, M247L, and M317L retained 10%, 28%, 46%, 28%, n
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29 72%, and 43% of the original activity, respectively. Concomitantly, the alkaline stability, thermal
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30 stability, and catalytic efficiency of M247L were also improved. The pH stability of the mutants ro
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31 (M145L, M214L, M229L, and M317L) remained unchanged compared to that of the wild-type enzyme, h
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32 while the stable pH range of M247L was extended from 7.0-11.0 for the wild-type to 6.0-12.0 for the //
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33 mutant. The wild-type enzyme lost its activity after incubation at 50°C for 2 h, and the mutants M145L, .
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34 M214L, M229L, and M317L retained less than 14% of the activity; whereas, the mutant M247L
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35 retained 34% of the activity under the same condition. Compared to the wild-type enzyme, the k /
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36 value of M145L, M214L, M229L, and M317L decreased, while that of M247L increased slightly from A
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37 5.0×104 to 5.6×104 min-1. The mechanism responsible for the increased oxidative stability, alkaline il 3
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38 stability, thermal stability, and catalytic efficiency of M247L was further analyzed with a structure 0
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39 model. The combinational mutants were also constructed and their biochemical properties were y

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40 characterized. Resistance of the wild-type enzyme and the mutants to surfactants and detergents was e
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41 also investigated. Our results indicate that the mutant M247L has a great potential in the detergent and
42 textile industries.
43 Key words: Alkaline amylase; Oxidative stability; Alkaline stability; Thermal stability; Methionine;
44 Leucine
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45 INTRODUCTION
46 α-Amylases hydrolyze starch by cleaving α-1, 4-glucosidic linkages and have been widely used in
47 the food, textile, and pharmaceutical industries (10, 16, 20-22, 25). Alkaline α-amylases have high
48 catalytic efficiency and stability at alkaline pH from 9 to 11 (5, 9, 11, 17) and find applications where
49 starch is hydrolyzed under alkaline conditions in the starch and textile industries (5, 10, 16, 21, 22). D
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50 Oxidative stability is one of the most important quality parameters for alkaline amylase, especially n
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51 in detergents where the washing environment is oxidizing (13). Methionine (Met) residues in proteins
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52 are especially oxidation prone (4, 28). Oxidation of this residue has been shown to cause a decrease in ro
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53 activity or outright inactivation of amylases (12, 23). To reduce inactivation caused by oxidation, h
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54 replacing methionine by an oxidative-resistant amino acid may be effective. The non-oxidizable amino //
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55 acid residues include leucine (Leu), serine (Ser), isoleucine (Ile), threonine (Thr), and alanine (Ala). .
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56 For example, Met 197 of α-amylase from Geobacillus stearothermophilus US110 was substituted with
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57 Ala, and the mutant retained 70% of the activity in the presence of 1.8 M HO after 60 min of /
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58 treatment (15). A
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59 In previous work, the alkaline amylase (AmyA) from alkaliphilic Alkalimonas amylolytica N10 il 3
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60 was expressed in E. coli DH5α (27). However, AmyA is susceptible to oxidation by hydrogen peroxide. 0
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61 On the other hand, an oxidation-resistant AmyA could be an important candidate for the combined y

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62 desizing, bioscouring and bleaching (CDBB) process in the detergent and textile industries. In this e
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63 work, we aim to improve the oxidative resistance of this enzyme via site-directed mutagenesis of
64 methionine residues. Based on an analysis of a three-dimensional structure model of the enzyme, five
65 methionine residues (Met 145, Met 214, Met 229, Met 247, and Met 317) around the active site of
66 AmyA were selected for mutation. These methionines were individually replaced by leucine, and the
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67 mutant enzymes are designated as M145L, M214L, M229L, M247L, and M317L, respectively. The
68 combinational mutants with multiple mutation sites were also constructed. The anti-oxidant and other
69 biochemical properties (alkaline stability, thermal stability, catalytic efficiency, and specific activity) of
70 the mutants were characterized and compared with those of the wild-type enzyme, and the possible
71 mechanisms responsible for changes in the biochemical properties were explored by analyzing the D
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72 structure model. Our mutagenesis procedure here may be applied to improve the anti-oxidant ability of n
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73 other industrial enzymes.
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74 ro
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75 MATERIALS AND METHODS h
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76 Strains and plasmids //
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77 All strains and plasmids used were obtained from the Lab of Biosystem & Bioprocess Engineering .
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78 at Jiangnan University. The alkaline amylase gene (Accession No. AY268953) from alkaliphilic A.
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79 amylolytica N10 was synthesized based on the preferred codon usage of Escherichia coli by Sangon /

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80 Biotech Co., Ltd (Shanghai). Plasmid pET-20b (+) was used for subcloning, and E. coli JM109 was the A
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81 host for cloning work and E. coli BL21 (DE3) was the host for expression of the alkaline amylase. il 3
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82 0
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83 Media and culture conditions y

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84 Luria-Bertani (LB) medium was used for the seed culture of E. coli. The seed of E. coli was e
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85 cultured on a rotary shaker at 200 rpm and 37°C for 10 h. Terrific Broth (TB) medium was used for
86 production of alkaline amylase. E. coli transformants carrying recombinant plasmid were screened on
87 corn starch/agar plates. After incubation at 37°C for 10 h, the formation of a clear halo around
88 individual colonies indicated the expression of the alkaline amylase induced by the inducer isopropyl
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89 β-D-1-thiogalactopyranoside (IPTG) (0.4 mM) in the plate. Fermentation was performed in 250-mL
90 shaker flasks containing 25 mL medium, agitated at 200 rpm at 37°C; when the optical density at 600
91 nm (OD ) reached 0.6, IPTG was added to 0.4 mM for induction at 25°C for 48 h.
600
92 Site-directed mutagenesis
93 The recombinant plasmid was PCR amplified with mutagenic oligonucleotides (Table 1) using the D
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94 MutanBEST Kit (TaKaRa, Dalian, China). Fragments amplified by PCR were purified and isolated on n
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95 1% agarose gels after electrophoresis. The fragments were blunted by Blunting Kination Enzyme Mix
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96 (TaKaRa, Dalian, China) through the Blunting Kination reaction. The blunt-end fragments were ligated ro
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97 with Ligation Solution I (TaKaRa, Dalian, China). The reaction mixture was then transformed into h
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98 competent cells of E. coli JM109. The transformants were selected at 37oC on the LB agar plates //
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99 containing 50 μg/mL ampicillin. The alkaline α-amylase gene in the transformants was confirmed by .
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100 PCR and checked by sequencing, and finally the recombinant plasmids were transformed into
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101 competent cells of E. coli BL21 (DE3) for expression. /

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102 A
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103 Purification of alkaline amylase il 3
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104 Solid ammonium sulfate was added to the E. coli supernatant to 70% saturation at 0°C. The 0
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105 precipitate was collected and dissolved in glycine/NaOH buffer (pH 9.0, 20 mM) and dialyzed y

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106 overnight against the same buffer. After dialysis, the enzyme solution was filtered. The enzyme e
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107 solution was then injected into an AKTA purifier (GE Healthcare, USA) through anion exchange
108 (Q-Sepharose HP). Buffer A (equilibration buffer) was 20 mM phosphate buffer (pH 6.0). Buffer B
109 (elution buffer) contained 20 mM phosphate buffer (pH 6.0) and 1 M NaCl. The flow rate was 1.0
110 mL/min. When the sample was injected, buffer A was used to absorb the target enzyme to the column;
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111 and after eluting the unbound proteins, a linear elution was done by ramping buffer B from 0% to
112 100%. The fractions were collected for activity assay and SDS-PAGE analysis.
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114 Enzyme assays
115 Alkaline amylase activity was determined by measuring the amount of reducing sugar released D
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116 during hydrolysis of 1% soluble starch in glycine/NaOH buffer (pH 10.0, 20 mM) at 55°C for 5 min. A n
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117 control without enzyme addition was used. The amount of reducing sugar was measured with a
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118 modified dinitrosalicylic acid method (7). One unit of alkaline amylase activity was defined as the ro
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119 amount of enzyme that released 1 μmol of reducing sugar as glucose per min at the assay condition. h
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120 The protein concentration was measured with the Bradford method (27), with bovine albumin (Sangon //
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121 Biotech, Shanghai, China) as the standard. .
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122
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123 Computer-aided modeling of the tertiary structure /

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124 The theoretical structure of alkaline α-amylase was obtained from homology modeling from the A
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125 Swiss Model server (1, 8). Amino acid mutations were inserted into the structure with the mutation tool il 3
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126 of the Swiss-Viewer followed by side chain reconstruction for neighboring amino acids and energy 0
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127 minimization (2). The root-mean-square deviation (RMSD) for the modeling structure was calculated y

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128 with Swiss-PdbViewer 4.0.4. The number of hydrogen bonds in the enzyme was calculated by e
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129 Discovery Studio 2.5.
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131 Influence of mutation on oxidative stability
132 The wild-type and engineered enzymes were incubated with 500 mM HO in glycine/NaOH
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133 buffer (pH 9.0, 20 mM) at 35°C for 5 h. The choice of the HO concentration is dictated by that
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134 (approximately 500 mM) typically employed in the bleaching process in the textile industry when
135 combined with desizing and bioscouring (17). After the 5 h HO incubation period, catalase (10,000
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136 U/mL) was immediately added to the samples to a final concentration of 2000 U/mL to quench the
137 remaining HO. The starting concentration for the alkaline α-amylase was 16 U/mL when incubated D
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138 with HO. Samples (200 μL) were then withdrawn to measure the residual activity under the standard n
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139 assay condition.
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140 ro
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141 Effect of temperature and pH on stability h
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142 The thermal stability of alkaline α-amylase was determined from 30 to 50°C in glycine/NaOH //
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143 buffer (pH 9.0, 20 mM). The enzyme was incubated at 30°C for 10 h, and was incubated at 40 or 50°C .
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144 for 2 h. The activation energy (E) was determined in the temperature range from 20 to 50oC from the
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145 Arrhenius equation ln (k) = (-E/RT) + ln (A). The reaction rate constant (k) was calculated from the /
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146 equation ln (c) = -kt + ln (c), where c is the concentration of the substrate (soluble starch), and c is the A
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147 initial concentration of the substrate. To estimate the optimal pH of alkaline α-amylase, the purified il 3
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148 protein was incubated in sodium phosphate buffer (pH 6.0-8.0, 20 mM), Tris/HCl buffer (pH 8.0-9.5, 0
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149 20 mM), glycine/NaOH buffer (pH 9.5-11.0, 20 mM), and Ca(OH)2 buffer (pH 12.0, 20 mM). The pH y

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150 stability of alkaline α-amylase was determined at pH ranging from 6.0 to 12.0 at 25°C for 24 h. After e
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151 incubation, the alkaline α-amylase activity was measured at pH 10.0 and 55°C. The starting
152 concentration for the alkaline α-amylase was 16 U/mL.
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154 Influence of mutation on kinetic parameters
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155 The kinetic parameters (K , V , k , and k /K ) of the wild-type enzyme and mutants were
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156 determined in glycine/NaOH buffer (pH 10.0, 20 mM) at 55°C. Assays were performed with active
157 enzyme at a fixed starting level of 16 U/mL and soluble starch at concentrations ranging from 1 to 10
158 g/L. Enzyme kinetic parameters K and V were estimated from Eadie-Hofstee plots (6).
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159 D
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160 Effect of surfactants and detergents on stability and activity n
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161 The effect of surfactants Tween 20, Tween 60, Tween 80, Triton X-100, and sodium dodecyl
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162 sulfate (SDS) on enzyme stability was studied by pre-incubating enzyme at 40°C for 1 h. The ro
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163 concentration of surfactants was 10% (w/v). The starting concentration for the alkaline α-amylase was h
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164 16 U/mL. Residual enzyme activity was measured under standard assay conditions. The activity of //
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165 enzyme without surfactant addition was taken as 100%. Compatibility of enzyme with solid detergents .
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166 was studied with laundry solid soap (Nice, China), toilet soap (Safeguard, USA), washing powder-1
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167 (Tide, USA), and washing powder-2 (Nice, China). Compatibility of enzyme with liquid detergents /

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168 was studied with laundry detergent-1 (Blue noon, China), laundry detergent-2 (Liby, China), liquid A
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169 detergent-1 (Nice, China), and liquid detergent-2 (Liby, China). The detergents were first diluted in tap il 3
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170 water to give a concentration of 70 mg/mL for solid detergents and 10% for liquid detergents. The 0
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171 endogenous proteases from the detergents were inactivated by incubating the detergents at 65oC for 1 h by

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172 before addition. When enzyme activity was determined, the detergents were diluted to give a final e
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173 concentration of 7 mg/mL for solid detergents and 1% for liquid detergents to simulate washing
174 conditions (15). The enzyme activity of the control without detergent addition was taken as 100%.
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176 Circular dichroism (CD)
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177 CD spectra were measured on MOS-450/AF-CD-STP-A (Bio-Logic, France) at a protein
178 concentration of 0.1 mg/mL in a 1 cm path-length quartz cuvette in 20 mM Tris/HCl buffer (pH 9.0).
179 In order to minimize signal baseline drift, the spectropolarimeter and xenon lamp were warmed up for
180 at least 30 min prior to each experiment. Ellipticity data of the enzymes in the band of 190-250 nm
181 were collected, from which the spectrum of a buffer blank was subtracted. D
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182 n
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183 RESULTS AND DISCUSSION
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184 Selection of mutation sites based on structure analysis ro
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185 Oxidative stability of enzymes is one of the most important parameters for their use in the h
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186 presence of bleach-containing detergents in textile processing. Studies of oxidative inactivation of //
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187 some industrial enzymes indicate that oxidation of methionine residues to their sulfoxide derivatives is .
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188 a main problem (28). Oxidation of methionine residues situated in the cavity of the active site increases
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189 the size of the side-chain, leading to steric hinderance of the active site (15). /

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190 To determine the mutation sites of alkaline amylase, a 3-D (three-dimensional) model of alkaline A
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191 amylase was constructed based on the known structure of α-amylase B (3bc9) with the Swiss Model il 3
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192 server (Fig. 1(A)) (26). The amino acid sequence of alkaline amylase includes four conserved regions 0
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193 (I through IV) that have been previously identified in the α-amylase family: (I) 137DVVFNH142, (II) by

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194 244GFRMDAIAH252, (III) 278EAWV281, and (IV) 335FVDNHD340 (27). The four conserved regions are e
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195 necessary for the catalytic activity and they are present in the catalytic domain of the (β/α)-barrel
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196 structure of α-amylases in Family 13. Region I contains three fairly conserved amino acids (Asp137,
197 Asn141, and His142) that are important for the stability and activity of the enzyme. Region II contains
198 the catalytic nucleophiles Asp248 and the invariant residue Arg246. The two residues are found in all
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199 α-amylases and are believed to be indispensable for catalytic activity. Region III includes the catalytic
200 proton donor Glu278, which is the only totally conserved residue in this region. Region IV contains the
201 only fully conserved residue Asp340 (27). The Na/Ca binding site in AmyA corresponds to Asn142,
202 Pro211, and Asp217; whereas, the active site of AmyA includes Asp248, Glu278, and Asp340.
203 As suggested by the constructed model (Fig. 1), Met 145, Met 214, Met 229, and Met 247 are D
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204 situated in the cavity of the active site, with the Met side chains pointing toward the substrate. n
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205 Oxidation of these methionine residues could increase the size of the side-chain and cause steric
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206 obstruction of the active site (15). And replacing these methionine residues with leucine may improve ro
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207 anti-oxidative resistance of the enzyme to HO. Surface-exposed methionine residues in proteins have h
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208 been also shown to be susceptible to oxidation (14). Although Met 317 is not in the cavity of the active //
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209 site, it is sufficiently close to the active site and exposed to the surface (Fig. 1) and may also contribute .
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210 to oxidation stability of the enzyme. Thus, structure analysis of the enzyme suggests that these five
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211 methionine residues may be the key amino acids for improving anti-oxidative properties. /

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212 A
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213 Expression of mutant proteins il 3
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214 Based on the above structure analysis, five methionine residues were replaced with leucine 0
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215 through site-directed mutagenesis. After verification of the gene sequence, the mutant genes were y

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216 recombined with pET-20b (+) and expressed in E. coli BL21 (DE3). SDS-PAGE analysis shows that e
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217 the protein after mutation was approximately 60 kDa (Fig. 2).
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219 Oxidative stability of the wild-type and mutant proteins
220 The wild-type and mutant enzymes were evaluated for their tolerance to HO. The wild-type
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