Báo cáo khoa học: Enhancing thermostability of maltogenic amylase from Bacillus thermoalkalophilus ET2 by DNA shuffling pdf

Enhancing thermostability of maltogenic amylase from Bacillus thermoalkalophilus ET2 by DNA shuffling Shuang-Yan Tang 1 , Quang-Tri Le 1 , Jae-Hoon Shim 1 , Sung-Jae Yang 1 , Joong-Huck Auh 1 , Cheonseok Park 2 and Kwan-Hwa Park 1 1 Center for Agricultural Biomaterials, and Department of Food Science and Biotechnology, School of Agricultural Biotechnology, Seoul National University, South Korea 2 Department of Food Science and Biotechnology and Institute of Life Sciences and Resources, Kyunghee University, Yongin, South Korea In recent years, many thermostable amylases have been cloned from thermophilic archaea and bacteria [1–4]. The physical basis for the protein stability at high tem- peratures has also been widely investigated [5,6]. Ther- mostability appears to be conferred by a variety of strategies. Studies of thermostability have been carried out by comparing the atomic structure of a thermophi- lic protein with its mesophilic homologues [7–9] or by comparing between thermophilic and mesophilic genomic sequences in a large scale [10–13]. It is believed that surface electrostatics, amino acid compo- sition, shorter loops, increased charged dipole on heli- ces, and interactions between cations and aromatic rings (cation–p interaction) are important overall factors for increased thermostability of proteins. Many different forces and interactions contribute to thermostability. The challenge is to pinpoint which of the different factors and interactions are the most crit- ical for the specific proteins [14]. Studies of Bacillus licheniformis a-amylase (BLA) revealed that the ther- mostability of this highly thermostable enzyme could be further enhanced through better hydrophobic pack- ing and cavity-filling effects, introduction of favorable aromatic–aromatic interactions on the surface, improved formation of secondary structures and release of conformational strain, stabilization of an intrinsic metal binding site, and removal of possible deamidating residues [15]. Site-directed mutagenesis has been consistently used to enhance thermostability of various enzymes based on sequence comparison between thermophilic and mesophilic counterparts or on tertiary structural Keywords DNA shuffling; maltogenic amylase (MAase); neopullulanase; site-directed mutagenesis; thermostability Correspondence K H. Park, Department of Food Science and Biotechnology, Seoul National University, Shillim-dong, Kwanak-gu, Seoul 151–742, Korea Fax: +82 2 8735095 Tel: +82 2 8804854 ⁄ 8804852 E-mail: parkkh@plaza.snu.ac.kr (Received 28 March 2006, revised 22 May 2006, accepted 24 May 2006) doi:10.1111/j.1742-4658.2006.05337.x DNA shuffling was used to improve the thermostability of maltogenic amylase from Bacillus thermoalkalophilus ET2. Two highly thermostable mutants, III-1 and III-2, were generated after three rounds of shuffling and recombination of mutations. Their optimal reaction temperatures were all 80 °C, which was 10 °C higher than that of the wild-type. The mutant enzyme III-1 carried seven mutations: N147D, F195L, N263S, D311G, A344V, F397S, and N508D. The half-life of III-1 was about 20 times greater than that of the wild-type at 78 °C. The mutant enzyme III-2 car- ried M375T in addition to the mutations in III-1, which was responsible for the decrease in specific activity. The half-life of III-2 was 568 min while that of the wild-type was <1 min at 80 °C. The melting temperatures of III-1 and III-2, as determined by differential scanning calorimetry, increased by 6.1 °C and 11.4 °C, respectively. Hydrogen bonding, hydro- phobic interaction, electrostatic interaction, proper packing, and deamida- tion were predicted as the mechanisms for the enhancement of thermostability in the enzymes with the mutations. Abbreviations BLA, Bacillus licheniformis a-amylase; BTMA, MAase from Bacillus thermoalkalophilus ET2; b-CD, b-cyclodextrin; DSC, differential scanning calorimetry; MAase, maltogenic amylase. FEBS Journal 273 (2006) 3335–3345 ª 2006 The Authors Journal compilation ª 2006 FEBS 3335 information [16,17]. Compared with site-directed mutagenesis, which is a more conventional approach, directed evolution allows us to explore enzyme thermo- stability for which the molecular basis is poorly under- stood. DNA shuffling is an evolutionary protocol wherein cycles of selection, recombination, mutation, and amplification are employed to evolve DNA sequences and corresponding protein structures. It is a powerful molecular evolution technology which enables in vitro generation of large libraries of chimeric and mutated hybrids genes. In addition to recombina- tion, DNA shuffling also introduces point mutations at a controlled rate, which broadens the possibilities for evolving improved genes [18–21]. DNA shuffling has been used to improve protein properties such as stabil- ity, catalytic activity, and substrate specificity [6,22,23]. Maltogenic amylase (EC 3.2.1.133, MAase) is a multifunctional enzyme able to catalyze both hydro- lysis and transglycosylation activities. MAase shares similar catalytic properties with neopullulanases (EC 3.2.1.135) and cyclomaltodextrinases (EC 3.2.1.54), and together these enzymes constitute a subfamily in the glycoside hydrolase family 13. The specific sequence in the fifth conserved region distinguishes this subfamily from the oligo-1,6-glucosidase subfamily [24]. It has been suggested that these three enzymes are nearly the same enzymes in terms of their structures and catalytic properties [25]. They are able to hydro- lyze multiple carbohydrate substrates including starch, cyclodextrin, and pullulan, with the main hydrolysis product of starch and cyclodextrin being maltose while that of pullulan is panose. They are also capable of simultaneously transferring the hydrolyzed sugar moi- ety to another sugar acceptor molecule [26]. The most widely used thermostable enzymes are the amylases in the starch industry [27,28]. Thermostable amylases have such an extremely large application in the starch industry, as solubilized starch is a better substrate for amylases and starch gelatinization only occurs at high temperatures. Amylases from fungi and bacteria have dominated the applications in industry [29]. In addition to starch processing, for some high- value compounds with poor solubility, maltogenic amylase can transfer sugar residues to them to increase their solubilities [30]. In these cases, thermostable mal- togenic amylase can work at high temperatures under which the solubility of substrates is better. In this study, we generated a thermostable mutant of MAase from Bacillus thermoalkalophilus ET2 (BTMA), which had an optimal reaction temperature of 80 °C, 10 °C higher than that of the wild-type, and had 85% activity left at 85 °C. The half-life of the mutant was about threefold greater than that of the mutant of maltogenic amylase from Thermus strain (ThMA) at 85 °C. The hypothetical mechanism of each mutation contributing to the thermostability was predicted based on the modeled tertiary structures of the mutant enzymes. Results Screening of mutants with improved optimal reaction temperature and thermostability Under each round at the screening temperature, the parental enzyme showed only a very faint halo on the starch-containing plate when stained with iodine solu- tion. Mutants that showed a larger and stronger halo were selected, and the optimal reaction temperatures and half-lives of thermal inactivation of their purified enzymes were examined. Mutants with higher optimal reaction temperatures or longer half-lives than the par- ental enzyme were selected and changes in their DNA sequences were investigated. Approximately 12 000 colonies were screened after heat treatment at 90 °C for 60 min in the first round of shuffling reaction. Two mutant enzymes, I-1–79 and I-5–49, whose optimal reaction temperatures were 75 °C, corresponding to 5 °C higher than the wild-type BTMA, were selected. Their half-lives were 1.7- and three-fold longer than that of the wild-type at 75 °C (Table 1). I-1–79 contained N147D and I-5–49 con- tained F397S according to DNA sequencing analysis (Fig. 1). These mutant enzymes were used as parental enzymes for the second round of shuffling. Approximately 8000 colonies were screened after heat treatment at 93 °C for 45 min and II-1–56, which has an optimal reaction temperature of 80 °C and a 1.3-fold longer half-life compared with I-5–49 at 75 °C, was selected (Table 1). I95M, S550P, and A569V were found to be introduced in addition to N147D and F397S (Fig. 1). Through recombination using restriction enzymes, II (N147D, F397S), II-1–56A (N147D, F397S, I95M), and II-1–56B (N147D, F397S, S550P, A569V) were constructed from II-1–56. The optimal reaction temperatures of II, II-1–56A, and II-1–56B were all 80 °C, and no significant differences in their thermosta- bilities were detected (Table 1). From these results, it was therefore thought that the combination of N147D and F397S mutations mainly contributed to the increase in thermal resistance of II-1–56. The mutant II was used as the parental enzyme for the third round of shuffling. In this round, about 8000 colonies were screened after heat treatment at 96 °C for 30 min, and four mutants with longer half- lives than that of II (III-1–18, III-1–19, III-4–85, and Enhancing MAase thermostability by DNA shuffling S Y. Tang et al. 3336 FEBS Journal 273 (2006) 3335–3345 ª 2006 The Authors Journal compilation ª 2006 FEBS III-3–13) were selected. Their half-lives at 75 °C were 11.7-, 4.6-, 4.0-, and 1.8-fold longer than that of the parental enzyme, respectively (Table 1). Two point mutations (Q343H and M375T) in III-1–18, four point mutations (Y18C, V51I, F195L, and N263S) in III-1– 19, two mutations (D311G and A344V) in III-4–85, and two mutations (N508D and L549S) in III-3–13 were newly introduced mutations in addition to the existing mutations in the parental gene (Fig. 1). To investigate the contribution of the mutations generated in the third round shuffling mutants, various additional mutants were constructed by general recom- bination using restriction enzymes or by site-directed mutation. Two mutants were constructed from III-4– 85: mutants III-4–85A and III-4–85B carried D311G and A344V, respectively, in addition to N147D and F397S. Both mutants had shorter half-lives than III-4– 85, but had 2.9- and 2.0-fold longer half-lives than the parental enzyme II, suggesting both D311G and A344V contributed to the thermostability to some extent (Table 1). Mutations in III-1–18A and III-1–18B were Q343H and M375T, respectively, in addition to N147D and F397S. It was found that the half-life of III-1–18B at 75 °C was 56.02 ± 4.49 min, roughly two times higher than that of III-1–18 (29.41 ± 2.41 min); however, III-1–18A showed very short half-life (1.06 ± 0.02 min) compared with III-1–18 (Table 1). These results suggested that M375T positively contributed to the thermostability, whereas Q343H had little effect. Interestingly, the specific activity of III-1–18 and III-1– 18B significantly decreased to about 13% of the wild- type level, indicating that M375T negatively affected the enzyme activity of BTMA, even though it stabil- ized the enzyme at high temperature. Four mutations (Y18C, V51I, F195L, and N263S) added in III-1–19 were separated into two mutants, III-1–19A (Y18C, V51I, N147D, F397S) and III-1–19B (F195L, N263S, N147D, F397S). The thermostability of III-1–19B was similar to III-1–19, while that of III-1–19A was analogous to the parental enzyme II (Table 1). To examine the contribution of F195L and N263S further, III-1–19B1 (F195L, N147D, F397S) and III-1–19B2 (N263S, N147D, F397S) were con- structed. Although III-1–19B1 and III-1–19B2 had shorter half-lives than III-1–19B, they were 3.8- and 3.0-fold longer than that of II, respectively, suggesting that F195L and N263S both individually contributed to the thermostability of BTMA (Table 1). Two mutants were constructed from III-3–13: III-3– 13A (N508D, N147D, F397S) and III-3–13B (L549S, N147D, F397S). The thermostability of III-3–13A was comparable to that of III-3–13, while that of III-3–13B Wild-type N147D F397S N147D, F397S N147D, F397S D311G* A344V* N147D, F397S Y18C* V51I* F195L* N263S* N147D, F397S N508D* L549S* N147D, F397S Q343H* M375T* N147D, F397S D311G, A344V, F195L , N263S N508D N147D, F397S D311G, A344V, F195L , N263S N508D, M375T III-4-85 III-1-19 III-3-13 III-1-18 II I-1-79 I-5-49 III-1 III-2 N147D, F397S I95M* S550P* A569V* II-1-56 1st round 2nd round 3rd round Fig. 1. Lineage of BTMA shuffling mutants. Newly introduced mutations in each generation are marked with asterisks. Positive mutations are highlighted in bold. Table 1. Thermostabilities of BTMA wild-type and mutants obtained from DNA shuffling. Enzymes Mutations t 1 ⁄ 2 (min) a Wild-type 0.63 ± 0.10 I-1–79 N147D 1.06 ± 0.14 I-5–49 F397S 1.86 ± 0.18 II-1–56 N147D, F397S, I95M, S550P, A569V 2.34 ± 0.08 II N147D, F397S 2.51 ± 0.20 II-1–56 A N147D, F397S, I95M 2.46 ± 0.16 II-1–56B N147D, F397S, S550P, A569V 2.43 ± 0.17 III-1–18 N147D, F397S, Q343H, M375T 29.41 ± 2.41 III-1–18 A N147D, F397S, Q343H 1.06 ± 0.02 III-1–18B N147D, F397S, M375T 56.02 ± 4.49 III-1–19 N147D, F195L, Y18C, V51I, N263S, F397S 11.43 ± 0.40 III-1–19 A N147D, F397S, Y18C, V51I 1.76 ± 0.15 III-1–19B N147D, F397S, F195L, N263S 11.09 ± 0.69 III-1–19B1 N147D, F397S, F195L 9.47 ± 0.37 III-1–19B2 N147D, F397S, N263S 7.54 ± 0.36 III-3–13 N147D, F397S, N508D, L549S 4.63 ± 0.38 III-3–13 A N147D, F397S, N508D 4.43 ± 0.52 III-3–13B N147D, F397S, L549S 2.01 ± 0.14 III-4–85 N147D, F397S, D311G, A344V 10.05 ± 0.45 III-4–85 A N147D, F397S, D311G 7.17 ± 0.41 III-4–85B N147D, F397S, A344V 4.96 ± 0.41 a t 1 ⁄ 2 , half-life, determined with 0.04 mgÆmL )1 protein concentration at 75 °C. S Y. Tang et al. Enhancing MAase thermostability by DNA shuffling FEBS Journal 273 (2006) 3335–3345 ª 2006 The Authors Journal compilation ª 2006 FEBS 3337 was analogous to the parental enzyme II. Conse- quently, we suspected that N508D was responsible for the increase of thermostability in III-3–13 (Table 1). Finally, two mutants with all positively affecting mutations were made by general recombination using restriction enzymes and site-directed mutation. Mutant III-2 carried all positive mutations found in each shuf- fling mutant, whereas mutant III-1 included all muta- tions except M375T, which negatively affects the specific activity of the enzyme. As expected, the specific activity of III-1 was similar to that of the wild-type and other shuffling mutants, except III-1–18; however, the specific activity of III-2 decreased to 20% of the specific activity of the wild-type (Table 2). The optimal reaction temperature of both mutants was 80 °C. The half-lives of III-1 and III-2 observed at 78, 80, and 85 °C were all longer than those of other shuffling mutants and wild-type BTMA. The half-life of III-1 was 20 times greater than that of the wild-type at 78 °C. The thermostability of III-2 was much higher than that of III-1. The half-life of III-2 was 568 min while that of wild-type was less than 1 min at 80 °C. When the thermostability of the shuffling mutants of BTMA was compared with that of the ThMA mutant, the half-life of III-2 was about three times longer than that of the ThMA shuffling mutant at 85 °C (Table 2). T m of BTMA mutants The melting temperatures (T m ) of BTMA wild-type enzyme and various mutants, II, III-1, and III-2, obtained from shuffling reactions were determined by differential scanning calorimetry (DSC). A variety of shuffling mutants exhibited higher T m than the wild- type. The T m of II was 3.5 °C higher than that of the wild-type, while the T m values of III-1 and III-2 were 6.1 and 11.4 °C higher than that of wild-type, respect- ively. The enthalpy change at T m also increased when the T m increased (Table 3, Fig. 2), which showed that higher temperatures and more energy were needed for the denaturation of the mutants. Predicting thermostabilization mechanisms The homology-based models of the BTMA wild-type and shuffling mutants were constructed by the swiss- model program [31] with reference to the known Table 2. Specific activities and thermostabilities of BTMA wild-type and mutants. Enzymes Specific activity (UÆmg )1 ) t 1 ⁄ 2 (min) a 78 80 85 Wild-type 452.9 1.94 ± 0.07 ND ND II 484.0 14.31 ± 0.69 3.57 ± 0.25 ND III-3–13 A 481.3 22.17 ± 1.36 3.90 ± 0.13 ND III-4–85 474.1 30.39 ± 2.98 6.62 ± 0.36 ND III-1–19B 474.3 31.84 ± 2.29 7.41 ± 0.16 ND III-1–18B 64.6 ND 157.02 ± 4.11 6.50 ± 0.17 III-1 484.3 37.29 ± 2.90 8.35 ± 0.35 ND III-2 84.4 ND 567.78 ± 67.94 12.45 ± 0.47 ThMA-DM (6) 20.0 ND ND 4.47 ± 0.46 a t 1 ⁄ 2 , half-life, determined with 0.12 mgÆmL )1 protein concentration; ND, not determined. Table 3. Differential scanning calorimetry analysis of BTMA wild- type and mutants. Enzymes T m (°C) Enthalpy (10 4 kJÆmol )1 ) Wild-type 76.68 132.9 II 80.22 148.6 III-1 82.76 167.3 III-2 88.10 171.6 Temperature ( o C) 60 70 80 90 / elo m/lack(p C o )C -2.0e+4 0.0 2.0e+4 4.0e+4 6.0e+4 8.0e+4 1.0e+5 1.2e+5 1.4e+5 1.6e+5 wild-type II III-1 III-2 Fig. 2. Differential scanning calorimetry analysis of BTMA wild-type and mutants. Wild-type and mutant BTMAs were concentrated to 1mgÆmL )1 in 50 mM glycine–NaOH buffer (pH 8.0) using a Microcon filter (Millipore Co., Billerica, MA, USA). The samples were scanned at temperatures from 40 to 110 °C with a scan rate of 1°CÆmin )1 . Enhancing MAase thermostability by DNA shuffling S Y. Tang et al. 3338 FEBS Journal 273 (2006) 3335–3345 ª 2006 The Authors Journal compilation ª 2006 FEBS structure of wild-type ThMA [32] and neopullulanases from Bacillus stearothermophilus [33], whose amino acid sequences were 64 and 70% identical to BTMA, respectively. In hyperthermophilic proteins, there is a reduction in the number of glutamines and asparagines, as these two amino acids are easily deamidated at elevated temperatures [34–36]. Examination of amino acid compositions of the total protein content of eight mesophilic and seven hyperthermophilic microorgan- isms, for which genome sequences data are available, revealed a 55% reduction in the number of glutam- ines and a 28% reduction in asparagines in hyper- thermophilic proteins [37]. N263S might contribute to thermostability for this reason. Deamidation of N263 and N261, N264 surrounding it could lead to a pack- ing of negative charges and a destabilization of the native structure (Fig. 3A). When the asparagine was mutated to serine, however, the hydroxyl group of serine could form a hydrogen bond with the lysine at position 268, while in the wild-type, this hydrogen bond could not form between K268 and N263 (Fig. 3A). It has been shown that improved calcium binding could help the thermostability of the enzyme in many cases, for example, a-amylase from B. licheniformis [38] and Bacillus amyloliquefaciens [39], and influenza virus neuraminidase [40]. N147 was one of the residues in the calcium-binding site in BTMA (Fig. 3B). After N147D substitution, the negatively charged aspartate could interact better with a calcium ion than aspara- gine and therefore enhance the thermostability of the enzyme. The peptide chain between V490 and I509 was flex- ible, so when N508 was mutated to aspartate, the neg- atively charged aspartate might adjust its direction and interact with the positively charged arginine at position 26 (Fig. 3C). The newly formed electrostatic interac- tion might improve the interaction between the N-domain and C-domain and subsequently contribute to the thermostability of the enzyme. D311 was located at a-helix 3 in the (a ⁄ b) 8 domain. The surrounding amino acids, Y308, L309, L310, V312, A313, and Y315, were all hydrophobic amino acids, whereas aspartate is hydrophilic. The mutation of aspartate to glycine enhanced the hydrophobicity of this area, and subsequently enhanced the thermostabil- ity of the enzyme (Fig. 4A). The same mechanism was predicted for A344V, on which many hydrophobic amino acids (L310, I317, F337, F341, and I348) were surrounded, forming a strong hydrophobic area. A substitution of alanine with valine made this area more hydrophobic (Fig. 4B). N261 N264 N263S K268 2.75Å A N153 G172 D174 N147D N149 B C R26 N508D N-domain C-domain Fig. 3. (A) Predicted thermostabilization mechanism with N263S mutation. Deamidation of N261, N263 and N264 could lead to a crowding of negative charges. S263 could form a hydrogen bond with K268. (B) Calcium-binding site in BTMA involving N147D muta- tion. The negatively charged aspartate could improve the calcium binding. (C) Predicted electrostatic interaction between the N- and C-domains of BTMA involving N508D mutation. S Y. Tang et al. Enhancing MAase thermostability by DNA shuffling FEBS Journal 273 (2006) 3335–3345 ª 2006 The Authors Journal compilation ª 2006 FEBS 3339 F195 is a residue buried inside the enzyme, and a hydrophobic core was formed, with the hydrophobic amino acids I178, I198, L241, V239, and I193 sur- rounding it. The size of phenylalanine is much larger than those of the other amino acids (isoleucine, leu- cine, and valine), possibly decreasing the ease with which they could be packed. The mutation of phenyl- alanine to leucine, an amino acid smaller in size, might therefore help in the packing of the hydrophobic core and subsequently enhance the thermostability of the enzyme (Fig. 5A). By computing the total occurrences of all possible mutations in 16 families, Gromiha et al. [41] gave the differences in preference of residue substi- tutions from mesophilic to thermophilic proteins. The sixth strongest preference of replacement was observed for the mutation F fi L, as the steric hindrance of phenylalanine might make it less preferable than leu- cine in thermostable proteins. The M375T mutation was located in the middle of the barrel at the C-terminal of b-strand 6, close to the catalytic center of the enzyme (Fig. 5B). This mutation not only enhanced the thermostability dramatically but also caused a decrease in specific activity of the enzyme (Tables 1 and 2). D328, E357, and D424 were A Y315 A313 V312 Y308 L309 L310 D311G B I317 I348 A344V F341 L310 F337 Fig. 4. (A) Predicted hydrophobic interactions with D311G mutation. D311G enhanced the hydrophobicity of the area formed by Y308, L309, L310, V312, A313, and Y315. (B) Predicted hydrophobic inter- actions with A344V mutation. A344V enhanced the hydrophobicity of the area surrounded by L310, I317, F337, F341, and I348. I178 I193 F195L V239 I198 L241 A B D424 E357 D328 M375T I358 3.5Å Fig. 5. (A) Predicted packing with F195L mutation. F195L might help the packing of the hydrophobic core formed by I178, I198, L241, V239, and I193. (B) Predicted thermostabilization mechanism with M375T mutation. The small size of threonine and the newly formed hydrogen bond between it and I358 could make the pack- ing of this area tighter, which could be responsible for the enhanced thermostability and decreased catalytic activity. The cata- lytic residues at the active site are shown. Enhancing MAase thermostability by DNA shuffling S Y. Tang et al. 3340 FEBS Journal 273 (2006) 3335–3345 ª 2006 The Authors Journal compilation ª 2006 FEBS identified as the catalytic residues of the enzyme [32]. As the size of sulfur atom is larger than other atoms, the bulky side chain of methionine at position 375 might assist the overall folding of the active site and provide enough space and flexibility for the catalytic activity. When methionine was mutated to the relat- ively smaller residue, threonine, the conformation around the active site might have changed so that the hydroxyl of threonine might form a hydrogen bond with the nitrogen atom of I358. The small size of thre- onine and the newly formed hydrogen bond could make the packing of this area tighter, which could be responsible for the enhanced thermostability and decreased catalytic activity. Discussion With three rounds of DNA shuffling method and recombination of mutations, two types of thermostable mutants of maltogenic amylase from B. thermoalkalo- philus ET2 were obtained. While the specific activity of one type of mutant remained unchanged relative to the wild-type enzyme, the other type of mutant decreased its catalytic capacity. In total, seven amino acid residue substitutions were found to enhance the thermostabili- ty of the enzyme without affecting the activity while one mutation caused the specific activity to decrease, but at the same time improved thermostability. Hydrogen bonding is one of the major mechanisms for protein thermostability. Substantial free energies of interaction have been observed in enzyme–ligand inter- actions, even in the presence of water. In Fersht’s experiments involving mutations in tyrosyl-tRNA syn- thetase, which compared ligand affinities as reflected by k cat ⁄ K m , hydrogen bonds between neutral partners typically seemed to have free energies of formation of 0.5 to 1.8 kcal, and hydrogen bonds involving one charged partner attain effective values of 3.5– 4.5 kcalÆmol )1 [42]. Even at the solvent-accessible molecular surface, intramolecular hydrogen bonding is favorable. In a mutational analysis of the protein bar- nase, it was found that a side chain to a backbone hydrogen bond at the N-terminus of either helix stabil- ized the protein by up to 2.5 kcalÆmol )1 relative to a nonhydrogen-bonding residue in the corresponding position [43]. In this study, M375T and N263S substi- tutions generated new hydrogen bonds that did not exist in the wild-type. In M375T, the hydrogen bond was created with neutral partners while in N263S, it was established with one charged partner. Hydrophobic interaction is another powerful strat- egy for protein thermostability. Removal of nonpolar molecules or groups from water is accompanied by an increase in entropy and a compensating uptake of heat from the surroundings. Hydrophobic bonds are thus distinguished by a tendency to become stronger with increasing temperature [44]. In free energy contribu- tions due to hydrophobic, electrostatic, hydrogen bonding, disulfide bonding, and van der Waals interac- tions, hydrophobic free energy due to carbon and nitrogen atoms and such combinations of free energy components play a vital role in the thermostability of proteins [45]. In many cases, the correlation between hydrophobic interactions and protein stability has been demonstrated [5,46–49]. In the cases of D311G and A344V, it was thought that the increased hydrophobic- ity of the environment after the substitutions contribu- ted to the enhanced thermostability of the enzyme. Electrostatic interactions, such as salt bridges and their networks, have important roles in protein folding [50,51]. A correlation between salt bridge networks and melting temperature has been observed for the d-glyceraldehyde-3-phosphate dehydrogenase family [52]. A similar correlation was also observed between salt bridge networks and thermostabilities of glutamate dehydrogenases from several hyperthermophilic, ther- mophilic, and mesophilic sources [53]. Grimsley et al. [54] have shown that the stability of RNase T1 can be increased by improving long-range electrostatic interac- tions among charged groups on the protein surface. In this study, N508D might belong to this category, in which the electrostatic interaction between aspartate and arginine could tighten the interaction between the N- and C-domains of the enzyme. Packing is also an undeniable factor in protein sta- bilization. The molecular interior of a folded protein should be well packed; otherwise dispersion forces would favor denaturation, since efficient packing can presumably be achieved between protein and solvent in the denatured state [55]. We postulate that the improved thermostability by F195L was due to the better packing of the protein after the residue substitu- tion. Experimental procedures Bacterial strains and plasmid The vector p6xHTKXb119, which carried the B. lichenifor- mis maltogenic amylase promoter and N-terminal hexahisti- dine tag, was used for over-expression of the six-His tagged wild-type and mutant of MAase gene from B. thermoalkalo- philus ET2 [6,56]. Escherichia coli MC1061 [F – , araD139, recA13, D(araABC-leu)7696, galU, galK, DlacX74, rpsL, thi, hsdR2, mcrB] was used as the host. The E. coli trans- formants were cultured at 37 °C in Luria–Bertani medium S Y. Tang et al. Enhancing MAase thermostability by DNA shuffling FEBS Journal 273 (2006) 3335–3345 ª 2006 The Authors Journal compilation ª 2006 FEBS 3341 [LB; 1% (w ⁄ v) Bacto–tryptone, 0.5% (w ⁄ v) yeast extract, and 0.5% (w ⁄ v) NaCl]. DNA shuffling for random mutagenesis of BTMA DNA shuffling was carried out according to the method of Zhao and Arnold [18]. The wild-type or mutant BTMA gene on p6xHTKXb119 vector was isolated by digestion with XbaI and HindIII and degraded randomly by DNaseI inreac- tion buffer at 15 °C for 7 min. The fragments smaller than 300 bp were isolated from 2% (w ⁄ v) agarose gel and used for self-assembly PCR with Taq polymerase (Takara Bio Inc., Shiga, Japan). The thermocycling reaction was carried out as follows: one initial denaturation step at 94 °C for 3 min and 40 cycles of denaturation at 94 °C for 1 min, annealing at 55 °C for 1 min, and extension at 72 ° C for 1 min, with one final extension step at 72 °C for 7 min. To isolate the products of full-length self-assembly, 3 lL of the self-assembled DNA mixture was amplified using BTMA1, the 5¢-flanking primer (5¢-caccattctagaatgttgaaagaagccatt tatca-3¢), and BTMA2, the 3¢-flanking primer (5¢-gcgagag gaagcttccttctcgcctttta-3¢). The PCR was conducted using the conditions mentioned above. PCR products of full-length assembly were purified, digested with XbaI and HindIII, and ligated into p6xHTKXb119. Screening of mutants with enhanced thermostability The transformants were picked onto LB agar plates con- taining kanamycin (20 lgÆmL )1 ) and incubated at 37 °C for 12 h. The cells were then transferred onto nylon membranes (Hybond-N; Amersham Pharmacia Biotech, Uppsala, Swe- den), lysed, and fixed according to the method described by Song and Rhee [57]. The membrane was incubated under the appropriate conditions for each DNA shuffling round (specific screening conditions for each round are described in Results). The heat-treated membranes were placed on fresh LB plates that contained 1% (w ⁄ v) soluble starch (Showa Manufacturing Co., Fukuoka, Japan) and incuba- ted at 60 °C for 12 h. Finally, mutants showing larger clear zones than the parental enzyme when stained with an iod- ine solution (0.203 g I 2 and 5.2 g KI in 100 mL H 2 O) were selected. The mutations were verified by DNA sequencing using the BigDye terminator cycle sequencing kit for the ABI 377 Prism (Applied Biosystems, Foster City, CA, USA). Enzyme purification and activity assays Wild-type BTMA and various mutant proteins tagged with six-histidine residues were purified from E. coli harboring the corresponding genes on p6xHTKXb119 using a nickel– nitrilotriacetic acid (Ni–NTA) column (Qiagen Inc., Valen- cia, CA, USA) as described previously [2]. The purity of the enzyme was determined by SDS ⁄ PAGE [10%] as described by Laemmli [58]. The hydrolytic activities of wild-type and mutant enzymes were assayed in 0.5% (w ⁄ v) b-cyclodextrin (b-CD) (Sigma Chemical Co., St Louis, MO, USA) in 50 mm glycine–NaOH buffer (pH 8.0) according to the 3,5-dinitrosalicylic acid method using maltose as the standard [59]. One unit of enzyme activity was defined as the amount of enzyme that produced 1 lmol of maltose per minute. The protein concentration was measured using the Bradford method [60] with bovine serum albumin (Sigma Chemical Co.) as the standard. Analysis of the thermostability of enzymes The thermostability of enzymes was measured by determin- ing the half-life of thermal inactivation and the melting temperature (T m ). Enzyme samples at certain protein con- centrations were incubated in water baths at different tem- peratures (75, 78, 80, and 85 °C). Aliquots were taken at various time points and placed immediately in an ice bath. The residual b-CD hydrolyzing activities of the aliquots were measured at optimal reaction temperatures. The first- order rate constant, k d , of irreversible thermal denaturation was obtained from the slope of the plots of ln(residual activity) vs. time, and the half-life was calculated as ln2 ⁄ k d . To determine the T m of the mutant enzymes, DSC was performed with a VP-DSC MicroCalorimeter (MicroCal, Northampton, MA, USA). 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(1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar Anal Chem 31, 426– 428 60 Bradford MA (1976) Rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding Anal Biochem 72, 248–254 FEBS Journal 273 (2006) 3335–3345 ª 2006 The Authors Journal compilation ª 2006 FEBS 3345 . Enhancing thermostability of maltogenic amylase from Bacillus thermoalkalophilus ET2 by DNA shuffling Shuang-Yan Tang 1 , Quang-Tri. 2006) doi:10.1111/j.1742-4658.2006.05337.x DNA shuffling was used to improve the thermostability of maltogenic amylase from Bacillus thermoalkalophilus ET2. Two highly thermostable mutants,