Dimerization Mediates Thermo-Adaptation, Substrate Affinity and Transglycosylation in a Highly Thermostable Maltogenic Amylase of Geobacillus thermoleovorans

Background Maltogenic amylases belong to a subclass of cyclodextrin-hydrolyzing enzymes and hydrolyze cyclodextrins more efficiently than starch unlike typical α-amylases. Several bacterial malto-genic amylases with temperature optima of 40–60°C have been previously characterized. The thermo-adaption, substrate preferences and transglycosylation aspects of extremely thermostable bacterial maltogenic amylases have not yet been reported. Methodology/Principal Findings The recombinant monomeric and dimeric forms of maltogenic α-amylase (Gt-Mamy) of the extremely thermophilic bacterium Geobacillus thermoleovorans are of 72.5 and 145 kDa, which are active optimally at 80°C. Extreme thermostability of this enzyme has been explained by analyzing far-UV CD spectra. Dimerization increases T1/2 of Gt-Mamy from 8.2 h to 12.63 h at 90°C and mediates its enthalpy-driven conformational thermostabilization. Furthermore, dime-rization regulates preferential substrate binding of the enzyme. The substrate preference switching of Gt-Mamy upon dimerization has been confirmed from the substrate-binding affinities of the enzyme for various high and low molecular weight substrates. There is an alteration in Km and substrate hydrolysis efficiency (Vmax/Km) of the enzyme (for cyclodex-trins/starch) upon dimerization. N-terminal truncation indicated the role of N-terminal 128 amino acids in the thermostabilization and modulation of substrate-binding affinity. This has been confirmed by molecular docking of β-cyclodextrin to Gt-Mamy that indicated the requirement of homodimer formation by the interaction of a few N-terminal residues of chain A with the catalytic residues of (α/β)8 barrel of chain B and vice-versa for stable cyclodextrin binding. Site directed mutagenesis provided evidence for the role of N-terminal D109 at the dimeric interface in substrate affinity modulation and thermostabilization. The dimeric Gt-Mamy transglycosylates hydrolytic products of G4/G5 and acarbose, while the truncated form does not because of the lack of extra sugar-binding space formed due to dimerization. Conclusion/Significance N-terminal domain controls enthalpy-driven thermostabilization, substrate-binding affinity and transglycosylation activity of Gt-Mamy by homodimer formation.


Introduction
Cyclodextrins are the cyclic oligosaccharides of glucose linked via a-(1,4)-glycosidic linkages. Among carbohydrases that hydrolyze cyclodextrins are the maltogenic amylases (glucan-1,4-amaltohydrolase, EC 3.2.1.133), which belong to a subclass of cyclodextrin hydrolyzing enzymes along with neopullulanases and cyclomaltodextrinases in the family13 (a-amylase family) of glycosyl hydrolases. A few properties make them distinguishable from the typical a-amylases. Firstly, they hydrolyze cyclodextrins more efficiently than starch and produce maltose and glucose as major hydrolytic products. Secondly, they are catalytically versatile, that is, they hydrolyze a-(1,4)as well as a-(1,6)linkages of the substrate molecule and transglycosylate the hydrolytic products. Thirdly, they possess an additional 130 residues at the N-terminus that are absent in the typical a-amylases. Fourthly, they can hydrolyze acarbose, a potent inhibitor of a-amylases, to produce glucose and acarviosine-glucose [pseudotrisacc-haride]. Maltogenic amylases are, therefore, known as multifunctional amylases [1].
To date maltogenic amylases with temperature optima of 40-60uC have been reported from the bacteria [2][3][4][5][6]. Only a few extremely thermostable maltogenic amylases have been reported from archaea with temperature optima in the range of 70-100uC [7][8][9][10]. This investigation is the first thorough insight into an extremely thermostable maltogenic amylase (T opt 80uC) from the extremely thermophilic bacterium Geobacillus thermoleovorans. Higher thermostability of the dimeric maltogenic amylase of G. thermoleovorans (Gt-Mamy) than the known bacterial maltogenic amylases [2][3][4][5][6] encouraged us to investigate the role of dimerization as a thermo-adaptive mechanism. We have biochemically and biophysically characterized this enzyme and correlated dimeric state of Gt-Mamy to the substrate preference switch and transglycosylation activity by N-terminal truncation and site directed mutagenesis. To our knowledge, this is the first detailed investigation on the role of dimerization as a thermo-adaptive mechanism, regulation of substrate-preference switching and transglycosylation activity of a highly thermostable maltogenic amylase.

PCR amplification and sequence analysis of Gt-Mamy
Using the primers Mal_int_F/R, a ,300 bp amplicon was sequenced, which shares 88-93% identity with the a-cyclodextrinase of G. stearothermo-philus (GenBank ID AB070710.1), aamylase catalytic region of Geobacillus sp. Y412MC52 (GenBank ID CP002442.1), maltogenic amylase of Geobacillus sp. Gh6 (GenBank ID GQ884176.1) and maltogenic amylase of G. thermoleovorans (GenBank ID CP003125.1). Using the end regions of maltogenic amylase sequences and end regions of the region I (300 bp) sequence, external and internal primers were designed and PCR amplified and sequenced. All the sequences were overlapped for obtaining the full-length Gt-Mamy gene ( Figure S1). The sequence analysis revealed an ORF of 1767 bp (GenBank ID JQ999960). The in silico translated protein sequence (588 amino acids) has the predicted pI and molecular mass of 5.52 and 68.5 kDa, respectively.

Cloning and expression, and purification and characterization of the recombinant Gt-Mamy
When the 1767 bp Gt-Mamy gene was amplified and cloned into pCOLD TM I vector and pCOLD-gt-Mamy construct was transformed into E. coli BL21 (DE3), Gt-Mamy was expressed at 15uC using 0.5 mM IPTG. Figure 1 presents the over expression of Gt-Mamy on SDS-PAGE. The recombinant strain produces 25461.4 U of Gt-Mamy mg 21 total intracellular protein in the recombinant E. coli.
The purified protein forms one and two protein bands (72.5 and 145 kDa) on 12% SDS-PAGE under denatured and nondenatured conditions, respectively. The two monomeric and dimeric forms have been separated by the Sephacryl TM S-200 gel-filtration column, and their molecular masses have been confirmed to be 72.560.7 kDa and 14561.2 kDa, respectively ( Figure 1).
Recombinant Gt-Mamy is optimally active at 80uC, and it declined at 90 and 100uC (Figure 2a). The enzyme is active over a wide range of pH between 5 and 9 ( Figure 2b), but the activity drastically declined at pH 4 and 10. The recombinant Gt-Mamy is, therefore, extremely thermostable and pH stable. The pH stability profile of monomeric Gt-Mamy is shown in Figure 2c. There is no loss in the activity of the enzyme till 14 days of incubation at pH 7 and 8, but it decreased drastically after 8 days at pH 5 and 6. The high thermostability of Gt-Mamy over a wide pH range is an advantage for its applicability in starch saccharification, formation of prebiotics via transglycosylation, baking and detergent industries.
Among the metal ions tested, Co 2+ , Mn 2+ , Al 3+ and Na + stimulated the Gt-Mamy activity (Table 1), while K + , Ni 2+ , Ca 2+ and Ba 2+ did not exert any observable effect on the Gt-Mamy. The activity was slightly inhibited by Mg 2+ , while Cu 2+ and Hg 2+ strongly inhibited (Table 1). Strong inhibition by Cu 2+ and Hg 2+ indicates the role of thiol/carboxyl group and indole-containing amino acid residues, respectively, in the enzyme function and catalysis [12]. EGTA [ethylene glycol-bis(2-aminoethylether)-N,N,N9,N9-tetraacetic acid] and EDTA [ethylenedia-mine tetraacetic acid] had no observable effect on the activity, suggesting that the enzyme does not require Ca 2+ or any other metal ion for its activity (Table 1). Slight inhibition by diethylpyro-carbonate (DEPC) and Woodward's reagent K (WRK) indicated the role of histidine and acidic amino acids, respectively in the catalysis and/ or substrate binding [13,14]. Complete inhibition of Gt-Mamy activity by N-bromosuccinamide (NBS) signifies the possible role of tryptophan in maintaining the secondary structure [12].
Gt-Mamy displays less hydrolytic action on raw starches than on soluble starch and that of a-amylase Gt-amy [12]. The enzyme hydrolyzed soluble potato, corn, water chestnut, tapioca, rice, wheat, sago, oat and gram starches to a varied extent ( Table 2), but failed to hydrolyze pullulan. Other substrates such as acyclodextrins, b-cyclodextrins, amylose and amylopectin were hydrolyzed to a varied extent.
Maltogenic amylases are distinguished from the typical aamylases in the hydrolysis of cyclodextrins and acarbose. TLC analyses of the end-products ( Figure 3) showed that the enzyme liberates maltose as the major end product on hydrolyzing starch, amylose and amylopectin (Lanes 1, 2, and 3). The hydrolysis of a-, b-, and c-cyclodextrins led to the release of maltose and glucose as the major end-products, a property typical of maltogenic amylases [13]. Gt-Mamy hydrolyzes acarbose, a well-known inhibitor of aamylases, to glucose and pseu-dotrisaccharide (PTS), and G5/G4/ G3 to G2 and G1, suggesting that G3 is the smallest oligosaccharide that can be hydrolyzed by Gt-Mamy. Similar observations have been recorded for those from Thermophilus pendans and Thermoplasma volcanicum GSS1 [2,8].

Temperature and pH inactivation of Gt-Mamy
The thermal inactivation of Gt-Mamy follows first-order kinetics, and the inactivation rate constant (K d ) was calculated from the linear regression analysis. Thermal denaturation at elevated temperature is evident by the increase in K d values and a sharp decline in T 1/2 values with increase in temperature ( Table 3). The T 1/2 values of Gt-Mamy at 80, 90 and 100uC are 35.7, 8.2 and 3.12 h, respectively. The decrease in the T 1/2 value with increase in temperature occurs as a result of irreversible thermal inactivation of the enzyme due to heat exposure.
Dimeric Gt-Mamy exhibits a markedly higher thermostability, and hence, displays lower thermal inactivation as compared to the monomeric form ( Table 3). The T 1/2 values of the dimeric enzyme at 80, 90 and 100uC are 55.7, 12.63 and 3.58 h, respectively. High T 1/2 and lower K d values of dimeric Gt-Mamy in comparison with the monomeric enzyme indicates thermal stabilization as a result of conformational stabilization and lesser thermal inactivation. Higher thermostability of dimer has also been indicated by the higher E a value (energy required for the thermal denaturation process) for dimer (150.23 KJ mol 21 ) than the monomer (133.69 KJ mol 21 ).  As visualized from figure 4a, far-UV CD spectra display large CD bands with negative ellipticity at 208 and 222 nm, a distinct property of a-helix rich proteins. Deconvolution of the CD spectra for the secondary structure estimation revealed that the enzyme has a-helix, b-sheet and random coil contents of 67.4, 9.2 and 23.4%, respectively.
Thermal and pH stability of Gt-Mamy was also explained by studying far-UV CD spectra. The negative signal at 222 nm ( Figure 4b) remains unchanged in the pH range 6-10 at 40-80uC. There is a slight increase in the ellipticity values at 90 and 100uC, which indicate minor structural changes or shifts. As the deconvoluted CD spectra under these conditions do not show any major change in the a-helix and b-sheet content, this confirms the conformational stability of Gt-Mamy at high pH and temperatures. Sharp increase in the ellipticity values towards zero at pH values 4, 5 and 10, with rise in temperature to 90 and 100uC indicates structural transition of the a-helices to denatured state (Table 4, Figure 4b). The comparison of CD spectra at pH 4, 5 and 10 indicated that pH shift to extremes at lower temperatures (40 to 70uC) does not lead to a major alteration in the enzyme structure. Major structural transition of a-helix content of the enzyme begins as the temperature increases to 80uC, and further, at pH 4 (Table 4). At pH 10, this transition initiates at a much lower temperature. These results confirmed that neither pH nor temperature alone is sufficient for the alteration of its structural conformation. Simultaneous the presence of extreme pH and temperature is necessary for complete denaturation of the enzyme, which confirms its high thermostability as well as pH stability.

Thermo-adaptation of Gt-Mamy
The dimeric Gt-Mamy is more thermostable than the monomer. Moreover, the sequence analysis of Gt-Mamy suggested a high degree of similarity to the currently known bacterial cyclodextrin hydrolyzing enzymes (88-93%), but it is not clear as to why Gt-Mamy is extremely thermostable, while others are not. The thermodynamics of thermal inactivation of monomeric and dimeric forms of Gt-Mamy have, therefore, been studied by applying the first order kinetics to thermal inactivation data, to assess its thermo-adaptation. The energy required for the process is expressed as DH. Opening of the enzyme structure is accompanied by the increase in the disorder of the enzyme structure (DS). From the data in table 3, it is clear that there is an insignificant change in the values of DH and DS with increase in temperature, while DG increased. Positive DG values for the thermal inactivation of Gt-Mamy indicated non-spontaneity of the process of thermal inactivation, and the enzyme to be thermostable. The changes in DH and DS become significant when thermal inactivation is related to dimerization. As shown in table 3, values of DG, DH and DS increased upon dimerization.
As DH is a measure of enzyme inactivation, higher DH signifies greater numbers of non-covalent bonds that are present in the enzyme structure, which need to be broken for thermal inactivation. Increase in DH as a result of dimerization makes overall DG of the dimer greater than the monomer, making it more stable to thermal denaturation, and hence, greater thermal stability than its monomeric counterpart. Thermostability of an enzyme is a balance of stabilizing and destabilizing forces, which is affected by several factors like number of non-covalent bonds in the enzyme structure, overall hydrophobicity of the enzyme and others [15]. Dimerization causes slight increase in the DS of the Gt-Mamy. The slight increase in the DS is nullified by a large increase in DH as a result of dimerization. In summary, DH contributes to the thermostability of the enzyme upon dimerization, making this process enthalpy-driven.

Kinetics of substrate hydrolysis
Monomeric Gt-Mamy shows higher affinity towards starch as compared to cyclodextrins. Dimerization modulates the affinity of Note: Results are shown as mean 6 standard deviation with n = 6.  enzyme towards cyclodextrins as can be seen from the K m values, which decrease from 2.01 and 1.87 mg mL 21 (monomer) to 0.2 and 0.61 mg mL 21 (dimer) for aand b-cyclodextrin, respectively. The substrate hydrolysis efficiency can be calculated as V max /K m ratio [16]. There is 139-and 17-fold increase in the substrate hydrolysis efficiency of Gt-Mamy for aand b-cyclodextrins, respectively upon dimerization ( Table 5). The preference for oligomeric state to bind and hydrolyze cyclodextrins has been described for maltogenic amylases from Thermoplasma volcanicum GSS1 and Thermus sp. [8,17]. The hydrolysis efficiency for starch, amylose and amylopectin decreases by a factor of 63, 65 and 35, respectively (Table 5). Sequence comparison of Gt-Mamy and ThMA (1SMA) showed the presence of I431, a bulky amino acid, instead of V431 in ThMA [18] at the substrate entry site. This substitution might explain a decrease in the substrate-binding affinity, turnover   number (k cat ) as well as substrate hydrolysis efficiency (V max /K m ) of Gt-Mamy for starch, amylose and amylopectin upon dimerization. These observations clearly indicate that there is a switch in Gt-Mamy activity from starch hydrolysis to cyclodextrin hydrolysis upon dimerization.

N-terminal truncation of Gt-Mamy
The comparison of amino acid sequence of Gt-Mamy and Gt-amy (a-amylase) reveals the presence of an extra N-terminal 128 amino acids (N-domain) in Gt-Mamy. To determine the impact of N-terminus on the dimeric state of Gt-Mamy, the first 128 amino acids corresponding to the N-domain were deleted. The truncated Gt-Mamy is (designated as Gt-MamyT) was overexpressed and purified, with a molecular mass of ,59 kDa, which confirms that the truncation leads to monomerization of this enzyme. Similarly N-domain has been shown to play a role in the oligomerization of other cyclodextrin hydrolyzing enzymes [19].
The biochemical characterization of Gt-MamyT suggested that its temperature optima and pH optima are identical to those of Gt-Mamy. Modulators also exhibit similar effects on Gt-MamyT, as reported by Li et al. [1] for the multifunctional amylase of Bacillus sp. ZW25311. However, there is a 16-and 3.5-fold reduction in the affinity of Gt-MamyT to aand b-cyclodextrins (Table 6) as compared to the dimeric form, an effect similar to that of the monomer. There is also a decrease in aand b-cyclodextrin hydrolysis efficiency of Gt-MamyT by a factor of 232 and 19, respectively, to that of the native dimer. Decrease in the substratebinding affinity and substrate hydrolytic efficiency of Gt-MamyT towards aand b-cyclodextrins as compared to that of the native dimer also suggests a structural modification in the enzyme active site. This further suggests the role of N-domain in the modulation of substrate specificity of Gt-MamyT for cyclodextrins.
Indirect or direct influence of N-domain in the alteration of thermostability of Gt-MamyT has been elucidated by monitoring its thermal denaturation pattern. The truncated enzyme displays a decreased thermostability as compared to both monomeric and dimeric Gt-Mamy (Table 3), as evident from the lower T 1/2 and greater K d values at elevated temperatures. The decreased thermal stability also explains the lower E a value of Gt-MamyT. These observations further validate N-terminus to play a role in homodimer formation and thermosta-bilization of Gt-Mamy. When thermodynamic parameters of Gt-MamyT inactivation were calculated, a decrease in DH and DG values was recorded (Table 3); this explains the decreased thermostability of the truncated enzyme due to the loss of dimerization, which is an indirect effect like that of the monomeric Gt-Mamy.  Table 4. a-Helix, b-sheets and random coils content in the secondary structure of Gt-Mamy.  TVA II (Thermoactinomyces vulgaris amylase II) [21]. This narrow and deep groove ( Figure S3g,h) is particularly different from the wide and shallow active site cleft of other a-amylases and maltogenic amylase of Thermoactinomyces vulgaris.

Molecular docking of b-cyclodextrin to mal_amy model
The molecular docking of b-cyclodextrin to Gt-Mamy model was carried out to interpret the observations explained earlier, and to locate N-terminal amino acid residues responsible for binding to cyclodextrin, and modulating its substrate-binding affinity and thermostability via dimerization. The free energy of b-cyclodextrin binding to dimeric Gt-Mamy is more negative (24.51 kcal mol 21 ) than the monomeric Gt-Mamy (22.05 kcal mol 21 ). Furthermore, the frequency of docking runs resulting in the same geometry is more (80%) for dimer than the monomeric form (30%).
Higher negative free energy of binding and frequency, obtained after docking of b-cyclo-dextrin to dimeric Gt-Mamy than the monomer, indicates that the docking of b-cyclo-dextrin to dimeric Gt-Mamy doesn't require reorientation of the protein chains and change in the torsion angle of the b-cyclodextrin molecule, ensuring that binding of b-cyclodextrin to dimer is reliable and specific as compared to the monomer. Amino acid residues involved in the binding of b-cyc-lodextrin to the dimeric Gt-Mamy are Y45, W47, D109, T111 of chain A and H205, F289, V292, M295, D328, E357, T423, D424, I431 of chain B. While residues Y45 and W47 make hydrophobic interaction with the inner ring of b-cyclodextrin, T111 and E357 form polar interaction. Amino acid residues involved in the binding of b-cyclodextrin to the monomeric Gt-Mamy are Y45, W47, D110, T111 and A112.
The docking model explains that b-cyclodextrin interacts with some residues from the (a/b) 8 barrel of one chain and N-terminal domain of the second chain, and vice-versa to form two substrate binding clefts as a result of its dimerization. Catalytic residues D424, D328 and E357, which come in the proximity of bcyclodextrin via dimerization, form hydrogen bond, polar interactions and polar interactions with b-cyclodextrin, respectively. Consequently, it forms the deep and narrow cyclodextrinbinding grove into which b-cyclodextrin molecule fits through the stable interactions ( Figure S3d, figure S3e, figure 5). Increased affinity of the dimer to cyclode-xtrins results from the formation of a deep and narrow cyclodextrin-binding catalytic pocket in comparison with the wide and shallow active site in the monomer. This deep and narrow catalytic pocket permits easy access of flat and thin cyclodextrins [14.6/15.4 Å (a/b-cyclodextrin) high and 7.9 Å wide] with more stabilizing interactions, and thus, causes steric hindrance for the entry of coiled helices of amylose (12-15 Å wide), amylopectin (100 Å wide) and starch, as reported for a few cyclodextrin-hydrolyzing enzymes ( Figure 5) [17,22]. The wide and shallow active site in the monomer eases the entry of larger molecules of amylose and amylopectin and makes more stable interactions than cyclodextrins that fit loosely; this explains higher affinity of monomeric enzyme for starch, amylose and amylopectin. The preferential affinity of Gt-Mamy to cyclodextrins is, therefore, related to its structural arrangement, homo-dimer formation.

Characterization of D109 mutants
According to docking results, an N-terminal amino acid residue out of four that mediates binding to b-cyclodextrin is D109. In a dimeric Gt-Mamy, I431 of chain A interacts with D109 and G50 of chain B as shown in the figure 5, forming the substrate entry site. Site directed mutagenesis was, therefore, carried out at this position to understand the role of D109 on the substrate affinity and dimerization state of Gt-Mamy. The mutated forms of Gt-Mamy (D109E and D109A) were generated. SDS-PAGE as well as zymogram analysis of the D109E mutein revealed the occurrence of dimeric form. Moreover, D109E mutein does not show any effect on the binding affinity and substrate hydrolytic efficiency towards aand b-cyclodextrins (Table 6). A strong decline in the affinity and substrate hydrolytic efficiency of the mutant enzyme towards amylopectin has been observed, as evident from the K m value of 21.43 mg mL 21 and V max /K m value of 0.19, which are 1.9 and 7.3 times less than the dimeric wild-type Gt-Mamy, respectively. Site directed mutagenesis at the substrate entry site resulted in 2.3-fold decrease in the affinity of ThMA to amylopectin [18].
In contrast, D109A mutant shows increased affinity towards amylose, amylopectin and starch, and a decreased affinity towards aand b-cyclodextrins. As summarised in the Table 6, there is 2.5, 2.7 and 1.5 fold increase in its affinity towards starch, amylose and amylopectin, and 4.7 and 2 fold decrease in the affinity of mutant enzyme to aand b-cyclodextrins, respectively. Increased affinity of D109A towards amylose, amylopectin and starch, and a slight decreased affinity towards aand b-cyclodextrins, might have occurred as a consequence of destabilization effect of this mutation on the enzyme structure. Due to this destabilization effect, the mutant enzyme dissociates into the monomeric form and is present in monomer-dimer equilibrium in the solution, and therefore, K m values of this mutein to different substrates lie in between the values for monomer and dimer, and hence, the substrate binding affinities too.
D109E mutant enzyme shows insignificant changes in T 1/2 at the elevated temperatures, while D109A exhibits decreased T 1/2 values. The T 1/2 values of D109A at 80, 90 and 100uC are 44.6, 8.9 and 2.82 h, respectively. Decline in the T 1/2 values, with concomitant decrease in the E a required for its thermal denaturation to 141.4 KJ mol 21 , explains decreased thermostability as a result of partial loss of dimerization state of the enzyme  and its dissociation to the monomeric form. These results indicate the role of D109 in the dimerization of Gt-Mamy and its indirect role in the modulation of substrate preferences and thermostability.

Conclusions
Bacterial maltogenic amylase with high thermostability and broad pH range for activity is being reported for the first time. The enzyme is distinct from the a-amylase (Gt-amy) of G. thermoleovorans reported earlier. The maltogenic amylase has greater affinity for cyclodextrins as compared to starch, and further, hydrolyzes acarbose, an inhibitor of a-amylase. Gt-Mamy forms a homodimer and dimerization mediates enthalpy-driven conformational thermostabilization of the enzyme. N-terminal truncation has indicated that dimerization modulates substrate-binding affinity and transglycosylation activity of the enzyme. Molecular docking of b-cyclodextrin to Gt-Mamy and SDM aided in confirming the role of N-terminal D109 at the substrate entry site in dimer formation.

Materials
Escherichia coli DH5a and E. coli BL21 (DE3) (Novagen) were used as the host strains for cloning and expression of maltogenic amylase gene from Geobacillus thermoleovorans (MTCC 4220, 16S rDNA sequence accession no. JN871595). Vector pCOLD TM I was procured from Takara Bio Inc., Japan. Primer oligonucleotides and Ni 2+ -NTA agarose resin were purchased from Sigma (St Louis, USA) and Novagen, respectively.

DNA manipulation
Genomic DNA was extracted according to Bazzicalupo and Fani [23]. A set of internal oligonucleotide primers (Mal_int_F/R, table 7) were designed from the conserved regions of the maltogenic amylases of the known bacterial species. Approximately 50 ng of genomic DNA was used as a template for the amplification of this internal region of the Gt-Mamy in a Thermocycler (Bio-Rad, USA) in 50 ml reaction mixture (initial denaturation at 95uC for 5 min followed by 30 cycles of denaturation at 95uC for 50 sec, annealing at 59uC for 30 sec, and extension at 72uC for 1 min, and a final extension of 5 min at 72uC) using Taq DNA polymerase (New England Biolabs, MA). This amplicon was cloned into pGEMH-T easy vector and sequenced at Nucleic Acid Sequencing Facility, University of Delhi South Campus, New Delhi. This amplicon shares significant homology with the a-cyclodextrinase of G. stearothermophilus (GenBank ID AB070710.1), a-amylase catalytic region of Geobacillus sp. Y412MC52 (GenBank ID CP002442.1) and maltogenic amylase of Geobacillus sp. Gh6 (GQ884176.1).
The end primers (Mal_GT_F/R, table 7) were designed from the maltogenic amylase sequences and the full-length Gt-Mamy was amplified in a 50 ml reaction mixture (initial denaturation at 95uC for 5 min followed by 30 cycles of denaturation at 95uC for 50 sec, annealing at 64uC for 30 sec and extension at 72uC for 1 min 30 sec, and a final extension of 5 min at 72uC) using Taq DNA polymerase and sequenced by automated sequencing at Nucleic Acid Sequencing Facility at University of Delhi South Campus, New Delhi. The compatible restriction enzyme sites of SacI and XhoI were created at the end of the Mal.GT.F/R to create Mal_GT_Sa_F/Xh_R ( Table 7). The digested PCR amplicon and pCOLD TM I vector were purified using Qiagen PCR purification kit, ligated and transformed into E. coli DH5a competent cells. Positive clones were confirmed by colony PCR and double digestion of the construct by SacI and XhoI, and were sequenced.

Bioinformatic analysis and homology modeling of Gt-Mamy
Multiple sequence alignment (MSA) was carried out using ClustalW program [24]. The template selection for the homology model was carried out based on the analysis of scores, E-values and percent identities of the gapped-BLAST generated sequences that produced significant alignment with the in silico translated sequence of Gt-Mamy. Automated homology modelling was carried out using the template using SWISS-MODEL [25], and the model was visualized by the DeepView -Swiss-PdbViewer 4.1.0 [26].
Molecular docking of b-cyclodextrin to Gt-Mamy model b-cyclodextrin ligand (CID 444041, SID 57280122) was downloaded from the PubChem Compound Database at NCBI (National Center for Biotechnology Information). Docking calculations were carried out using Docking Server [27]. Gasteiger partial charges were added to the ligand atoms. Non-polar hydrogen atoms were merged, and rotatable bonds were defined. Docking calculations were carried out on b-cyclodextrin Mal_amy model. Essential hydrogen atoms, Kollman united atom type charges, and solvation parameters were added with the aid of AutoDock tools [28]. Affinity (grid) maps of 20620620 Å grid points and 0.375 Å spacing were generated using the Autogrid program [28]. AutoDock parameter set-and distance-dependent dielectric functions were used in the calculation of the van der Waals and the electrostatic terms, respectively.
Docking simulations were performed using the Lamarckian genetic algorithm (LGA) and the Solis & Wets local search method [29]. Initial position, orientation, and torsions of the ligand molecules were set randomly. Each docking experiment was derived from 10 different runs that were set to terminate after a maximum of 250000 energy evaluations. The population size was set to 150. During the search, a translational step of 0.2 Å , and quaternion and torsion steps of 5 were applied.

N-terminal truncation of Gt-Mamy
To truncate the Gt-Mamy gene from the N-terminus, primer Trn_amy_F (59 CCCGAGCTCCC-GGACTGGGTAAAAGA-CAC 39) was designed deleting the 128 amino acids from the Nterminus region. This forward primer and Mal_GT_Xh_R was used to amplify the truncated Gt-Mamy gene (designated as Gt-MamyT). This amplicon was digested with SacI and XhoI and cloned in the pCOLD TM I vector as mentioned previously.

Site-Directed Mutagenesis (SDM)
PCR-based SDM was carried out using the GENEART site directed mutagenesis kit (Invitrogen, Carsband, USA). A set of overlapping oligonucleotide primer pairs with the desired mutations (Table 7) were designed to generate the mutant constructs for D109A and D109E mutants. Approximately 30-40 ng of recombinant vector was used as a template for the amplification of the  Expression of the recombinant Gt-Mamy and its mutants, and their purification Plasmid construct was transformed into E. coli BL21 (DE3), and the transformed cells were cultured at 37uC for 16-18 h in LB medium supplemented with ampicillin (100 mg mL 21 ). The expression of Gt-Mamy gene under the control of cspA promoter was performed as described by Qing et al. [30]. The whole-cell protein profiles of the induced and uninduced cultures were analysed on 12% SDS-PAGE. The gel was run at 35 mA constant current till the dye front reaches the end. The recombinant enzyme was purified using Ni 2+ -NTA affinity chromatography under non-denaturing conditions at pH 7.0. Elution was carried out with 150 mM imidazole and analysed on SDS-PAGE as described by Laemmli [31]. Active fractions were dialyzed against 100 mM sodium phosphate buffer (pH 7) and stored at 4uC. The protein concentration was determined according to Lowry et al. [32]. Zymogram analysis was carried out using SDS-PAGE as described earlier [12].
Monomeric and dimeric forms of the purified Gt-Mamy were separated by FPLC (AKTAPrime plus). The Sephacryl TM S-200 high resolution column (Pharmacia) was pre-equilibrated with sodium phosphate buffer (100 mM, pH 7) containing 50 mM NaCl and the purified enzyme containing monomeric and dimeric forms were loaded, and eluted at a flow rate of 0.3 ml min 21 . Cytochrome c (12.4 kDa), carbonic anhydrase (29 kDa), bovine serum albumin (66 kDa), yeast alcohol dehydrogenase (150 kDa), and sweet potato bamylase (200 kDa) were used as standards to calculate the molecular mass of the purified enzymes. Molecular mass of the monomeric and dimeric forms of Gt-Mamy were determined using a plot of molecular weight Vs Ve/Vo.

Amylase assay
To 0.5 mL appropriately diluted enzyme (1.5 U), 0.5 mL 0.5% potato starch or b-cyclodextrin (Sigma, St. Louis, MO) prepared in 100 mM sodium phosphate buffer (pH 7.0) were added, mixed and incubated at 80uC for 10 min. The reducing sugars liberated were determined using 3, 5-dinitrosalicylic acid (DNSA) reagent [33]. One unit of a-amylase is defined as the amount of enzyme that liberates 1 mmol of reducing sugars as maltose per min under the assay conditions.

Biochemical characterization and enzyme kinetics
The optimum pH for the activity of pure recombinant enzyme was determined by incubating the reaction mixtures at 80uC in buffers of varying pH. The optimum temperature for the enzyme activity was determined by incubating the reaction mixtures (pH 7.0) at 40uC-100uC. The reducing sugars liberated were quantified. Biochemical characterization, endproduct analysis and substrate spectrum analysis of Gt-Mamy were carried out at pH 7.0 and 80uC [12]. Soluble potato starch (Sigma, St. Louis, USA) solutions of varying concentrations of starch (0.1%-1.0%) and diluted enzyme (1.5 U) were prepared in 100 mM sodium phosphate buffer (pH 7.0). The enzyme activities determined at 80uC were used in calculating K m , V max and k cat .

Kinetics and thermodynamics parameters of enzyme inactivation
Thermal stability was assessed by incubating the enzyme (pH 7.0) at different temperatures (80uC-100uC) followed by assay at the desired intervals. The residual enzyme activities were determined at each temperature. This data was fit into the firstorder kinetics and K d (deactivation rate constant), half-life (T 1/2 ) and energy required for thermal inactivation (E a ) of the Gt-Mamy inactivation were calculated [12].
Thermodynamic parameters of thermal inactivation of the Gt-Mamy were calculated by rearranging the Eyring's absolute rate equation from the transition state theory.
Eyring's absolute rate equation, Effect of temperature and pH on the secondary structure of Gt-Mamy The secondary structure of purified Gt-Mamy (0.25 mg mL 21 ) was determined by CD spectroscopy in a JASCO-815 Spectropolarimeter equipped with in-built Peltier controlled thermostat cell holder (CDF-423S). Nitrogen was flushed continuously through the instrument at the rate of 5.0 ml min 21 , and the path length of cuvette used was 10 mm. The effect of temperature and pH on Gt-Mamy was investigated by performing the scans at different temperatures (30 to 100uC) and different pH (4)(5)(6)(7)(8)(9)(10)(11). The changes in the structural conformation were recorded in the wavelength range 200 to 240 nm at a scanning rate of 50 nm min 21 . Each CD spectrum is an average of six scans, and the data acquisition and analysis were performed on a computer interfaced to the instrument. Data analysis was done by K 2 D 2 software.

Transglycosylation of acceptor molecules by Gt-Mamy and its mutants
Applicability of Gt-Mamy and Gt-MamyT was studied in the transglycosylation of sugars to various acceptor molecules. 5 mg each of the donor and acceptor sugars were mixed in a 50 ml reaction and incubated at 70uC for 1 h. After 1 h, 5.0 U of the purified enzyme was added to the mixture and the incubation was continued for 24 h. The products were analyzed by thin layer chromatography [12].

Sequence accession numbers
The G. thermoleovorans gt-Mamy sequence was submitted to the GenBank database and assigned accession no. JQ999960. The protein ID of the amino acid sequence is AFM43699. Figure S1 Schematic representation of sequencing of full-length gt-Mamy gene by primer walking. Full-length gene was sequenced in three parts; Region I was sequenced first and then extended towards both the directions for the sequencing of regions II and III; forward and reverse primers are shown by forward and backward arrows.