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Applied and Environmental Microbiology, August 2008, p. 5183-5194, Vol. 74, No. 16
0099-2240/08/$08.00+0     doi:10.1128/AEM.00181-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Isomaltose Production by Modification of the Fructose-Binding Site on the Basis of the Predicted Structure of Sucrose Isomerase from "Protaminobacter rubrum" ^,

Hyeon Cheol Lee,^1* Jin Ha Kim,¹ Sang Yong Kim,¹ and Jung Kul Lee²

BioNgene Co., Ltd., 10-1, 1 Ka, Myungryun-Dong, Jongro-Ku, Seoul 110-521,¹ Department of Chemical Engineering, Konkuk University, Seoul 143-701, Republic of Korea²

Received 20 January 2008/ Accepted 7 June 2008

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"Protaminobacter rubrum" sucrose isomerase (SI) catalyzes the isomerization of sucrose to isomaltulose and trehalulose. SI catalyzes the hydrolysis of the glycosidic bond with retention of the anomeric configuration via a mechanism that involves a covalent glycosyl enzyme intermediate. It possesses a ³²⁵RLDRD³²⁹ motif, which is highly conserved and plays an important role in fructose binding. The predicted three-dimensional active-site structure of SI was superimposed on and compared with those of other -glucosidases in family 13. We identified two Arg residues that may play important roles in SI-substrate binding with weak ionic strength. Mutations at Arg³²⁵ and Arg³²⁸ in the fructose-binding site reduced isomaltulose production and slightly increased trehalulose production. In addition, the perturbed interactions between the mutated residues and fructose at the fructose-binding site seemed to have altered the binding affinity of the site, where glucose could now bind and be utilized as a second substrate for isomaltose production. From eight mutant enzymes designed based on structural analysis, the R³²⁵Q mutant enzyme exhibiting high relative activity for isomaltose production was selected. We recorded 40.0% relative activity at 15% (wt/vol) additive glucose with no temperature shift; the maximum isomaltose concentration and production yield were 57.9 g liter^–1 and 0.55 g of isomaltose/g of sucrose, respectively. Furthermore, isomaltose production increased with temperature but decreased at a temperature of >35°C. Maximum isomaltose production (75.7 g liter^–1) was recorded at 35°C, and its yield for the consumed sucrose was 0.61 g g^–1 with the addition of 15% (wt/vol) glucose. The relative activity for isomaltose production increased progressively with temperature and reached 45.9% under the same conditions.

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Isomaltose (6-O--D-glucopyranosyl-D-glucose) is a disaccharide that comprises an -1,6-glycosidic bond between two glucose molecules. Its caloric value is less than 50% that of sucrose, and its viscosity is lower than that of maltose (8). Further, its properties are similar to those of other well-known low-cariogenic sugar alcohols or sucrose isomers, since much less acid and glucan are formed by Streptococcus mutans than sucrose. Isomaltose is a member of the isomalto-oligosaccharide group, whose members contain at least one -1,6-glycosidic linkage, such as pannose, isomaltotriose, isomaltotetraose, nigerose, and isopanose. Isomalto-oligosaccharides are known to improve the general well-being of humans and animals when ingested orally on a daily basis. Isomaltose has potential applications, not only in the food industry, such as in confections, processed fruits and vegetables, and canned and bottled food, but also as an ingredient in animal feed, cosmetics, and medicines. It is generated as a by-product during chemical and enzymatic reactions that use liquefied starch or a glucose-containing solution as a reactant. However, it has not yet been possible to obtain a disaccharide-enriched product by industrial processes (8).

Sucrose isomerase (SI) is widely used in industries for the production of isomaltulose and trehalulose (4, 5, 9, 18). The bacteria known to produce these sugar isomers include Serratia plymuthica, Erwinia rhapontici, Klebsiella planticola, Pseudomonas mesoacidophila, "Protaminobacter rubrum," Pantoea dispersa, and Enterobacter species (5, 9, 13, 15). In addition to its function in the isomerization of sucrose to isomaltulose and trehalulose, SI produces small amounts of glucose and fructose as by-products. P. rubrum CBS 547.77, S. plymuthica NCIB 8285, and E. rhapontici NCPPB 1578 primarily produce isomaltulose (75 to 85%), whereas P. mesoacidophila MX45 and Agrobacterium radiobacter MX-232 mainly produce trehalulose (90%) (5, 9, 13, 15, 17). The ratios of these products vary among bacterial strains.

SI produced by P. rubrum is a member of the -amylase family and has two functions, the hydrolysis of sucrose at the -1,2 bond and the separate formation of an -1,6 bond for isomaltulose and an -1,1 bond for trehalulose. SI produced by P. rubrum comprises 628 amino acids, and its molecular mass is 69.8 kDa. SI produced by P. rubrum exhibits 70.9% and 80.0% similarity with those produced by E. rhapontici and Klebsiella sp. strain LX3, respectively, in terms of the amino acid sequence (3, 29). Because of its substantial differences in sequence and enzymatic properties, different names are used to distinguish SI genes in various organisms: smuA for P. rubrum, palI for Erwinia-Klebsiella-Enterobacter, sim for P. dispersa, and mutB for P. mesoacidophila sucrose-trehalulose isomerase (1, 9, 30). All SIs that have been sequenced thus far exhibit similar secondary and tertiary structures, having an N-terminal triose phosphate isomerase barrel (β/)₈.

Recently, SI-encoding genes were isolated from E. rhapontici, P. rubrum, Klebsiella sp. strain LX3, and P. dispersa, and molecular-level studies that predicted the enzyme structures by sequence alignment and X-ray crystallography have been conducted (2, 26, 28). The SI produced by P. rubrum belongs to the group of -glucosidases, which includes many important digestive enzymes from E. rhapontici and Klebsiella sp. strain LX3 (1, 6, 7, 14, 19, 20). These enzymes catalyze the hydrolysis of the glycosidic bond while retaining the anomeric configuration via a mechanism that usually involves a covalent glycosyl-enzyme intermediate. Also, they contain a potential catalytic triad of amino acid residues (Asp²⁴¹, Glu²⁹⁵ and Asp³⁶⁹), two histidine residues (His¹⁴⁵ and His³⁶⁸), and a fructosyl moiety-binding motif (³²⁵RLDRD³²⁹), all of which are highly conserved (2, 3, 10, 12, 13, 24, 28).

A unique RLDRD motif in proximity to the active site was identified and was shown to be responsible for sucrose isomerization (21, 24, 27, 28). A two-step reaction mechanism for hydrolysis and isomerization, which occur in the same pocket, is proposed on the basis of both structural and biochemical data (24, 27). An identical sequence is also found in the peptide sequences of SIs from E. rhapontici, Enterobacter sp. strain SZ62, and Klebsiella sp. strain LX3 (2, 29). On the other hand, the SI from P. mesoacidophila MX-45, which is known to produce more than 90% trehalulose and a small amount of isomaltulose, contains a different corresponding sequence (³¹¹RYDRA³¹⁵), and the SI from P. dispersa contains a still another corresponding sequence (³²⁴RLDRY³²⁸) (15, 16). According to the proposed reaction mechanism of SI, the fructosyl moiety is cleaved from sucrose and then is rearranged into isomaltulose (23, 27). Further, glucose and fructose are produced as by-products and were reported to act as competitive inhibitors for SI under standard conditions (24).

In this study, we performed secondary-structure analysis by using sequence alignment tools with known SIs and glucosidase family enzymes. A reasonable SI three-dimensional (3D) structure was determined from sequence alignment data using modeling and simulation programs. Arg³²⁵ and Arg³²⁸ in the fructose-binding site (FBS) of SI were located at the interface of the fructosyl moiety and were thus considered to be easily able to interact with O-6 of fructose via H bonds. Therefore, we focused on these two Arg residues for isomaltose production and investigated the changes in the reaction mechanism and the ratio of the products formed using mutant enzymes obtained by site-directed mutagenesis. Finally, the relationship between the enzyme activity and the transition state energy [(G)] was studied on the basis of the predicted FBS 3D structures.

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Materials.
The restriction endonucleases SacI and HindIII, T4 DNA ligase, and calf intestinal alkaline phosphatase were obtained from Takara (Kyoto, Japan). Pfu DNA polymerase was obtained from Roche Molecular Biomedicals (Basel, Switzerland). The expression vector pQE80L was obtained from Qiagen (Valencia, CA) and the cloning vector pSTBlue-1 from Novagen (Madison, WI). Electrophoresis grade agarose was purchased from Intron (Seoul, Korea). Plasmids and PCR products were purified by using DNA minipreparation kits (Qiagen, Valencia, CA). Escherichia coli BL21(DE3)(pLysS) (Invitrogen, Groningen, The Netherlands) was used for protein expression, and E. coli XL1-Blue (Stratagene, La Jolla, CA) was used during cloning procedures. P. rubrum CBS 547.77 was obtained from the Centraalbureau voor Schimmelcultures.

Standard sugars (sucrose, glucose, fructose, and isomaltose) for high-performance liquid chromatography (HPLC) were purchased from Sigma (St. Louis, MO). Isomaltulose and trehalulose were obtained from Südzucker Co. Ltd (Obrigheim/Pfalz, Germany).

Expression of the mutated SI genes in E. coli. (i) Cloning of smuA into expression vector pQE80L.
Based on the sequences of P. rubrum CBS 547.77 SI (GenBank accession number CQ765963), PCR primers were designed for subcloning the SI genes (without noncoding regions and signal sequences) into expression vector pQE80L. The forward primer included a SacI restriction site and a start codon. The reverse primer included a HindIII restriction site and a stop codon. The following primers were used; forward primer, 5'-GGG AGC TCA TGC CCC GTC AAG GA-3', and reverse primer, 5'-GGA AGC TTC TAT TTT GCG CTA AAA AAA C-3'.

PCR was performed with a PCR GeneAmp 2400 (Perkin-Elmer, Boston, MA) for 1 min at 95°C, followed by 30 cycles of 30 s at 95°C, 30 s at 60°C, 1 min at 72°C, and a 5-min final step at 72°C in a total volume of 50 µl, using 20 ng of template DNA and 2.5 U of Taq DNA polymerase. The PCR product was directly cloned into pSTBlue-1 (pSTBlue::PrSI). The expression vector pQE::PrSI was constructed by joining pQE80L and the P. rubrum SI gene (smuA) cloned from pSTBlue::PrSI.

(ii) Site-directed mutagenesis of smuA.
Site-directed mutagenesis of smuA was performed using the QuikChange site-directed mutagenesis kit (Stratagene), with pQE::PrSI as a template. Each desired amino acid replacement was generated by using two synthetic oligonucleotide primers. After 12 amplification cycles (95°C for 30 s, 55°C for 1 min, and 68°C for 12 min) with Pfu Turbo DNA polymerase (Stratagene), the PCR products were treated with 1 U DpnI, and then the nicked plasmid DNA with the desired mutation was transformed into competent cells of E. coli BL21(DE3)(pLysS). The sequences of the mutated SIs were confirmed by DNA sequencing.

(iii) Expression of SmuA in E. coli.
Five cultures per construct were set up in 5 ml of LB medium with 50 µg ml^–1 kanamycin in 30-ml flasks. The cells were grown at 37°C with shaking at 250 rpm. Three cultures per construct (at an optical density at 600 nm of approximately 1.0) were chosen for induction. IPTG (isopropyl-β-D-thiogalactopyranoside) was added to a final concentration of 0.5 mM with a 3-h incubation time at 30°C. After the incubation, an appropriate volume of culture was used for protein quantification, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and quantification of the efficiency of conversion from sucrose to isomaltulose, trehalulose, and isomaltose. For SI protein purification, the culture volume was increased to 250 ml in a 1-liter flask.

(iv) Molecular modeling and analysis.
The protein sequences of SIs (GenBank accession numbers CQ765963, AF279281, AY040843, AY223549, AY223550, AY227804, and AAP57083) and glucosidases (GenBank accession numbers A45860, AF279283, and AF279285) were aligned by using ClustalW (http://www.ebi.ac.uk/Tools/clustalw2/index.html). The most similar enzymes with known crystal structures (Protein Data Bank 1M53, 1UOK, and 1JGI) were aligned initially by using VAST Search (http://www.ncbi.nlm.nih.gov/Structure/VAST/vastsearch.html), and then the alignments were viewed in Cn3D (http://www.ncbi.nih.gov/Structure/CN3D/cn3d.shtml).

Secondary-structure predictions for P. rubrum SI were obtained by using the BMERC protein sequence analysis server (http://bmerc-www.bu.edu/psa/) with minimal assumptions. 3D structure predictions were obtained by using ESyPred3D (http://www.fundp.ac.be/urbm/bioinfo/esypred/) with the obtained alignment data and Protein Data Bank template 1M53 (SI from Klebsiella sp. strain LX3; 2.2-Å resolution with an R factor of 19.4%). (The standard crystallographic R factor is a measure of how well the refined structure predicts the observed data.)

The root mean square deviation (RMSD) values were calculated for all atoms of the protein backbone for the entire predicted SI structure, in order to characterize the amount by which a given selection of predicted molecules deviates from a defined position in space. NAMD 2.6 (http://www.ks.uiuc.edu/Research/namd/) input files for RMSD analysis were prepared with the VMD program (http://www.ks.uiuc.edu/Research/vmd/). The VMD program was also used for viewing the simulation results. The NAMD output files from minimization and equilibration of SI in a water sphere were used in order to calculate RMSD values and to analyze the extent of equilibration of the simulation. Each mutant model was predicted from a reasonable refined SI model using the VMD mutator plug-in.

The PROCHECK program (http://swissmodel.expasy.org/workspace/index.php) was used to assess the stereochemical quality (2.5-Å resolution) of the predicted protein structure. The superimposition of enzyme active-site structure was performed by ProSup analysis (11).

Enzyme extraction and purification.
Cells were harvested by centrifugation (3,000 x g; 4°C; 10 min) and washed with 50 mM Tris (pH 8.0)/2 mM EDTA three times. The cell pellets were resuspended in extraction buffer (20 mM Tris [pH 7.4], 200 mM NaCl, 1 mM EDTA, 1 mM azide, 10 mM β-mercaptoethanol) and then lysed by sonication (six 30-s pulses at 50 W with a Sonoplus sonifier [Bandelin Electronic, Berlin, Germany]).

The pQE80L vector introduced an N-terminal six-His tag into expressed proteins, and the proteins were purified by adsorption to nickel-nitrilotriacetic acid agarose (Qiagen) and by elution with 25 mM NaH₂PO₄, 150 mM NaCl, 125 mM imidazole buffer (pH 8.0) following the manufacturer's instructions. The purity of SI proteins was determined by SDS-PAGE. A batch procedure in which nickel-nitrilotriacetic acid agarose in suspension was used yielded target proteins predominantly (95%). Unless otherwise specified, this was the form of the purified SI enzymes used for biochemical characterization. The protein concentration was determined by the bicinchoninic acid method using bovine serum albumin (Sigma) as the standard. The reducing sugar was determined by the dinitrosalicylic acid method using isomaltulose (Sigma) as the standard.

Determination of SI activities. (i) Assay of SI enzyme activity.
The main activity of SI is the conversion of sucrose into isomaltulose. Enzyme activities were measured by incubating 200 µl of 25 µg ml^–1 of the purified enzyme with 50 µl of 20 mM calcium acetate buffer (pH 5.5) containing 5% (wt/vol) sucrose at 25°C for 5 min with gentle agitation. The quantitation of individual sugars formed from sucrose was analyzed by HPLC (Waters 2690; Waters Co., Milford, MA) using a carbohydrate column (4.6 by 250 mm; Waters Co.) and a refractive-index detector (Waters 2410; Waters Co.) (15, 22). Samples were eluted isocratically with 80% (vol/vol) acetonitrile at a flow rate of 1 ml min^–1. The unit activity is defined as the amount of enzyme that can convert 1 µmole sucrose per min at the initial stage under the standard assay conditions. The data presented are the means of three individual experiments. Data were analyzed by using double-reciprocal plots to calculate k_cat/K_m values, and (G) values were calculated from the k_cat/K_m values.

(ii) Total sucrose conversion.
The conversion of sucrose was performed in an Eppendorf tube containing 250 µl of 20% (wt/vol) sucrose solution and 5 µg of enzyme at 25°C in a shaking water bath until the sucrose could not be converted into any other products (approximately 12 h). Quantitative analysis of individual sugars formed from sucrose was performed by HPLC. The amount of each sugar produced for consumed sucrose in a steady state of reaction is represented.

(iii) Effects of SI activities in the absence or presence of additive glucose.
SI activities for sucrose conversion were measured by incubating the purified enzyme in 20% (wt/vol) sucrose solution with different glucose concentrations (2.5, 5, 10, 15, 20, 25, and 30% [wt/vol]) under standard assay conditions.

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Predicted secondary and 3D structures of P. rubrum SI.
The sequence determined for SI obtained from P. rubrum revealed that the SI gene is substantially different from those of other species and contains a 1,884-bp open reading frame encoding 628 amino acids, including a 51-amino-acid signal sequence (see GenBank accession number CQ765963). For convenience, we designated the structural element domains (the N-terminal domain [N], subdomain [S], and C-terminal domain [C]) and features (β-sheets and -helices). The features within domains were numbered beginning at the N terminus, and the loops were assigned the same number as the preceding β-sheet. Sequence alignment with glucosidases revealed that all the SIs have N1-8 helices and Nβ1-8 sheets, collectively termed the barrel (β/)₈, and the catalytic-triad residues (Asp²⁴¹, Glu²⁹⁵, and Asp³⁶⁹) at the Nβ4, Nβ4, and Nβ7 sheet positions (Fig. 1A). The additive Pcβ8 and Pc1 at the C-terminal end of the SI of P. rubrum were not found in other SIs and were expected to contribute to substrate stabilization in the active pocket. The FBS (³²⁵RLDRD³²⁹) in SI is located between the Nβ6 sheet and the N6 helix.

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FIG. 1. Predicted secondary structure and 3D structure of the P. rubrum SI. (A) Comparison of amino acid sequences among SIs produced by P. rubrum, E. rhapontici and Klebsiella sp. strain LX3. S1 and S2 and Sβ1 to Sβ4, subdomain -helix and β-sheet; N1 to N8 and Nβ1 to Nβ8, N-terminal domain (β/)8 structure; Cβ1 to Cβ7, C-terminal domain β-sheets; Pc1 and Pcβ1, the estimated C-terminal -helix and β-sheet of P. rubrum SI; Prub, P. rubrum SI; Erha, E. rhapontici SI; Klx3, Klebsiella sp. strain LX3 SI; and Pdis, P. dispersa SI. Black backgrounds indicate active site residues, asterisks indicate well-conserved residues, colons indicate similarly conserved residues, and periods indicate partially conserved residues. (B) For the 3D structure, the same numbering and coding are used. The core of the triose phosphate isomerase barrel is located in the center of the 3D structure. Prominent -helices and β-sheets are numbered by domain from the N terminus.

Structural P. rubrum SI models obtained by energy minimization suggest the relative positions of -helices and β-sheets in the 3D structure of the proteins (Fig. 1B). The P. rubrum SI amino acid sequence shares 80% identity with the major template (1M53; 2.2 Å) submitted to ESyPred3D. The predicted model was simulated in an explicit water environment for 420 ps in an effort to refine the structure and to establish the stability of the model in general. The RMSD from the initial model-built structure is displayed in Fig. 2. The overall deviation increased with time for the first 40 ps and then remained relatively constant, indicating that the structure was not changing in a systematic manner. The time history for the RMSD reached a maximum deviation of approximately 1.5 Å after 40 ps. This is reasonable for a model-built structure and suggested that the system had converged to a stable structure, or at least a stable local minimum, which was close to the starting structure.

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FIG. 2. RMSD time history obtained from the molecular-dynamics simulation. RMSD values were calculated for all atoms of the protein backbone for the entire predicted SI structure.

An analysis of the Ramachandran plot (/ values for each residue) was performed by using the PROCHECK program. Analysis of the predicted SI structures indicated that the percentages of residues in the most favored regions, additional allowed regions, generously allowed regions, and disallowed regions were 81.6%, 15.6%, 1.4%, and 1.4%, respectively (see the supplemental material). The score expressing how well the backbone conformations of all residues were corresponding to the populated areas in the Ramachandran plot was within expected ranges for a well-refined structure (Ramachandran Z score, –0.334), and the standard deviation of values agreed with this expectation, around 5.5 (standard deviation of values, 5.783). The score of how well the -1/-2 angles of all residues corresponded to the populated areas in the database was within expected ranges for well-refined structures (-1/-2 correlation Z score, –0.315). The distribution of residue types over the inside and the outside of the protein was normal (inside/outside root mean square Z score, 1.062).

The predicted 3D model of P. rubrum SI seemed to share conserved barrel (β/)₈ domains for sucrose-binding and glycosidase activities with all other SIs and glucosidases. A unique FBS (³²⁵RLDRD³²⁹) motif that is in proximity to the active site is also well conserved in the predicted 3D model of P. rubrum SI. This motif is considered to be responsible for sucrose isomerization, similar to other SIs. The structural features of the FBS were considered to contribute to the positioning and binding of the fructose unit for isomerization after the hydrolysis of sucrose. Thus, the modification of its residues near the substrate pocket of the SI structure had high probability of changing the substrate preference or different product specificities of SI.

Analysis of the SI active-site structure.
In order to investigate the structural features of the FBS, Bacillus cereus oligo-1,6-glucosidase, P. dispersa SI, and P. rubrum SI were superimposed on the amylosucrase-sucrose complex from Neisseria polysaccharea based on the atoms of the five conserved residues (³²⁵RLDRD³²⁹) determined by ProSup analysis (Fig. 3). Comparing the FBS structures revealed that Arg³²⁵ and Arg³²⁸ of P. rubrum SI are located in close proximity to the fructosyl moiety of sucrose and thus may be easily able to interact with O-6 of fructose via H bonds. These two residues are positively charged and possess a relatively large side chain. Our model of the binding-site structure suggested that the function of two Arg residues is to stabilize the enzyme-sucrose complex. It seems that these two Arg residues are well conserved in an atypical SI from P. mesoacidophila MX-45, which possesses a different corresponding sequence (³¹¹RYDRA³¹⁵) in the FBS and produces more than 90% trehalulose and a small amount of isomaltulose. Together, the data indicated that these two Arg residues may play an important role in stabilizing the enzyme-sucrose complex until the reaction is terminated.

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FIG. 3. Superimposition of SIs and oligo-1,6-glucosidase onto the amylosucrase-sucrose complex based on the atoms of the five conserved active-site residues (11, 14, 25, 26). Based on this model of the binding-site structure, the residues that are involved directly in the hydrolysis of the glycosidic bond, i.e., His145, Asp241, Glu295, His368, and Asp369, were observed to be highly conserved in -glucosidase family 13. In contrast, two Arg residues in the FBS were unique to SIs and were observed to be closely associated with the stability of the enzyme-sucrose intermediate complex.

Changes in the FBS structures of mutated SIs.
The two positively charged Arg residues of the ³²⁵RLDRD³²⁹ motif in the SI were replaced with negatively charged Asp (R³²⁵D and R³²⁸D), neutral Gln (R³²⁵Q and R³²⁸Q), positively charged Lys (R³²⁵K and R³²⁸K), or hydrophobic Ala (R³²⁵A and R³²⁸A) residues. His-tagged SI (302IU) had activity similar to that of mature SI (299IU) in an activity assay with total cell lysate. The level of expression did not appear to be affected by point mutations, as shown in Fig. 4.

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FIG. 4. SDS-PAGE analysis of the purified His-tagged SI and its derivatives. For convenience of purification, the expression of SI with a His tag was performed. His-tagged SI showed the same activity as mature SI in an activity assay with total cell lysate, and the expression level appeared not to be affected by point mutations.

Changes in the (G) were determined based on the kinetic parameters of the eight generated mutant enzymes (Table 1). We investigated the relationship between (G) and the FBS structure (³²⁵RLDRD³²⁹) by analyzing the predicted active-site structures of the mutant SIs. In order to investigate the interactions between the substrate and each amino acid residue that had been mutated in the model of P. rubrum SI, the distance between each residue and the fructosyl moiety was calculated from the predicted model using molecular-dynamic simulations (Fig. 5). In the wild-type SI, the average distance between Arg³²⁵ and O-6 in the predicted model was estimated to be approximately 2.5 Å. As expected, the catalytic efficiency (7.7 mM^–1 s^–1) of the R³²⁵D mutant enzyme was reduced and the (G) (12.7 kJ mol^–1) was increased. As shown in Fig. 5A, the interference of the H bond and the repulsion between the Asp residue and the substrate seemed to be important reasons for a decrease in the sucrose-binding affinity and in the stability of the enzyme-sucrose complex. In the R³²⁵A and R³²⁵Q mutant enzymes, the average distances between the mutated amino acid residue in the FBS and the fructosyl moiety were increased 2.4- and 3.3-fold, respectively. Compared to the wild-type SI, R³²⁵A and R³²⁵Q exhibited lower catalytic efficiencies (68.4 and 125.0 mM^–1 s^–1, respectively) and higher (G) values (7.3 and 5.8 kJ mol^–1, respectively), probably due to the weakened H bonds caused by increases in the distances between atoms (6.0 and 8.5 Å, respectively). However, the R³²⁵A and R³²⁵Q mutant enzymes had less reduced affinities for sucrose than the R³²⁵D mutant enzyme due to the charge-charge repulsion between the mutated amino acid residues and sucrose. In the R³²⁵K mutant enzyme with a lower (G) than the others (4.7 kJ mol^–1), some degree of interaction was maintained between Arg³²⁵ and the fructose moiety within 3.8 Å via a weakened H bond. It is possible that R³²⁵K may interact with fructose via H bonds in a manner similar to that of wild-type SI. However, the catalytic efficiency for isomaltulose of R³²⁵K (193.8 mM^–1 s^–1) was considerably lower than that of wild-type SI (1,301 mM^–1 s^–1). As shown in Table 1, charge-charge interaction and maintenance of the H bond at the Arg³²⁵ position were key players in influencing the stability of the transition state and the catalytic efficiency.

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TABLE 1. Kinetic parameters and (G)s for mutant SIs

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FIG. 5. Changes in the predicted FBS structures of mutated SIs. Each model was predicted from a reasonable refined SI model by the VMD mutator plug-in. The black dotted lines represent the average distances between the fructose moiety and each functional group with attraction, and the red dotted lines represent the average distances between the fructose moiety and each functional group with charge repulsion. Each inset graph shows the distance profile obtained by molecular dynamic simulations for 220 ps. (A) Average distances between atoms in the Arg325 residues and the sucrose O-6 atom. (B) Average distances between atoms in Arg328 residues and the sucrose O-6 atom.

Although the Arg³²⁸ mutation decreased the stability of the enzyme-sucrose complex, all the Arg³²⁸ mutant enzymes produced some amount of isomaltulose and trehalulose. This indicates that Arg³²⁸ may influence the stability of the enzyme-sucrose complex of SI less than Arg³²⁵ does. Removal of the H bond in R³²⁸A and R³²⁸K caused slight perturbations in the enzyme structure, but these changes were small in comparison to the other structural changes (Fig. 5B). As expected, these mutants had higher catalytic efficiencies (210.3 and 130.1 mM^–1 s^–1, respectively) and lower (G) values (4.5 and 5.7 kJ mol^–1, respectively) than R³²⁸D (catalytic efficiency, 25.1 mM^–1 s^–1; (G) value, 9.8 kJ mol^–1). In the cases of R³²⁸D and R³²⁸Q, relatively high (G) values (9.8 and 8.2 kJ mol^–1, respectively) and low catalytic-efficiency values (25.1 and 46.7 mM^–1 s^–1, respectively) were recorded, which might have been due to charge repulsion arising at relatively close proximity to the units involved (3.3 and 4.6 Å, respectively) (Table 1). Even though our model of the predicted FBS based on charge repulsion and the H bond alone cannot explain why the catalytic efficiency of R³²⁸K was lower and its (G) value was higher than those of R³²⁸A, it is possible that the effect of the Arg³²⁸ mutation is due to some steric hindrance in the active pocket.

Isomaltose production by mutant enzymes having a modified FBS.
Although significant catalytic activity was lost when a mutation was introduced into the catalytic-triad residues (Asp²⁴¹, Glu²⁹⁵, and Asp³⁶⁹) and glucose moiety-binding residues (His¹⁴⁵ and His³⁶⁸) of SI (data not shown), the replacement of the two Arg residues altered the ratio of product species formed instead of completely abolishing the isomerization activity, with a reduction of sucrose consumption as revealed by a total sucrose assay (Table 2).

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TABLE 2. Sugar compositions of reaction mixtures of the purified enzymes in the absence and the presence of 5% (wt/vol) glucosea

Table 2 shows the conversion of sucrose into isomaltulose, trehalulose, isomaltose, glucose, and fructose in the absence or in the presence of glucose. Both monosaccharides were produced in the same concentration during the initial stage of the reaction; however, the glucose concentration became lower than the fructose concentration as the incubation time increased. The deviation increased as isomaltose production rose.

As shown in Table 2 (in the absence of glucose), the Arg³²⁵ mutation resulted in a significant decrease in isomaltulose production and an increase in trehalulose production and the release of monosaccharides. Further, isomaltose, which was not produced by the wild-type enzyme, was produced by R³²⁵A, R³²⁵Q, R³²⁵K, and R³²⁵D. Among these mutant enzymes, the main function of SI, i.e., isomaltulose conversion, was more severely inhibited in R³²⁵D, where Arg³²⁵ was replaced with a negatively charged Asp, than in R³²⁵K, where Arg³²⁵ was replaced with a positively charged Lys.

In the case of the Arg³²⁸ mutation, more isomaltulose was produced and fewer monosaccharides were released than with the Arg³²⁵ mutation, although the amount of isomaltulose production was significantly low compared to that of the wild-type SI. Isomaltose was produced when Arg³²⁸ was replaced with a neutral Ala or with a positively charged Lys, but not with a negatively charged Asp or with a partially negatively charged Gln (Table 2).

These results suggested that Arg³²⁵ is not directly involved in the hydrolytic activity of the enzyme but rather plays a more important role than Arg³²⁸ in the appropriate isomerization of sucrose to isomaltulose and trehalulose by stabilizing the intermediate binding. This finding was consistent with the structural analysis (Fig. 5 and Table 1). Under these circumstances, we consider that the ratio of products generated by a mutated SI varies depending on its ability to form a stable complex with a fructose unit in the transition state, where the glycosidic bond of sucrose is hydrolyzed for the isomerization of sucrose to isomaltulose or trehalulose, and on its ability to stably accept a glucose unit.

In order to demonstrate that the isomaltose production by each mutant enzyme depends on the presence of glucose in the reaction mixture, we performed the same reaction described above but with an initial glucose concentration of 5% (wt/vol). The results showed an increase in the amount of isomaltose production, although the yields of isomaltulose and trehalulose in the presence of 5% (wt/vol) glucose were similar to or even slightly less than in absence of glucose (Table 2).

We also investigated the kinetic mechanism of the isomaltose production by R³²⁵Q using initial velocity measurements in which the concentrations of both substrates were varied systematically and the results were analyzed assuming steady-state conditions. In this experiment, the concentration of sucrose varied in the presence of several different fixed concentrations of glucose. When the data were plotted in double-reciprocal form as a function of the sucrose concentration, a series of near-parallel lines was obtained (Fig. 6).

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FIG. 6. Steady-state kinetic analysis of R325Q catalysis with various sucrose concentrations. The concentrations of glucose were 1% (•), 5% (), 10% (), and 15% () (wt/vol), respectively. The inset is a replot of the intercepts versus the reciprocals of the corresponding glucose concentrations.

Based on a sucrose conversion assay and steady-state kinetics results, the schematic reaction mechanism of R³²⁵Q was suggested (Fig. 7). The isomerization of sucrose to isomaltulose and trehalulose occurred at the enzyme-sucrose complex stage; however, for the conversion of sucrose to isomaltose, free glucose in the reaction mixture was reused following the liberation of fructose by the enzyme-glucose complex. Together, the data suggested that this mechanism of transfer product formation is possibly a ping-pong bi-bi system, where the enzyme binds a first substrate (sucrose), releases a first product (fructose), and produces a second enzyme form (enzyme-glucose complex), which binds a second substrate (H₂O and glucose) and finally releases a second product (glucose and isomaltose).

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FIG. 7. Proposed reaction mechanism scheme for R325Q SI. Sucrose bound to the wild-type enzyme is converted into isomaltulose or trehalulose (shaded panel); sucrose bound to R325Q is degraded to glucose and fructose, and then free glucose in the reaction mixture is reused following the liberation of fructose by the enzyme-glucose complex (unshaded panel).

In order to investigate the effect of the glucose concentration on the activity of the purified R³²⁵Q mutant enzyme, which exhibited a relatively high activity for isomaltose production (9.3% with sucrose alone and 20.0% with sucrose containing 5% [wt/vol] glucose), 0 to 30% (wt/vol) glucose was added to the reaction mixture under the standard conditions (Fig. 8). With wild-type SI, sucrose was almost completely converted to isomaltulose; the isomaltose content in the reaction mixture increased to 13.0 g liter^–1 by using up 184 g liter^–1 sucrose, and the isomaltose yield for the consumed sucrose was 0.07 g of isomaltose g of sucrose^–1 under conditions of 30% (wt/vol) glucose (Fig. 8A). With the R³²⁵Q mutant enzyme, the sucrose consumption was slightly decreased compared to that of the wild-type SI, the isomaltose content in the reaction mixture increased to 57.9 g liter^–1 by using up 105 g liter^–1 sucrose, and the isomaltose yield for the consumed sucrose was 0.55 g of isomaltose g of sucrose^–1 under conditions of 15% (wt/vol) glucose. The relative activities were 22.1% for each monosaccharide, 9.1% for isomaltulose, 6.8% for trehalulose, and 40.0% for isomaltose. The production and the yield of isomaltose reached a plateau at 15% of the glucose concentration, showing that a maximum of 29 g liter^–1 free glucose can be reused by the R³²⁵Q mutant enzyme as a secondary substrate and then be converted to isomaltose (Fig. 8B).

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FIG. 8. Effects of the glucose concentration on isomaltose production in wild-type SI (A) and R325Q mutant SI (B). Sucrose, ; fructose, •; isomaltulose, ; trehalulose, ; isomaltose, . The conversion of sucrose was performed in 250 µl of reaction mixture containing 20% (wt/vol) sucrose, 5 µg of enzyme, and 0 to 30% (wt/vol) glucose at 25°C in a shaking water bath for 12 h.

Effect of temperature on isomaltose production by R³²⁵Q mutant enzymes.
Isomaltose can be produced efficiently by the R³²⁵Q mutant enzyme in the absence of a temperature shift (Fig. 8). Because temperature is an important factor related to the binding force between the substrate and the enzyme, we investigated the influence of temperature on isomaltose production in R³²⁵Q and determined the optimum temperature for the production of each sugar (Fig. 9). The increase in fructose produced meant an increase in enzyme-glucose intermediate produced, where glucose can be introduced in order to produce isomaltose. Under conditions of 15% (wt/vol) glucose, the release of fructose from the enzyme-glucose-fructose complex was increased by raising the temperature, thereby increasing isomaltose production. Although the production of fructose reached a saturation point at 25°C, the production of isomaltose increased up to 35°C, showing a maximum production of 75.7 g liter^–1 and a maximum yield of 0.61 g of isomaltose g of sucrose^–1. The relative activity for isomaltose production increased steadily with the temperature and reached 45.9% at 35°C. The production of isomaltose was decreased at 45°C due to the increase of glucose, which was released from an enzyme-glucose intermediate, even though the production of fructose was not decreased. R³²⁵Q was inactivated at 55°C.

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FIG. 9. Effects of temperature on isomaltose production in R325Q mutant SI. Sucrose, ; fructose, •; isomaltulose, ; trehalulose, ; isomaltose, . The conversion of sucrose was performed in 250 µl of reaction mixture containing 20% (wt/vol) sucrose, 5 µg of enzyme, and 15% (wt/vol) glucose at 15 to 45°C in a shaking water bath for 12 h.

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The finding that the mutations in two Arg residues led to the instability of the enzyme-sucrose complex could be explained by the increase in the H bond distance and by charge repulsion, which is consistent with the (G) value. Generally, the effects of mutations were dependent on the charge interactions between binding substrates and the substrate binding site of the enzyme.

Differences in the reaction kinetics and specificity of SI most probably reflect differences in its FBS (10, 23, 24, 26). In addition to glucose, used in this study, fructose is reported to increase the ratio of trehalulose production by SI, but it does not inhibit the activity of the purified forms of SI extracted from S. plymuthica, P. rubrum, and E. rhapontici (16, 17, 26). In contrast, fructose acts as a competitive inhibitor for the SIs from P. dispersa UQ68J and K. planticola UQ14S, implying that the binding of fructose to the active sites of these enzymes is stronger, although trehalulose production via the direct condensation of glucose to fructose is impossible. Further comparisons of the kinetics of these SIs in the presence of different monosaccharides, along with mutagenesis studies to test the functional significance of apparent differences in key amino acids, are likely to be informative.

In this study, the interactions between the amino acid residues in the FBS of P. rubrum SI and the substrate were investigated at the molecular level. We demonstrated a method for altering the ratio of the products of SI by introducing an environmental change around the bound substrate. We speculate that it is possible to reconstruct enzymes having different reaction mechanisms for use in isomaltose production by introducing mutations in the FBS residues in association with the environment around the bound substrate.

First, we predicted the 3D structure of the FBS by using a 3D modeling program to identify residues important for the adsorption and degradation of sucrose. We identified two positions that are critical for the fructose-binding ability of SI extract from P. rubrum. Further, 4 amino acid residues were introduced at these two positions, i.e., at Arg³²⁵ and Arg³²⁸. Among the five conserved amino acid residues, the two Arg residues were observed to be closely associated with the stability of the enzyme-sucrose intermediate complex. These findings indicated that the modification of these two Arg residues could alter the binding strength between the FBS of SI and the fructosyl moiety of sucrose. Further, modification of the FBS altered the ratio of products generated by the mutant enzymes. Our results revealed that the ratio of products formed by the mutant enzymes depends on the sucrose-binding abilities of the enzymes and that Arg³²⁵ was mainly involved in the interaction between the enzyme and the fructosyl moiety of sucrose. On the other hand, free glucose acted as a secondary substrate and was introduced with greater ease into the vacant pocket of the mutant enzyme leaving fructose than into that of the wild-type enzyme, thus finally producing isomaltose.

The relative activity for isomaltose production was 40.0% in the reaction with the purified R³²⁵Q mutant enzyme in the presence of 15% (wt/vol) glucose at 25°C and 9.1% in that with R³²⁵Q in the absence of glucose at 25°C. Further, the isomaltose yield was 0.55 g of isomaltose g of sucrose^–1 with 15% additive glucose. Véronèse et al. reported that a relative activity of 4.5% for isomaltose production was recorded by using purified S. plymuthica SI in the presence of 277 mM glucose (5% [wt/vol]) at 30°C (24). Further, they demonstrated that this intermolecular reaction was enhanced at high temperatures. In the case of wild-type SI extracted from P. rubrum, 2.9% relative activity for isomaltose production was recorded in the presence of 5% (wt/vol) glucose at 25°C. The relative activity of P. rubrum SI was similar to or slightly lower than those of SIs extracted from other sources, including S. plymuthica SI (3, 9, 17, 24, 28). Since most SIs are similar in structure and function, regions that are conserved in this class of enzymes and that are associated with the recognition of the fructosyl moiety of the substrate could be considered as candidates for the development of isomaltose-producing enzymes for use in industries.

His-tagged SI purified from E. coli was used in these experiments instead of SI purified from P. rubrum. Although the biochemical data were similar, the possible structural difference between wild-type SI and His-tagged SI remained and was not negligible. However, despite this limitation, our results suggest that the stability of fructose in the FBS is essential for sucrose isomerization and that direct condensation can be induced by modifications to 5 amino acids present in the FBS, by a temperature shift, or by a high environmental glucose concentration. Based on previous studies and the present one, engineering of the FBS of SI appears to have great potential for enhancing isomaltose production. In addition to the two Arg residues, modification of other binding residues could be considered for decreasing the threshold glucose saturation concentration for isomaltose production and thus maintaining the isomaltose productivity of SI under conditions of less than 10% (wt/vol) additive glucose.

Further, it may be possible to design a continuous mixed-enzyme reactor containing glucose isomerase that can be used for the conversion of fructose to glucose, if glucose isomerase with a high glucose/fructose ratio can be utilized and a threshold glucose saturation concentration for isomaltose production can be maintained.

 
* Corresponding author. Mailing address: BioNgene Co., Ltd., 10-1, 1 Ka, Myungryun-Dong, Jongro-Ku, Seoul 110-521, Republic of Korea. Phone: 82-2-747-9796. Fax: 82-2-747-0750. E-mail: churry{at}biongene.com

Published ahead of print on 13 June 2008.

Supplemental material for this article may be found at http://aem.asm.org/.

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Applied and Environmental Microbiology, August 2008, p. 5183-5194, Vol. 74, No. 16
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