Isomaltose Production by Modiﬁcation of the Fructose ... · Recently, SI-encoding genes were isolated from E. rhapon- tici, P. rubrum, Klebsiella sp. strain LX3, and P. dispersa,

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 2008, p. 5183–5194 Vol. 74, No. 160099-2240/08/$08.00�0 doi:10.1128/AEM.00181-08Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Isomaltose Production by Modification of the Fructose-Binding Site onthe 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 and Department of Chemical Engineering,Konkuk University, Seoul 143-701,2 Republic of Korea

Received 20 January 2008/Accepted 7 June 2008

“Protaminobacter rubrum” sucrose isomerase (SI) catalyzes the isomerization of sucrose to isomaltulose andtrehalulose. SI catalyzes the hydrolysis of the glycosidic bond with retention of the anomeric configuration viaa mechanism that involves a covalent glycosyl enzyme intermediate. It possesses a 325RLDRD329 motif, whichis 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. Weidentified 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 slightlyincreased trehalulose production. In addition, the perturbed interactions between the mutated residues andfructose at the fructose-binding site seemed to have altered the binding affinity of the site, where glucose couldnow bind and be utilized as a second substrate for isomaltose production. From eight mutant enzymes designedbased on structural analysis, the R325Q mutant enzyme exhibiting high relative activity for isomaltose pro-duction was selected. We recorded 40.0% relative activity at 15% (wt/vol) additive glucose with no temperatureshift; the maximum isomaltose concentration and production yield were 57.9 g liter�1 and 0.55 g of isomalt-ose/g of sucrose, respectively. Furthermore, isomaltose production increased with temperature but decreasedat a temperature of >35°C. Maximum isomaltose production (75.7 g liter�1) was recorded at 35°C, and its yieldfor the consumed sucrose was 0.61 g g�1 with the addition of 15% (wt/vol) glucose. The relative activity forisomaltose production increased progressively with temperature and reached 45.9% under the same conditions.

Isomaltose (6-O-�-D-glucopyranosyl-D-glucose) is a disac-charide that comprises an �-1,6-glycosidic bond between twoglucose molecules. Its caloric value is less than 50% that ofsucrose, and its viscosity is lower than that of maltose (8).Further, its properties are similar to those of other well-knownlow-cariogenic sugar alcohols or sucrose isomers, since muchless acid and glucan are formed by Streptococcus mutans thansucrose. Isomaltose is a member of the isomalto-oligosaccha-ride group, whose members contain at least one �-1,6-glyco-sidic linkage, such as pannose, isomaltotriose, isomalto-tetraose, nigerose, and isopanose. Isomalto-oligosaccharidesare known to improve the general well-being of humans andanimals when ingested orally on a daily basis. Isomaltose haspotential applications, not only in the food industry, such as inconfections, processed fruits and vegetables, and canned andbottled food, but also as an ingredient in animal feed, cosmet-ics, and medicines. It is generated as a by-product duringchemical and enzymatic reactions that use liquefied starch or aglucose-containing solution as a reactant. However, it has notyet been possible to obtain a disaccharide-enriched product byindustrial processes (8).

Sucrose isomerase (SI) is widely used in industries for the

production of isomaltulose and trehalulose (4, 5, 9, 18). Thebacteria known to produce these sugar isomers include Serratiaplymuthica, Erwinia rhapontici, Klebsiella planticola, Pseudomo-nas mesoacidophila, “Protaminobacter rubrum,” Pantoea dis-persa, and Enterobacter species (5, 9, 13, 15). In addition to itsfunction in the isomerization of sucrose to isomaltulose andtrehalulose, SI produces small amounts of glucose and fructoseas by-products. P. rubrum CBS 547.77, S. plymuthica NCIB8285, and E. rhapontici NCPPB 1578 primarily produce iso-maltulose (75 to 85%), whereas P. mesoacidophila MX45 andAgrobacterium radiobacter MX-232 mainly produce trehalulose(90%) (5, 9, 13, 15, 17). The ratios of these products varyamong bacterial strains.

SI produced by P. rubrum is a member of the �-amylasefamily and has two functions, the hydrolysis of sucrose at the�-1,2 bond and the separate formation of an �-1,6 bond forisomaltulose and an �-1,1 bond for trehalulose. SI produced byP. rubrum comprises 628 amino acids, and its molecular mass is69.8 kDa. SI produced by P. rubrum exhibits 70.9% and 80.0%similarity with those produced by E. rhapontici and Klebsiellasp. strain LX3, respectively, in terms of the amino acid se-quence (3, 29). Because of its substantial differences in se-quence and enzymatic properties, different names are used todistinguish SI genes in various organisms: smuA for P. rubrum,palI for Erwinia-Klebsiella-Enterobacter, sim for P. dispersa, andmutB for P. mesoacidophila sucrose-trehalulose isomerase (1,9, 30). All SIs that have been sequenced thus far exhibit similarsecondary and tertiary structures, having an N-terminal triosephosphate isomerase barrel (�/�)8.

* 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: [email protected].

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

� Published ahead of print on 13 June 2008.

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Recently, SI-encoding genes were isolated from E. rhapon-tici, P. rubrum, Klebsiella sp. strain LX3, and P. dispersa, andmolecular-level studies that predicted the enzyme structures bysequence alignment and X-ray crystallography have been con-ducted (2, 26, 28). The SI produced by P. rubrum belongs to thegroup of �-glucosidases, which includes many important diges-tive enzymes from E. rhapontici and Klebsiella sp. strain LX3(1, 6, 7, 14, 19, 20). These enzymes catalyze the hydrolysis ofthe glycosidic bond while retaining the anomeric configurationvia a mechanism that usually involves a covalent glycosyl-en-zyme intermediate. Also, they contain a potential catalytictriad of amino acid residues (Asp241, Glu295 and Asp369), twohistidine residues (His145 and His368), and a fructosyl moiety-binding motif (325RLDRD329), all of which are highly con-served (2, 3, 10, 12, 13, 24, 28).

A unique RLDRD motif in proximity to the active site wasidentified and was shown to be responsible for sucrose isomer-ization (21, 24, 27, 28). A two-step reaction mechanism forhydrolysis and isomerization, which occur in the same pocket,is proposed on the basis of both structural and biochemicaldata (24, 27). An identical sequence is also found in the pep-tide sequences of SIs from E. rhapontici, Enterobacter sp. strainSZ62, and Klebsiella sp. strain LX3 (2, 29). On the other hand,the SI from P. mesoacidophila MX-45, which is known toproduce more than 90% trehalulose and a small amount ofisomaltulose, contains a different corresponding sequence(311RYDRA315), and the SI from P. dispersa contains a stillanother corresponding sequence (324RLDRY328) (15, 16). Ac-cording to the proposed reaction mechanism of SI, the fructo-syl moiety is cleaved from sucrose and then is rearranged intoisomaltulose (23, 27). Further, glucose and fructose are pro-duced as by-products and were reported to act as competitiveinhibitors for SI under standard conditions (24).

In this study, we performed secondary-structure analysis byusing sequence alignment tools with known SIs and glucosi-dase family enzymes. A reasonable SI three-dimensional (3D)structure was determined from sequence alignment data usingmodeling and simulation programs. Arg325 and Arg328 in thefructose-binding site (FBS) of SI were located at the interfaceof the fructosyl moiety and were thus considered to be easilyable to interact with O-6 of fructose via H bonds. Therefore,we focused on these two Arg residues for isomaltose produc-tion and investigated the changes in the reaction mechanismand the ratio of the products formed using mutant enzymesobtained by site-directed mutagenesis. Finally, the relationshipbetween the enzyme activity and the transition state energy[�(�G)] was studied on the basis of the predicted FBS 3Dstructures.

MATERIALS AND METHODS

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). Plas-mids and PCR products were purified by using DNA minipreparation kits (Qia-gen, Valencia, CA). Escherichia coli BL21(DE3)(pLysS) (Invitrogen, Groningen,The Netherlands) was used for protein expression, and E. coli XL1-Blue (Strat-agene, La Jolla, CA) was used during cloning procedures. P. rubrum CBS 547.77was obtained from the Centraalbureau voor Schimmelcultures.

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

Expression of the mutated SI genes in E. coli. (i) Cloning of smuA intoexpression vector pQE80L. Based on the sequences of P. rubrum CBS 547.77 SI(GenBank accession number CQ765963), PCR primers were designed for sub-cloning the SI genes (without noncoding regions and signal sequences) intoexpression vector pQE80L. The forward primer included a SacI restriction siteand a start codon. The reverse primer included a HindIII restriction site and astop codon. The following primers were used; forward primer, 5�-GGG AGCTCA TGC CCC GTC AAG GA-3�, and reverse primer, 5�-GGA AGC TTCTAT 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 at72°C, and a 5-min final step at 72°C in a total volume of 50 �l, using 20 ng oftemplate DNA and 2.5 U of Taq DNA polymerase. The PCR product wasdirectly cloned into pSTBlue-1 (pSTBlue::PrSI). The expression vectorpQE::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 wasperformed using the QuikChange site-directed mutagenesis kit (Stratagene),with pQE::PrSI as a template. Each desired amino acid replacement was gen-erated by using two synthetic oligonucleotide primers. After 12 amplificationcycles (95°C for 30 s, 55°C for 1 min, and 68°C for 12 min) with Pfu Turbo DNApolymerase (Stratagene), the PCR products were treated with 1 U DpnI, andthen the nicked plasmid DNA with the desired mutation was transformed intocompetent cells of E. coli BL21(DE3)(pLysS). The sequences of the mutated SIswere confirmed by DNA sequencing.

(iii) Expression of SmuA in E. coli. Five cultures per construct were set up in5 ml of LB medium with 50 �g ml�1 kanamycin in 30-ml flasks. The cells weregrown at 37°C with shaking at 250 rpm. Three cultures per construct (at anoptical density at 600 nm of approximately 1.0) were chosen for induction. IPTG(isopropyl-�-D-thiogalactopyranoside) was added to a final concentration of 0.5mM with a 3-h incubation time at 30°C. After the incubation, an appropriatevolume of culture was used for protein quantification, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and quantification of the effi-ciency of conversion from sucrose to isomaltulose, trehalulose, and isomaltose.For SI protein purification, the culture volume was increased to 250 ml in a1-liter flask.

(iv) Molecular modeling and analysis. The protein sequences of SIs (GenBankaccession numbers CQ765963, AF279281, AY040843, AY223549, AY223550,AY227804, and AAP57083) and glucosidases (GenBank accession numbersA45860, AF279283, and AF279285) were aligned by using ClustalW (http://www.ebi.ac.uk/Tools/clustalw2/index.html). The most similar enzymes with knowncrystal structures (Protein Data Bank 1M53, 1UOK, and 1JGI) were alignedinitially 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 theBMERC protein sequence analysis server (http://bmerc-www.bu.edu/psa/) withminimal assumptions. 3D structure predictions were obtained by usingESyPred3D (http://www.fundp.ac.be/urbm/bioinfo/esypred/) with the obtainedalignment 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 crystal-lographic R factor is a measure of how well the refined structure predicts theobserved data.)

The root mean square deviation (RMSD) values were calculated for all atomsof the protein backbone for the entire predicted SI structure, in order to char-acterize the amount by which a given selection of predicted molecules deviatesfrom 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 forviewing the simulation results. The NAMD output files from minimization andequilibration of SI in a water sphere were used in order to calculate RMSDvalues and to analyze the extent of equilibration of the simulation. Each mutantmodel was predicted from a reasonable refined SI model using the VMD mu-tator plug-in.

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

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Enzyme extraction and purification. Cells were harvested by centrifugation(3,000 � g; 4°C; 10 min) and washed with 50 mM Tris (pH 8.0)/2 mM EDTAthree 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 �-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 expressedproteins, and the proteins were purified by adsorption to nickel-nitrilotriaceticacid agarose (Qiagen) and by elution with 25 mM NaH2PO4, 150 mM NaCl, 125mM imidazole buffer (pH 8.0) following the manufacturer’s instructions. Thepurity of SI proteins was determined by SDS-PAGE. A batch procedure in whichnickel-nitrilotriacetic acid agarose in suspension was used yielded target proteinspredominantly (95%). Unless otherwise specified, this was the form of thepurified SI enzymes used for biochemical characterization. The protein concen-tration was determined by the bicinchoninic acid method using bovine serumalbumin (Sigma) as the standard. The reducing sugar was determined by thedinitrosalicylic acid method using isomaltulose (Sigma) as the standard.

Determination of SI activities. (i) Assay of SI enzyme activity. The mainactivity of SI is the conversion of sucrose into isomaltulose. Enzyme activitieswere measured by incubating 200 �l of 25 �g ml�1 of the purified enzyme with50 �l of 20 mM calcium acetate buffer (pH 5.5) containing 5% (wt/vol) sucroseat 25°C for 5 min with gentle agitation. The quantitation of individual sugarsformed 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 isocrati-cally with 80% (vol/vol) acetonitrile at a flow rate of 1 ml min�1. The unit activityis defined as the amount of enzyme that can convert 1 �mole sucrose per min atthe initial stage under the standard assay conditions. The data presented are themeans of three individual experiments. Data were analyzed by using double-reciprocal plots to calculate kcat/Km values, and �(�G) values were calculatedfrom the kcat/Km values.

(ii) Total sucrose conversion. The conversion of sucrose was performed in anEppendorf tube containing 250 �l of 20% (wt/vol) sucrose solution and 5 �g ofenzyme at 25°C in a shaking water bath until the sucrose could not be convertedinto any other products (approximately 12 h). Quantitative analysis of individualsugars formed from sucrose was performed by HPLC. The amount of each sugarproduced for consumed sucrose in a steady state of reaction is represented.

(iii) Effects of SI activities in the absence or presence of additive glucose. SIactivities for sucrose conversion were measured by incubating the purified en-zyme 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.

RESULTS

Predicted secondary and 3D structures of P. rubrum SI. Thesequence determined for SI obtained from P. rubrum revealedthat the SI gene is substantially different from those of otherspecies and contains a 1,884-bp open reading frame encoding628 amino acids, including a 51-amino-acid signal sequence(see GenBank accession number CQ765963). For conve-nience, we designated the structural element domains (theN-terminal domain [N], subdomain [S], and C-terminal do-main [C]) and features (�-sheets and �-helices). The featureswithin domains were numbered beginning at the N terminus,and the loops were assigned the same number as the preceding�-sheet. Sequence alignment with glucosidases revealed thatall the SIs have N�1-8 helices and N�1-8 sheets, collectivelytermed the barrel (�/�)8, and the catalytic-triad residues(Asp241, Glu295, and Asp369) at the N�4, N�4, and N�7 sheetpositions (Fig. 1A). The additive Pc�8 and Pc�1 at the C-terminal end of the SI of P. rubrum were not found in other SIsand were expected to contribute to substrate stabilization inthe active pocket. The FBS (325RLDRD329) in SI is locatedbetween the N�6 sheet and the N�6 helix.

Structural P. rubrum SI models obtained by energy minimiza-tion suggest the relative positions of �-helices and �-sheets in the3D 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 modelwas simulated in an explicit water environment for 420 ps in aneffort to refine the structure and to establish the stability of themodel in general. The RMSD from the initial model-built struc-ture is displayed in Fig. 2. The overall deviation increased withtime for the first 40 ps and then remained relatively constant,indicating that the structure was not changing in a systematicmanner. The time history for the RMSD reached a maximumdeviation of approximately 1.5 Å after 40 ps. This is reasonablefor a model-built structure and suggested that the system hadconverged to a stable structure, or at least a stable local minimum,which was close to the starting structure.

An analysis of the Ramachandran plot (/ values for eachresidue) was performed by using the PROCHECK program.Analysis of the predicted SI structures indicated that the per-centages of residues in the most favored regions, additionalallowed regions, generously allowed regions, and disallowedregions were 81.6%, 15.6%, 1.4%, and 1.4%, respectively (seethe supplemental material). The score expressing how well thebackbone conformations of all residues were corresponding tothe populated areas in the Ramachandran plot was withinexpected ranges for a well-refined structure (Ramachandran Zscore, �0.334), and the standard deviation of � values agreedwith this expectation, around 5.5 (standard deviation of �values, 5.783). The score of how well the �-1/�-2 angles of allresidues corresponded to the populated areas in the databasewas within expected ranges for well-refined structures (�-1/�-2correlation Z score, �0.315). The distribution of residue typesover 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 shareconserved barrel (�/�)8 domains for sucrose-binding and gly-cosidase activities with all other SIs and glucosidases. A uniqueFBS (325RLDRD329) motif that is in proximity to the active siteis also well conserved in the predicted 3D model of P. rubrumSI. This motif is considered to be responsible for sucroseisomerization, similar to other SIs. The structural features ofthe FBS were considered to contribute to the positioning andbinding of the fructose unit for isomerization after the hydro-lysis of sucrose. Thus, the modification of its residues near thesubstrate pocket of the SI structure had high probability ofchanging the substrate preference or different product speci-ficities of SI.

Analysis of the SI active-site structure. In order to investi-gate the structural features of the FBS, Bacillus cereus oligo-1,6-glucosidase, P. dispersa SI, and P. rubrum SI were super-imposed on the amylosucrase-sucrose complex from Neisseriapolysaccharea based on the atoms of the five conserved resi-dues (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 fructosylmoiety of sucrose and thus may be easily able to interact withO-6 of fructose via H bonds. These two residues are positivelycharged and possess a relatively large side chain. Our model ofthe binding-site structure suggested that the function of twoArg residues is to stabilize the enzyme-sucrose complex. Itseems that these two Arg residues are well conserved in anatypical SI from P. mesoacidophila MX-45, which possesses adifferent corresponding sequence (311RYDRA315) in the FBS

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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. S�1 and S�2 and S�1 to S�4, subdomain �-helix and �-sheet; N�1 to N�8 and N�1 to N�8, N-terminal domain (�/�)8structure; C�1 to C�7, C-terminal domain �-sheets; Pc�1 and Pc�1, the estimated C-terminal �-helix and �-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-conservedresidues, colons indicate similarly conserved residues, and periods indicate partially conserved residues. (B) For the 3D structure, the same numbering and codingare used. The core of the triose phosphate isomerase barrel is located in the center of the 3D structure. Prominent �-helices and �-sheets are numbered bydomain from the N terminus.

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and produces more than 90% trehalulose and a small amountof isomaltulose. Together, the data indicated that these twoArg residues may play an important role in stabilizing theenzyme-sucrose complex until the reaction is terminated.

Changes in the FBS structures of mutated SIs. The twopositively charged Arg residues of the 325RLDRD329 motif inthe SI were replaced with negatively charged Asp (R325D andR328D), neutral Gln (R325Q and R328Q), positively chargedLys (R325K and R328K), or hydrophobic Ala (R325A andR328A) residues. His-tagged SI (302IU) had activity similar tothat of mature SI (299IU) in an activity assay with total celllysate. The level of expression did not appear to be affected bypoint mutations, as shown in Fig. 4.

Changes in the �(�G) were determined based on the kineticparameters of the eight generated mutant enzymes (Table 1).We investigated the relationship between �(�G) and the FBSstructure (325RLDRD329) by analyzing the predicted active-site structures of the mutant SIs. In order to investigate theinteractions between the substrate and each amino acid resi-due that had been mutated in the model of P. rubrum SI, thedistance between each residue and the fructosyl moiety wascalculated from the predicted model using molecular-dynamic

simulations (Fig. 5). In the wild-type SI, the average distancebetween Arg325 and O-6 in the predicted model was estimatedto be approximately 2.5 Å. As expected, the catalytic efficiency(7.7 mM�1 s�1) of the R325D mutant enzyme was reduced andthe �(�G) (12.7 kJ mol�1) was increased. As shown in Fig. 5A,the interference of the H bond and the repulsion between theAsp residue and the substrate seemed to be important reasonsfor a decrease in the sucrose-binding affinity and in the stabilityof the enzyme-sucrose complex. In the R325A and R325Q mu-tant enzymes, the average distances between the mutatedamino acid residue in the FBS and the fructosyl moiety wereincreased 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

FIG. 2. RMSD time history obtained from the molecular-dynamicssimulation. RMSD values were calculated for all atoms of the proteinbackbone for the entire predicted SI structure.

FIG. 3. Superimposition of SIs and oligo-1,6-glucosidase onto the amylosucrase-sucrose complex based on the atoms of the five conservedactive-site residues (11, 14, 25, 26). Based on this model of the binding-site structure, the residues that are involved directly in the hydrolysis ofthe 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 intermediatecomplex.

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



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weakened H bonds caused by increases in the distances be-tween atoms (6.0 and 8.5 Å, respectively). However, the R325Aand R325Q mutant enzymes had less reduced affinities forsucrose than the R325D mutant enzyme due to the charge-charge repulsion between the mutated amino acid residues andsucrose. In the R325K mutant enzyme with a lower �(�G) thanthe others (4.7 kJ mol�1), some degree of interaction wasmaintained between Arg325 and the fructose moiety within 3.8Å via a weakened H bond. It is possible that R325K mayinteract with fructose via H bonds in a manner similar to thatof wild-type SI. However, the catalytic efficiency for isomaltu-lose of R325K (193.8 mM�1 s�1) was considerably lower thanthat 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

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

Strain Kmsucrose

(mM)kcat

(103 s�1)kcat/Km

(mM�1 s�1)�(�G)a

(kJ mol�1)

Wild type 32.4 42.10 1,301.0 0.0R325D 85.7 0.66 7.7 12.7R325A 11.0 0.75 68.4 7.3R325Q 18.7 2.34 125.0 5.8R325K 8.5 1.66 193.8 4.7R328D 22.3 0.56 25.1 9.8R328A 11.7 2.46 210.3 4.5R328Q 12.1 0.56 46.7 8.2R328K 19.0 2.48 130.1 5.7

a �(�G) �RT ln�(kcat/Km)mut/(kcat/Km)wt�, where mut is mutant and wt iswild type.

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



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the Arg325 position were key players in influencing the stabilityof the transition state and the catalytic efficiency.

Although the Arg328 mutation decreased the stability of theenzyme-sucrose complex, all the Arg328 mutant enzymes pro-duced some amount of isomaltulose and trehalulose. This in-dicates that Arg328 may influence the stability of the enzyme-sucrose complex of SI less than Arg325 does. Removal of the Hbond in R328A and R328K caused slight perturbations in theenzyme structure, but these changes were small in comparisonto the other structural changes (Fig. 5B). As expected, thesemutants had higher catalytic efficiencies (210.3 and 130.1mM�1 s�1, respectively) and lower �(�G) values (4.5 and 5.7kJ mol�1, respectively) than R328D (catalytic efficiency, 25.1mM�1 s�1; �(�G) value, 9.8 kJ mol�1). In the cases of R328Dand R328Q, relatively high �(�G) values (9.8 and 8.2 kJ mol�1,respectively) and low catalytic-efficiency values (25.1 and 46.7mM�1 s�1, respectively) were recorded, which might have

been due to charge repulsion arising at relatively close prox-imity to the units involved (3.3 and 4.6 Å, respectively) (Table1). Even though our model of the predicted FBS based oncharge repulsion and the H bond alone cannot explain why thecatalytic efficiency of R328K was lower and its �(�G) value washigher than those of R328A, it is possible that the effect of theArg328 mutation is due to some steric hindrance in the activepocket.

Isomaltose production by mutant enzymes having a modi-fied FBS. Although significant catalytic activity was lost when amutation was introduced into the catalytic-triad residues(Asp241, Glu295, and Asp369) and glucose moiety-binding resi-dues (His145 and His368) of SI (data not shown), the replace-ment of the two Arg residues altered the ratio of productspecies formed instead of completely abolishing the isomeriza-tion activity, with a reduction of sucrose consumption as re-vealed by a total sucrose assay (Table 2).

FIG. 5—Continued.



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Table 2 shows the conversion of sucrose into isomaltulose,trehalulose, isomaltose, glucose, and fructose in the absence orin the presence of glucose. Both monosaccharides were pro-duced in the same concentration during the initial stage of thereaction; however, the glucose concentration became lowerthan the fructose concentration as the incubation time in-creased. The deviation increased as isomaltose productionrose.

As shown in Table 2 (in the absence of glucose), the Arg325

mutation resulted in a significant decrease in isomaltulose pro-duction and an increase in trehalulose production and therelease of monosaccharides. Further, isomaltose, which wasnot produced by the wild-type enzyme, was produced byR325A, R325Q, R325K, and R325D. Among these mutant en-zymes, the main function of SI, i.e., isomaltulose conversion,was more severely inhibited in R325D, where Arg325 was re-placed with a negatively charged Asp, than in R325K, whereArg325 was replaced with a positively charged Lys.

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

These results suggested that Arg325 is not directly involved inthe hydrolytic activity of the enzyme but rather plays a moreimportant role than Arg328 in the appropriate isomerization ofsucrose to isomaltulose and trehalulose by stabilizing the in-termediate binding. This finding was consistent with the struc-tural analysis (Fig. 5 and Table 1). Under these circumstances,we consider that the ratio of products generated by a mutatedSI varies depending on its ability to form a stable complex with

a fructose unit in the transition state, where the glycosidic bondof sucrose is hydrolyzed for the isomerization of sucrose toisomaltulose or trehalulose, and on its ability to stably accept aglucose unit.

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

We also investigated the kinetic mechanism of the isomalt-ose production by R325Q using initial velocity measurements inwhich the concentrations of both substrates were varied sys-tematically and the results were analyzed assuming steady-state conditions. In this experiment, the concentration of su-crose varied in the presence of several different fixedconcentrations of glucose. When the data were plotted in dou-ble-reciprocal form as a function of the sucrose concentration,a series of near-parallel lines was obtained (Fig. 6).

Based on a sucrose conversion assay and steady-state kinet-ics results, the schematic reaction mechanism of R325Q wassuggested (Fig. 7). The isomerization of sucrose to isomaltu-lose and trehalulose occurred at the enzyme-sucrose complexstage; however, for the conversion of sucrose to isomaltose,free glucose in the reaction mixture was reused following theliberation of fructose by the enzyme-glucose complex. To-gether, the data suggested that this mechanism of transferproduct formation is possibly a ping-pong bi-bi system, wherethe enzyme binds a first substrate (sucrose), releases a firstproduct (fructose), and produces a second enzyme form (en-zyme-glucose complex), which binds a second substrate (H2O

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

Strain Glucose(g liter�1)

Fructose(g liter�1)

Isomaltulose(g liter�1)

Trehalulose(g liter�1)

Isomaltose(g liter�1)

Consumedsucrose (g liter�1)

No glucose addedWild type 7.2 � 0.1 7.6 � 0.2 176.9 � 2.7 5.0 � 1.2 0.0 200.0 � 0.1R325D 37.5 � 2.3 43.9 � 3.0 11.3 � 0.1 20.0 � 2.0 11.5 � 1.8 124.8 � 1.2R325A 37.8 � 3.2 42.0 � 2.4 22.3 � 2.2 18.9 � 1.5 10.2 � 2.2 132.0 � 3.4R325Q 26.2 � 1.9 33.1 � 1.2 26.0 � 3.9 16.1 � 1.8 11.1 � 2.1 109.7 � 0.9R325K 34.2 � 0.1 42.3 � 2.7 72.3 � 1.5 30.7 � 1.2 16.4 � 1.8 200.0 � 0.1R328D 26.0 � 3.2 26.4 � 3.1 117.5 � 1.2 29.1 � 0.6 0.0 198.0 � 2.2R328A 18.9 � 1.4 21.9 � 1.7 126.9 � 2.5 25.3 � 1.9 5.0 � 2.0 200.0 � 0.1R328Q 21.1 � 2.6 21.3 � 2.2 126.3 � 1.1 29.5 � 1.1 0.9 � 0.1 200.0 � 0.1R328K 20.8 � 2.1 24.3 � 0.9 125.4 � 2.3 19.1 � 1.3 7.8 � 0.9 195.1 � 1.6

5% Glucose addedWild type 54.0 � 4.2b 7.0 � 2.1 175.5 � 3.2 5.1 � 1.1 4.4 � 1.4 200.0 � 0.1R325D 80.9 � 6.1 44.3 � 4.4 11.0 � 5.2 18.2 � 2.2 26.2 � 3.6 128.5 � 7.2R325A 76.3 � 5.7 39.6 � 4.0 21.3 � 4.1 16.2 � 2.9 26.9 � 2.4 133.2 � 2.5R325Q 71.8 � 5.0 35.5 � 2.7 16.5 � 2.4 14.3 � 1.7 25.4 � 1.5 110.7 � 3.7R325K 74.1 � 4.3 43.7 � 6.1 70.7 � 5.5 25.8 � 3.1 38.2 � 3.5 200.0 � 0.1R328D 67.8 � 5.1 25.3 � 4.2 116.7 � 4.1 26.7 � 4.2 15.1 � 2.3 197.0 � 3.5R328A 60.3 � 5.2 20.4 � 3.7 126.6 � 4.7 21.6 � 1.8 18.9 � 2.2 200.0 � 0.1R328Q 61.2 � 4.7 20.6 � 2.9 124.3 � 6.4 28.8 � 3.3 18.6 � 2.3 200.0 � 0.1R328K 65.7 � 4.6 26.9 � 2.1 119.1 � 7.9 16.0 � 2.1 20.4 � 3.4 194.8 � 4.3

a Samples (0.25 ml) of sucrose (20% �wt/vol�) in calcium acetate (pH 5.5) containing 10 �g of SI were incubated for 12 h at 25°C. The reaction mixture was boiledfor 5 min and analyzed by HPLC on a carbohydrate column.

b Glucose was added to 5% (wt/vol) final concentration.



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and glucose) and finally releases a second product (glucose andisomaltose).

In order to investigate the effect of the glucose concentra-tion on the activity of the purified R325Q mutant enzyme,which exhibited a relatively high activity for isomaltose pro-duction (9.3% with sucrose alone and 20.0% with sucrosecontaining 5% [wt/vol] glucose), 0 to 30% (wt/vol) glucose wasadded to the reaction mixture under the standard conditions(Fig. 8). With wild-type SI, sucrose was almost completelyconverted to isomaltulose; the isomaltose content in the reac-tion mixture increased to 13.0 g liter�1 by using up 184 g liter�1

sucrose, and the isomaltose yield for the consumed sucrose was0.07 g of isomaltose g of sucrose�1 under conditions of 30%(wt/vol) glucose (Fig. 8A). With the R325Q mutant enzyme, thesucrose consumption was slightly decreased compared to thatof the wild-type SI, the isomaltose content in the reactionmixture increased to 57.9 g liter�1 by using up 105 g liter�1

sucrose, and the isomaltose yield for the consumed sucrose was0.55 g of isomaltose g of sucrose�1 under conditions of 15%(wt/vol) glucose. The relative activities were 22.1% for each

FIG. 6. Steady-state kinetic analysis of R325Q catalysis with various su-crose concentrations. The concentrations of glucose were 1% (F), 5% (E),10% (�), and 15% (ƒ) (wt/vol), respectively. The inset is a replot of theintercepts versus the reciprocals of the corresponding glucose concentrations.

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



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monosaccharide, 9.1% for isomaltulose, 6.8% for trehalulose,and 40.0% for isomaltose. The production and the yield ofisomaltose reached a plateau at 15% of the glucose concen-tration, showing that a maximum of 29 g liter�1 free glucosecan be reused by the R325Q mutant enzyme as a secondarysubstrate and then be converted to isomaltose (Fig. 8B).

Effect of temperature on isomaltose production by R325Qmutant enzymes. Isomaltose can be produced efficiently by theR325Q mutant enzyme in the absence of a temperature shift(Fig. 8). Because temperature is an important factor related tothe binding force between the substrate and the enzyme, weinvestigated the influence of temperature on isomaltose pro-duction in R325Q and determined the optimum temperature

for the production of each sugar (Fig. 9). The increase infructose produced meant an increase in enzyme-glucose inter-mediate produced, where glucose can be introduced in orderto produce isomaltose. Under conditions of 15% (wt/vol) glu-cose, the release of fructose from the enzyme-glucose-fructosecomplex was increased by raising the temperature, therebyincreasing isomaltose production. Although the production offructose reached a saturation point at 25°C, the production ofisomaltose increased up to 35°C, showing a maximum produc-tion of 75.7 g liter�1 and a maximum yield of 0.61 g of iso-maltose g of sucrose�1. The relative activity for isomaltoseproduction increased steadily with the temperature andreached 45.9% at 35°C. The production of isomaltose wasdecreased at 45°C due to the increase of glucose, which wasreleased from an enzyme-glucose intermediate, even thoughthe production of fructose was not decreased. R325Q was in-activated at 55°C.

DISCUSSION

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

Differences in the reaction kinetics and specificity of SI mostprobably reflect differences in its FBS (10, 23, 24, 26). Inaddition to glucose, used in this study, fructose is reported toincrease the ratio of trehalulose production by SI, but it doesnot inhibit the activity of the purified forms of SI extractedfrom S. plymuthica, P. rubrum, and E. rhapontici (16, 17, 26). Incontrast, fructose acts as a competitive inhibitor for the SIsfrom P. dispersa UQ68J and K. planticola UQ14S, implying that

FIG. 8. Effects of the glucose concentration on isomaltose produc-tion in wild-type SI (A) and R325Q mutant SI (B). Sucrose, �; fructose,F; isomaltulose, E; trehalulose, �; isomaltose, ƒ. The conversion ofsucrose was performed in 250 �l of reaction mixture containing 20%(wt/vol) sucrose, 5 �g of enzyme, and 0 to 30% (wt/vol) glucose at 25°Cin a shaking water bath for 12 h.

FIG. 9. Effects of temperature on isomaltose production in R325Qmutant SI. Sucrose, �; fructose, F; isomaltulose, E; trehalulose, �;isomaltose, ƒ. The conversion of sucrose was performed in 250 �l ofreaction mixture containing 20% (wt/vol) sucrose, 5 �g of enzyme, and15% (wt/vol) glucose at 15 to 45°C in a shaking water bath for 12 h.



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the binding of fructose to the active sites of these enzymes isstronger, although trehalulose production via the direct con-densation of glucose to fructose is impossible. Further com-parisons of the kinetics of these SIs in the presence of differentmonosaccharides, along with mutagenesis studies to test thefunctional significance of apparent differences in key aminoacids, are likely to be informative.

In this study, the interactions between the amino acid resi-dues in the FBS of P. rubrum SI and the substrate were inves-tigated at the molecular level. We demonstrated a method foraltering the ratio of the products of SI by introducing anenvironmental change around the bound substrate. We spec-ulate that it is possible to reconstruct enzymes having differentreaction mechanisms for use in isomaltose production by in-troducing mutations in the FBS residues in association with theenvironment around the bound substrate.

First, we predicted the 3D structure of the FBS by using a3D modeling program to identify residues important for theadsorption and degradation of sucrose. We identified two po-sitions that are critical for the fructose-binding ability of SIextract from P. rubrum. Further, 4 amino acid residues wereintroduced at these two positions, i.e., at Arg325 and Arg328.Among the five conserved amino acid residues, the two Argresidues were observed to be closely associated with the sta-bility of the enzyme-sucrose intermediate complex. These find-ings indicated that the modification of these two Arg residuescould alter the binding strength between the FBS of SI and thefructosyl moiety of sucrose. Further, modification of the FBSaltered the ratio of products generated by the mutant enzymes.Our results revealed that the ratio of products formed by themutant enzymes depends on the sucrose-binding abilities ofthe enzymes and that Arg325 was mainly involved in the inter-action between the enzyme and the fructosyl moiety of sucrose.On the other hand, free glucose acted as a secondary substrateand was introduced with greater ease into the vacant pocket ofthe 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% inthe reaction with the purified R325Q mutant enzyme in thepresence of 15% (wt/vol) glucose at 25°C and 9.1% in that withR325Q in the absence of glucose at 25°C. Further, the isomalt-ose yield was 0.55 g of isomaltose g of sucrose�1 with 15%additive glucose. Veronese et al. reported that a relative activ-ity of 4.5% for isomaltose production was recorded by usingpurified S. plymuthica SI in the presence of 277 mM glucose(5% [wt/vol]) at 30°C (24). Further, they demonstrated thatthis intermolecular reaction was enhanced at high tempera-tures. In the case of wild-type SI extracted from P. rubrum,2.9% relative activity for isomaltose production was recordedin the presence of 5% (wt/vol) glucose at 25°C. The relativeactivity of P. rubrum SI was similar to or slightly lower thanthose of SIs extracted from other sources, including S.plymuthica SI (3, 9, 17, 24, 28). Since most SIs are similar instructure and function, regions that are conserved in this classof enzymes and that are associated with the recognition of thefructosyl moiety of the substrate could be considered as can-didates for the development of isomaltose-producing enzymesfor use in industries.

His-tagged SI purified from E. coli was used in these exper-iments instead of SI purified from P. rubrum. Although the

biochemical data were similar, the possible structural differ-ence between wild-type SI and His-tagged SI remained andwas not negligible. However, despite this limitation, our resultssuggest that the stability of fructose in the FBS is essential forsucrose isomerization and that direct condensation can be in-duced by modifications to 5 amino acids present in the FBS, bya temperature shift, or by a high environmental glucose con-centration. Based on previous studies and the present one,engineering of the FBS of SI appears to have great potentialfor enhancing isomaltose production. In addition to the twoArg residues, modification of other binding residues could beconsidered for decreasing the threshold glucose saturationconcentration for isomaltose production and thus maintainingthe isomaltose productivity of SI under conditions of less than10% (wt/vol) additive glucose.

Further, it may be possible to design a continuous mixed-enzyme reactor containing glucose isomerase that can be usedfor the conversion of fructose to glucose, if glucose isomerasewith a high glucose/fructose ratio can be utilized and a thresh-old glucose saturation concentration for isomaltose productioncan be maintained.

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Documents

Isomaltose Production by Modiﬁcation of the Fructose ... · Recently, SI-encoding genes were isolated from E. rhapon- tici, P. rubrum, Klebsiella sp. strain LX3, and P. dispersa,