Molecular imprinting of cyclodextrin glycosyltransferases from Paenibacillus sp. A11 and Bacillus macerans with c-cyclodextrin Jarunee Kaulpiboon 1,2 , Piamsook Pongsawasdi 3 and Wolfgang Zimmermann 2 1 Department of Pre-Clinical Science (Biochemistry), Faculty of Medicine, Thammasat University, Pathumthanee, Thailand 2 Department of Microbiology and Bioprocess Technology, Institute of Biochemistry, University of Leipzig, Germany 3 Starch and Cyclodextrin Research Unit, Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok, Thailand Cyclodextrin glycosyltransferase (EC 2.4.1.19; CGTase) catalyzes four different reactions: cyclization, disproportionation, coupling, and hydrolysis. Cyclo- dextrins (CDs, cyclic oligosaccharides of glucose resi- dues) are formed by the intramolecular circularization reaction, whereas a linear malto-oligosaccharide is Keywords cross-linked imprinted proteins; cyclodextrin glycosyltransferase; cyclodextrins; molecular imprinting; product specificity Correspondence W. Zimmermann, Department of Microbiology and Bioprocess Technology, Institute of Biochemistry, University of Leipzig, Leipzig 04103, Germany Fax: +49 341 97 36798 Tel: +49 341 97 36781 E-mail: wolfgang.zimmermann@ uni-leipzig.de Website: http://www.biochemie. uni-leipzig.de/agz (Received 3 November 2006, revised 11 December, accepted 13 December 2006) doi:10.1111/j.1742-4658.2007.05649.x Cyclodextrin glycosyltransferase catalyzes the formation of a mixture of cyclodextrins from starch by an intramolecular transglycosylation reaction. To manipulate the product specificity of the Paenibacillus sp. A11 and Bacillus macerans cyclodextrin glycosyltransferases towards the preferential formation of c-cyclodextrin (CD 8 ), crosslinked imprinted proteins of both cyclodextrin glycosyltransferases were prepared by applying enzyme imprinting and immobilization methodologies. The crosslinked imprinted cyclodextrin glycosyltransferases obtained by imprinting with CD 8 showed pH and temperature optima similar to those of the native and immobilized cyclodextrin glycosyltransferases. However, the pH and temperature stability of the immobilized and crosslinked imprinted cyclodextrin glyco- syltransferases were higher than those of the native cyclodextrin glycosyl- transferases. When the catalytic activities of the native, immobilized and crosslinked imprinted cyclodextrin glycosyltransferases were compared, the efficiency of the crosslinked imprinted enzymes for CD 8 synthesis was increased 10-fold, whereas that for cyclodextrin hydrolysis was decreased. Comparison of the product ratios by high-performance anion exchange chromatography showed that the native cyclodextrin glycosyltransferases from Paenibacillus sp. A11 and Bacillus macerans produced CD 6 :CD 7 : CD 8 : ‡ CD 9 ratios of 15 : 65 : 20 : 0 and 43 : 36 : 21 : 0 after 24 h of reaction at 40 °C with starch substrates. In contrast, the crosslinked imprinted cyclodextrin glycosyltransferases from Paenibacillus sp. A11 and Bacillus macerans produced cyclodextrin in ratios of 15 : 20 : 50 : 15 and 17 : 14 : 49 : 20, respectively. The size of the synthesis products formed by the crosslinked imprinted cyclodextrin glycosyltransferases was shifted towards CD 8 and ‡ CD 9 , and the overall cyclodextrin yield was increased by 12% compared to the native enzymes. The crosslinked imprinted cyclo- dextrin glycosyltransferases also showed higher stability in organic sol- vents, retaining 85% of their initial activity after five cycles of synthesis reactions. Abbreviations A11, Paenibacillus sp. A11; BM, Bacillus macerans; CD, cyclodextrin; CGTase, cyclodextrin glycosyltransferase; CLIP, crosslinked imprinted proteins; HPAEC, high-performance anion exchange chromatography; TNBS, 2,4,6-trinitrobenzene sulfonic acid. FEBS Journal 274 (2007) 1001–1010 ª 2007 The Authors Journal compilation ª 2007 FEBS 1001 transferred to an acceptor sugar molecule in the dis- proportionation reaction. CGTase also catalyzes the opening of a CD and transfer of the linear malto- oligosaccharide to an acceptor sugar molecule in the coupling reaction. Furthermore, CGTase can catalyze the hydrolysis of glycosidic linkages in starch [1]. The end-products, especially CD 6 ,CD 7 and CD 8 (a-CD, b-CD and c-CD), are extensively used in the food, cos- metic and pharmaceutical industries, owing to their ability to form inclusion complexes with appropriate guest compounds [1–5]. However, a major disadvan- tage of the synthesis of CD by CGTases is that all native enzymes usually produce a mixture of CD 6 , CD 7 ,CD 8 and large-ring CD (‡ CD 9 ), making proces- ses to separate each type of CD unavoidable. These are time-consuming, cost-intensive and potentially unsafe for the consumer and the environment. To resolve these problems, attempts to construct CGTases with higher product specificity have been made, using information on the three-dimensional structures of the enzymes and genetic engineering techniques [6–8]. However, until now, no CGTase with a product specif- icity for a single CD has been reported. Recently, a technique of crosslinking imprinted proteins (CLIP) has been described [9,10]. With a combination of imprinting and enzyme immobilization methods, this technique can be used for the production of recogni- tion sites with predetermined selectivity in the enzyme. In the first step, the enzyme is derivatized with itaconic acid anhydride and then imprinted with ligands such as substrate analogs or inhibitors in aqueous medium [10]. Subsequently, the manipulated enzyme conforma- tion is fixed by polymerizing it in a water-free organic solvent. The ligand is removed in the final step, and the CLIP enzyme can be used either in aqueous med- ium or organic solvent. The CLIP enzymes show altered substrate or product specificity and enhanced stability in high concentrations of organic solvents [11]. CLIP enzymes are also more enantioselective than the native enzyme [12]. Furthermore, they are insoluble and can be separated and recycled many times, increasing their productivity. These beneficial proper- ties are especially useful in the areas of synthetic organic chemistry, biomedical applications, and envi- ronmental catalysis. In this study, the modification of the product specif- icity and stability of two CGTases at the level of the mature protein is described. The native enzymes from Paenibacillus sp. A11 (A11) and Bacillus macerans (BM) form CD 7 and CD 6 as their major products, respectively [13,14]. The imprinting of the enzymes with CD 8 , resulting in high levels of the desired prod- uct being formed, is reported. Results and Discussion To provide enough attachment points for crosslinking of the enzymes, the CGTases were derivatized with ita- conic anhydride in an aqueous medium. Free amino groups of lysine, hydroxyl groups of tyrosine or sul- fhydryl groups of cysteine were covalently coupled with itaconic anhydride [12]. By varying the protein ⁄ itaconic anhydride ratio, different degrees of derivati- zation of the CGTases were obtained. The remaining activity of the enzyme depended on the degree of deri- vatization (Table 1). The optimum ratio (w ⁄ w) of both CGTases to itaconic anhydride was 1 : 5. The resulting degrees of derivatization of the A11 and BM CGTases determined by the 2,4,6-trinitrobenzene sulfonic acid (TNBS) assay were 62% and 65%, respectively. The remaining activities of the derivatized A11 and BM CGTases were 90% and 77%, respectively. With a ratio of 1 : 3, the remaining activity of both enzymes was not significantly different from a ratio of 1 : 5, but the derivatization degree was significantly lower. With the use of higher ratios, higher levels of derivatization were possible, but resulted in a further decrease in activity. In previous reports on the CLIP technique, Kronenburg et al. [12] succeeded in manipulating the enantioselectivity of epoxide hydrolase with a derivati- zation degree of 70%, whereas Peißker et al . [10] reported a 60% derivatization degree as optimum, con- sidering the remaining activity of the resulting deriva- tized protease. The derivatized A11 and BM CGTases were imprinted with CD 8 and crosslinked to obtain the cor- responding CLIP CGTases. The derivatized nonim- printed enzymes were also crosslinked to obtain immobilized enzyme preparations for comparison. The effect of pH on the activity of the different CGTase preparations from A11 and BM was Table 1. Degree of derivatization of the CGTases from A11 and BM obtained with different protein ⁄ itaconic anhydride ratios and remaining cyclization activity. Ratio a (w ⁄ w) Paenibacillus sp. A11 Bacillus macerans Derivatization degree (%) Remaining activity (%) Derivatization degree (%) Remaining activity (%) 1 : 0 0 100 0 100 1:3 54 97 52 80 1:5 62 90 65 77 1:7 71 80 73 75 1:9 80 76 76 74 1 : 11 90 72 79 72 1 : 13 94 66 86 67 a Protein ⁄ itaconic anhydride. Molecular imprinting of glycosyltransferases J. Kaulpiboon et al. 1002 FEBS Journal 274 (2007) 1001–1010 ª 2007 The Authors Journal compilation ª 2007 FEBS determined in the pH range 5–11, as shown in Fig. 1A,B. The optimum pH for the cyclization activ- ity of the native, immobilized and CD 8 -imprinted CLIP CGTases from A11 and BM was found to be 6.0. The pH activity profiles of the CGTases were sim- ilar, showing 60% activity at pH 5.0, and decreasing activity at higher pH values in the range of 7–11. However, the CGTase from A11 showed a broader pH optimum, extending from 6.0 to 8.0. When the immo- bilized and CD 8 -imprinted CLIP CGTases were com- pared with the native enzymes, a higher activity in the pH range from 8 to 11 was observed. This effect was more pronounced with the CGTase from BM. The immobilized and the CLIP CGTase from A11 were more stable than the native enzyme in the pH ranges from 3 to 6 and 8 to 11, whereas the immobilized and CLIP CGTases from BM showed higher stability in the ranges from 3 to 7 and 9 to 11 (data not shown). The activities of the native, immobilized and CD 8 - imprinted CLIP CGTases from A11 and BM were also determined at different temperatures in the range 30– 80 °C. The optimum temperature for the cyclization activities of the different enzyme preparations were 40– 50 °C for the A11 CGTase (Fig. 2A) and 60 °C for the BM CGTase (Fig. 2B). The similar temperature optima of the different forms indicate that there was no loss of enzyme activity through imprinting, immo- bilizing and crosslinking of the native enzyme. The temperature stability of the immobilized and CLIP CGTases from A11 and BM at 60 °C and 70 °C was considerably higher than that of the native enzymes (Fig. 3A,B). This could be explained by a stabilizing effect of the covalent crosslinking of the enzymes. The immobilized and CD 8 -imprinted CLIP CGT- ases from A11 showed 30% higher stability in phosphate buffer containing up to 50% ethanol or Relative activity (%)Relative activity (%) pH 0 4 5 6 7 8 9 10 11 12 456789101112 20 40 60 80 100 A B 0 20 40 60 80 100 Fig. 1. Effect of pH on the native (dotted line), immobilized (solid line) and CD 8 -imprinted CLIP CGTase (dashed line) activity at 40 °C. The CGTases were from A11 (A) and BM (B). The buffers used were 0.2 M potassium phosphate (pH 5.0–7.0) (s), Tris ⁄ HCl (pH 7.0–9.0) (x), and glycine ⁄ NaOH (pH 9.0–11.0) (D). 0 20 40 60 80 100 0 20 30 40 50 60 70 80 90 20 30 40 50 60 70 80 90 20 40 60 80 100 A B Temperature (°C) Relative activity (%) Relative activity (%) Fig. 2. Effect of temperature on native (dotted line), immobilized (solid line) and CD 8 -imprinted CLIP CGTase (dashed line) activity at pH 6.0. The CGTases were from A11 (A) and BM (B). J. Kaulpiboon et al. Molecular imprinting of glycosyltransferases FEBS Journal 274 (2007) 1001–1010 ª 2007 The Authors Journal compilation ª 2007 FEBS 1003 cyclohexane compared to the native enzyme, whereas the immobilized and CD 8 -imprinted CLIP CGTases from BM showed 15% higher stability. As ethanol and other cosolvents have been shown to increase the yield of CD produced by CGTases, the high stability of the CD 8 -imprinted CLIP CGTases in the presence of eth- anol could be used to further increase the product yields of the CLIP enzymes [15]. The effect of polar cosolvents has been explained by suppression of the intermolecular transglycosylation reaction, which cau- ses partial degradation of the CD products formed [16,17]. With nonpolar solvents, CDs could form an insoluble complex, resulting in their continuous removal from the reaction by precipitation and a shift of the equilibrium in favor of CD formation [18]. The reuse stability of the immobilized and CD 8 - imprinted CLIP CGTases, which is an important factor in the utilization of immobilized enzymes in large-scale applications, was also determined [19,20]. More than 80% of the initial immobilized and CLIP CGTase activities from A11 and BM were retained for up to five cycles of synthesis reactions. Comparison of the products obtained from the native, immobilized and CD 8 -imprinted CLIP CGTas- es revealed that the native CGTases from A11 and BM produced CD 6 :CD 7 :CD 8 : ‡ CD 9 in ratios of 15 : 65 : 20 : 0 and 43 : 36 : 21 : 0, respectively, after 24 h of reaction at 40 °C. In contrast, the CLIP CGTases from A11 and BM imprinted with CD 8 pro- duced CD in ratios of 15 : 20 : 50 : 15 and 17 : 14 : 49 : 20, respectively (Table 2). The CLIP CGTases showed an increase in product specificity towards preferential formation of CD 8 . In addition to a higher yield of CD 8 , the CD 8 -imprinted CLIP CGTases also produced a higher overall yield of CD compared with the native CGTases (Table 2). The immobilized and CD 8 -imprinted CLIP CGTases also produced larger amounts of large-ring CDs (‡ CD 9 ) after 24 h of reaction at 40 °C (Fig. 4A,B). As shown in Fig. 4C,D, large-ring CDs were predominantly pro- duced during the first 30 min of the reaction. After 24 h, the amount of large-ring CDs was reduced, owing to their conversion to smaller CDs. However, the conversion of large-ring CDs obtained with immo- bilized and CD 8 -imprinted CLIP CGTases was slower than with the native CGTases, indicating that the immobilized and CD 8 -imprinted CLIP enzymes had decreased hydrolysis and coupling activity. When the cyclization activities of the CGTase prepa- rations were compared, a 10-fold increase in the cata- lytic efficiency (k cat ⁄ K m ) of the CLIP CGTases from A11 and BM was observed, resulting from an increase in the turnover rate (k cat ) and the binding affinity (K m ) (Table 3). The K m values of the CLIP CGTases in the coupling reaction indicated stronger binding of the CD 8 substrate, whereas the turnover rates (k cat ) were 0 20 40 60 80 100 A 0 20 40 60 80 100 B 20 30 40 50 60 70 80 20 30 40 50 60 70 80 Temperature (°C) Remaining activity (%) Remaining activity (%) Fig. 3. Effect of temperature on native (dotted line), immobilized (solid line) and CD 8 -imprinted CLIP CGTase (dashed line) activity. The CGTases were from A11 (A) and BM (B). The incubation was performed at pH 6.0 for 30 min. Table 2. Yields and product ratios of the native, immobilized and CD 8 -imprinted CLIP CGTases from A11 and BM. CGTase preparation Yield (%) Product ratio (%) CD 6 CD 7 CD 8 ‡ CD 9 A11 CGTase Native 42 15 65 20 0 Immobilized 56 18 39 24 19 CLIP imprinted with CD 8 54 15 20 50 15 BM CGTase Native 44 43 36 21 0 Immobilized 57 28 27 22 23 CLIP imprinted with CD 8 56 17 14 49 20 Molecular imprinting of glycosyltransferases J. Kaulpiboon et al. 1004 FEBS Journal 274 (2007) 1001–1010 ª 2007 The Authors Journal compilation ª 2007 FEBS slower than with the native CGTases, resulting in higher yields of CD 8 after long reaction times. This result should, however, be interpreted with caution, as the catalytic efficiency of the enzymes in the coupling reaction was determined using cellobiose, which is not the natural acceptor in the starch transglycosylation reaction. The accumulation of large-ring CDs after 24 h of reaction of the immobilized and CD 8 -imprinted CLIP CGTases with starch can be explained by their chan- ged hydrolytic activities. The decreased k cat and overall catalytic efficiency of both CLIP enzymes in the hydro- lysis reaction clearly indicated their lower CD hydroly- sis activity. In summary, the CD 8 -imprinted CLIP CGTases had significantly higher catalytic efficiency for CD 8 cycliza- tion and lower efficiency for CD hydrolysis, whereas their efficiency in the CD 8 coupling reaction was slightly increased when compared with the native enzymes. These results correspond to the observed higher yield of CD 8 and large-ring CDs obtained with the CLIP CGTases. Whereas the immobilization of the CGTases alone resulted in increased yields of large-ring CDs, through reduction of their hydrolysis activity, the observed shift in product ratios of the CD 8 -imprinted CLIP CGTases suggests that the molecular imprinting had a pronounced effect on the structure of the active site of the enzymes. Imprinting of the CGTases with CD of different sizes should have similar effects on their preferential formation, which could, however, be expec- ted to be limited by the increasing flexibility of the ring structures of the larger CDs. rotceteD esnopser rotce t eD esnopser Native A11 CGTase Immobilized A11 CGTase CLIP A11 CGTase CD 8 -imprinted Native BM CGTase Immobilized BM CGTase CLIP BM CGTase CD 8 -imprinted 6 7 8 10 9 11 15 24 6 7 8 10 9 11 15 24 AB CD e snopser rotceteD e sno p s e r ro t ce t eD Native A11 CGTase Retention time (min) Immobilized A11 CGTase CLIP A11 CGTase CD 8 -imprinted Immobilized BM CGTase CLIP BM CGTase CD 8 -imprinted Retention time (min) Native BM CGTase 15 6 6 7 7 8 8 10 10 9 9 11 11 15 24 24 0 10 20 30 40 50 60 70 80 0 10 20 30 40 50 60 70 80 0 10 20 30 40 50 60 70 80 0 10 20 30 40 50 60 70 80 Fig. 4. HPAEC analysis of CD synthesized by different CGTase preparations from A11 (A, C) and BM (B, D) at 40 °C for 24 h (A, B) and 30 min (C, D). The numbers above the peaks indicate the degree of polymerization of the CD. J. Kaulpiboon et al. Molecular imprinting of glycosyltransferases FEBS Journal 274 (2007) 1001–1010 ª 2007 The Authors Journal compilation ª 2007 FEBS 1005 When the yields of CD 8 obtained after different reaction times with the enzyme preparations were com- pared, most of the CD 8 was found to be formed dur- ing the first 30 min of incubation (Fig. 5). After 24 h, the CD 8 -imprinted CLIP CGTases produced 32% (A11) and 25% (BM) CD 8 . In comparison to the native enzymes, the yield of CD 8 increased four-fold with the CLIP CGTase from A11, and three-fold with the CLIP CGTase from BM, after 24 h of reaction. The amount of CD 8 produced by the CLIP CGTase from BM slowly increased during the 24 h reaction time, whereas no further increase occurred after 6 h of reaction with the CLIP CGTase from A11. The differ- ences in the time course of CD 8 formation detected depended on the type of CGTase used, and are in accordance with previously reported results. Terada et al. [21] observed that the amount of CD 8 increased when a CGTase from Bacillus sp. A2-5a was incubated with starch for a long period of time, as a result of the conversion of large-ring CDs to smaller CDs. In con- trast, longer reaction times with CGTase from the bac- terial isolate BT3 resulted in a 10% decrease in CD 8 [22]. The highest yield (26% conversion to CD 8 ) with the CLIP CGTase from A11 was found when synthetic lin- ear amylose (molecular mass 280 kDa) was used as substrate (Fig. 6). Lower yields of CD 8 were obtained with starches from potato, pea, and rice. Corn starch (73% amylopectin) and corn amylopectin gave the lowest yields, owing to their branched structure. Low yields were also obtained with dextrins, glucose oligo- saccharides of short chain length (degree of polymer- ization 23), indicating the preference of the CGTases for long chains of unbranched glucose polymers. In conclusion, the CGTases from Paenibacillus sp. A11 and B. macerans could be imprinted with CD 8 , which is not the major CD produced by the native enzymes. CD 8 was produced by the CD 8 -imprinted and crosslinked CLIP CGTases at much higher levels than by the native enzymes. Moreover, the CLIP CGTases showed higher stability and yielded larger amounts of total CD in the synthesis reactions. Experimental procedures Materials and enzymes CD 6 ,CD 7 ,CD 8 , potato starch (molecular mass 296 kDa), corn starch (molecular mass 340 kDa), rice starch, corn Table 3. Comparison of the cyclization, coupling and hydrolysis activities catalyzed by native, immobilized and CD 8 -imprinted CLIP CGTases from A11 and BM. CGTase preparation CD 8 -cyclization activity CD 8 -coupling activity CD 6)24 -hydrolysis activity K m , starch (mgÆmL )1 ) k cat (s )1 ) k cat ⁄ K m (mgÆmL )1 Æs )1 ) K m, CD8 (mM) k cat (s )1 ) k cat ⁄ K m (M )1 Æs )1 ) K m, CD6)24 (mgÆmL )1 ) k cat (s )1 ) k cat ⁄ K m (mgÆmL )1 Æs )1 ) A11 CGTase Native 0.83 ± 0.03 1.6 · 10 2 1.9 · 10 2 0.90 ± 0.20 1.2 · 10 2 1.3 · 10 5 1.25 ± 0.03 3.2 · 10 1 2.9 · 10 1 Immobilized 0.50 ± 0.02 2.5 · 10 2 5.0 · 10 2 0.55 ± 0.02 1.1 · 10 2 2.0 · 10 5 1.08 ± 0.01 8.0 · 10 0 6.4 · 10 0 CLIP imprinted with CD 8 0.21 ± 0.01 4.9 · 10 2 2.3 · 10 3 0.26 ± 0.01 1.0 · 10 2 3.8 · 10 5 0.48 ± 0.04 5.5 · 10 0 1.1 · 10 1 BM CGTase Native 0.55 ± 0.02 6.7 · 10 1 1.2 · 10 2 1.60 ± 0.20 4.2 · 10 2 2.6 · 10 5 1.30 ± 0.02 1.6 · 10 1 1.4 · 10 1 Immobilized 0.50 ± 0.01 9.0 · 10 1 1.8 · 10 2 0.63 ± 0.04 2.1 · 10 2 3.3 · 10 5 1.11 ± 0.05 6.3 · 10 0 4.8 · 10 0 CLIP imprinted with CD 8 0.20 ± 0.01 2.1 · 10 2 1.1 · 10 3 0.25 ± 0.04 9.4 · 10 1 3.8 · 10 5 0.59 ± 0.02 5.3 · 10 0 9.0 · 10 0 A B Incubation time (h) Yield of CD 8 (% of starch) Yield of CD 8 (% of starch) 0 0 5 10 15 20 25 0 5 10 15 20 25 10 20 30 40 0 10 20 30 40 Fig. 5. Time course of CD 8 formation by native (m), immobilized (j) and CD 8 -imprinted CLIP CGTases (s) from A11 (A) and BM (B). Molecular imprinting of glycosyltransferases J. Kaulpiboon et al. 1006 FEBS Journal 274 (2007) 1001–1010 ª 2007 The Authors Journal compilation ª 2007 FEBS amylopectin, dextrin (degree of polymerization 23), cellobi- ose, BSA, phenolphthalein, itaconic anhydride, TNBS, 2,2¢-azobis(2-methylpropionitrile), ethylene glycol dimetha- crylate, water-free cyclohexane and n-propanol were pur- chased from Sigma-Aldrich Chemie GmbH (Munich, Germany). Pea starch (degree of polymerization 4000) was kindly provided by Emsland-Sta ¨ rke GmbH (Emlichheim, Germany). Synthetic amylose with an average molecular mass of 280.9 kDa was prepared by the method of Kitam- ura et al. [23]. Standards of large-ring CD (CD 9 to CD 24 ) were kindly provided by T Endo, Hoshi University, Tokyo, Japan. Rhizopus sp. glucoamylase was obtained from Toy- obo Co., Ltd (Osaka, Japan). BM CGTase was obtained from Amano Enzyme Inc. (Aichi, Japan) and had a specific activity of 1003 UÆmg )1 of dextrinizing activity [13]. A11 CGTase was purified using starch adsorption and ion exchange chromatography (DEAE-Toyopearl 650M col- umn; Tosoh Corporation, Tokyo, Japan). The enzyme had a specific activity of 5000 UÆmg )1 , as determined by its dextrinizing activity [13]. CGTase assays and protein determination Cyclization activity was determined as CD-forming activity by the phenolphthalein method [24]. CGTase (2.5 lg) was added to 0.6 mL of 2.0% (w ⁄ v) soluble potato starch in 0.2 m potassium phosphate buffer (pH 6.0). The reaction mixture was incubated for 30 min at 40 ° C. The reaction was stopped by boiling for 10 min. An aliquot (0.5 mL) was incubated with 2.0 mL of a solution containing 1.0 mL of 4 mm phenolphthalein in ethanol, 4 mL of ethanol and 100 mL of 125 mm Na 2 CO 3 in distilled water. The absorp- tion was measured at 550 nm, and the amount of CD 7 formed was calculated using a calibration curve. One unit of activity was defined as the amount of enzyme that pro- duced 1 lmol of CD 7 per min. The CD 8 -forming activity was determined by HPAEC. The coupling activity was determined by incubating CD 8 as donor with 50 mm cellobiose as glucosyl acceptor at 40 °C. Potassium phosphate buffer, 50 mm (pH 6.0), was added to obtain a total volume of 0.5 mL. CD 8 and cellobi- ose were preincubated for 5 min at 40 °C. The reaction was started by adding enzyme (2.5 lg). After 10 min, the reac- tion was stopped by boiling for 10 min. Subsequently, Rhiz- opus sp. glucoamylase (0.385 U) was added to convert linearized oligosaccharides to glucose at 40 °C for 30 min. The released reducing sugars were determined with the di- nitrosalicylic acid method [25]. One unit of activity was defined as the amount of enzyme that produced 1 lmol glu- coseÆmin )1 . The hydrolysis activity of the CGTase was determined by incubating the enzyme with a CD mixture (CD 6 to CD 24 , kindly provided by M N Mokhtar, Leipzig University) at 40 °C for 10 min. Subsequently, Rhizopus sp. glucoamylase (0.385 U) was added at 40 °C and incubated for 30 min. The amount of glucose formed was determined by HPAEC. One unit of activity was defined as the amount of enzyme that produced 1 lmol glucoseÆmin )1 . All kinetic experiments were carried out at 40 °Cin potassium phosphate buffer (pH 6.0). Lineweaver–Burk diagrams of the initial velocity against substrate concentra- tion were plotted, and kinetic parameters were determined using enzfitter software (Biosoft, Cambridge, UK). A reaction time of 30 min was used in the Lineweaver–Burk experiments. By varying the reaction time with fixed sub- strate concentration, it was confirmed that the reaction velocity was linear at this time point. The protein concentrations were determined according to Bradford [26], using BSA as standard. Analysis of cyclodextrins HPAEC with pulsed amperometric detection was performed using a DX-600 system (Dionex Corp., Sunnyvale, CA, USA) to analyze and quantify the CD products. A Carbo- pac PA-100 analytical column (4 · 250 mm; Dionex Corp.) was used. A sample (25 lL) was injected and eluted with a linear gradient of sodium nitrate (0–10 min, increasing from 0% to 4%; 10–12 min, 4%; 12–32 min, increasing from 4% to 8%; 32–48 min, increasing from 8% to 9%; 48–59 min, A 0 5 10 15 20 25 30 0 5 10 15 20 25 30 h c r a ts elb u los ot a t oP hcrats a e P h crats n r o C hc r ats e ci R e s ol y ma c i t e htnyS ni t ce p o l y ma er up n ro C )3 2=PD ( n i rtxeD B Substrates Yield of CD 8 (% ) Yield of CD 8 (% ) Fig. 6. Yield of CD 8 synthesized by the native (black bars), immobi- lized (gray bars) and CD 8 -imprinted CLIP CGTases (white bars) with different substrates at pH 6.0 for 30 min. The CGTases were from A11 (A) and BM (B). J. Kaulpiboon et al. Molecular imprinting of glycosyltransferases FEBS Journal 274 (2007) 1001–1010 ª 2007 The Authors Journal compilation ª 2007 FEBS 1007 increasing from 9% to 18%; 59–79 min, increasing from 18% to 28%) in 150 mm NaOH containing 2% acetonitrile with a flow rate of 1 mLÆmin )1 . The amounts of CD 6 to CD 24 were quantified by comparison with standard curves of authentic CD 6 to CD 24 samples. Derivatization of the CGTases by acylation with itaconic anhydride Six milligrams of A11 and BM CGTase in 10 mL of 50 mm potassium phosphate buffer (pH 6.0) was acylated by using various amounts of itaconic anhydride. The solution mix- tures with different ratios of itaconic anhydride per mg of protein were stirred at 4 °C for 60 min. The pH was monit- ored and maintained at 6.0 with 3 m NaOH. Nonreacted itaconic anhydride and other low molecular mass com- pounds were removed by gel filtration (HiTrap desalting column; Amersham Biosciences, Uppsala, Sweden) with dis- tilled water as the eluent. The fractions containing CGTase activity were combined and lyophilized. Determination of free amino groups of the CGTases The relative amounts of amino groups of the native and covalently derivatized CGTases were determined according to Habeeb [27] and Hall et al. [28] with TNBS. The extent of derivatization was calculated according to Shetty & Kin- sella [29]: Derivatization degree ð%Þ¼½1 ÀðA der =A nat Þ Â 100 where A der and A nat are the absorbance values obtained with derivatized and native protein solutions, respectively. To a sample (0.3 mL) of native or derivatized protein solution (0.5 mgÆ mL )1 ), 0.3 mL of NaHCO 3 (4%) and 0.3 mL of TNBS (0.1%) were added. The samples were placed in a thermomixer at 37 °C (1000 r.p.m.). After 60 min, 0.47 mL of 1 m HCl was added, and the absorption was measured at 335 nm against a blank treated as above but containing 0.3 mL of deionized water instead of the protein solution. Imprinting of the derivatized CGTases Dry derivatized enzyme (30 mg) and CD 8 (54 mg) were dis- solved in 1 mL of 10 mm potassium phosphate buffer (pH 5.5). The mixture was incubated at 25 °C for 30 min. The CGTase–CD 8 complex was precipitated by adding 4 mL of n-propanol () 20 °C) and kept on ice for 10 min. The precipitate was collected by centrifugation at 13 520 g for 15 min at 4 °C on a Hettich 46R with angle rotor (Het- tich GmbH & Co. KG, Tuttlingen, Germany). The pellet was washed with 1 mL of n-propanol () 20 °C), freeze- dried, and kept at ) 20 °C. Crosslinking of imprinted derivatized CGTases Imprinted derivatized CGTases (10 mg) were suspended in 1 mL of dry cyclohexane by using an ultrasonication bath for 15 min. Four milligrams of 2,2¢-azobis(2-methylpropio- nitrile) and 200 lL of ethylene glycol dimethacrylate were added to the suspension. The radical polymerization was initiated by UV irradiation (k ¼ 312 nm) at 25 °C for 2 h. The resulting polymer was kept in a refrigerator at 4 °C for 12 h. The white polymer was washed with 2 mL of cyclo- hexane and with 50 mm potassium phosphate buffer (pH 6.0) (3 · 10 mL) and lyophilized. The protein amounts and enzyme activities were monitored during the different steps. Effect of pH and temperature on native, immobilized and CD 8 -imprinted CLIP CGTase activity Each enzyme preparation (2.5 lg of protein) was incubated with 2% (w ⁄ v) soluble starch at various pH values and temperatures, and the cyclization activity of the enzymes was assayed by the phenolphthalein method. Potassium phosphate (0.2 m), Tris ⁄ HCl (0.2 m) and glycine ⁄ NaOH (0.2 m) were used as buffers for pH 5.0–7.0, 7.0–9.0 and 9.0–11.0, respectively. For determining the effect of tem- perature on the enzyme activity, the reactions were per- formed between 30 °C and 80 °C. Effect of pH on native, immobilized and CD 8 -imprinted CLIP CGTase stability Each enzyme preparation (2.5 lg of protein) was incubated at 4 °C for 24 h in 10 mm acetate buffer (pH 3.0–5.0), potassium phosphate buffer (pH 5.0–7.0), Tris ⁄ HCl buffer (pH 7.0–9.0), and glycine ⁄ NaOH buffer (pH 9.0–11.0). The remaining cyclization activity was assayed by the phenol- phthalein method. The results were expressed as a percent- age of the highest activity determined, which was defined as 100%. Effect of temperature on native, immobilized and CD 8 -imprinted CLIP CGTase stability The thermostability of the enzyme preparations was investi- gated over the range 30–80 °C. Each enzyme preparation (2.5 lg of protein) in 10 mm potassium phosphate buffer (pH 6.0) was incubated at temperatures between 30 °C and 80 °C for 30 min, and the residual cyclization activity was assayed by the phenolphthalein method. The results were expressed as a percentage of the highest activity determined, which was defined as 100%. Molecular imprinting of glycosyltransferases J. Kaulpiboon et al. 1008 FEBS Journal 274 (2007) 1001–1010 ª 2007 The Authors Journal compilation ª 2007 FEBS Stability of native, immobilized and CD 8 -imprinted CLIP CGTases in organic solvents The organic solvent tolerance of the native, immobilized and CLIP CGTases in ethanol and cyclohexane was determined by incubating the enzyme preparations (0.25 mgÆmL )1 )at30°C on a shaker with 10 mm phosphate buffer (pH 6.0) containing 10–50% of the solvents. After 1 h of incubation, the residual cyclization activity was assayed by the phenolphthalein method. Reuse stability of immobilized and CD 8 -imprinted CLIP CGTases The immobilized and CLIP CGTases were recovered after a synthesis reaction, and analyzed for their remaining cycli- zation activity during five cycles of synthesis reactions. After each cycle, the enzymes were filtered off and washed thoroughly with 10 mm potassium phosphate buffer (pH 6.0). Synthesis of CDs with native, immobilized and CD 8 -imprinted CLIP CGTases The native, immobilized and CLIP CGTases (2 U of cycli- zation activity) were incubated with 2.5 mL of 4% (w ⁄ v) soluble potato starch in 0.2 m potassium phosphate buffer (pH 6.0) at 40 °C for 24 h. The reaction was stopped by boiling for 10 min. Glucoamylase (10 lL, 38.5 UÆmL )1 ) was added for 3 h to convert the linear oligosaccharides to glucose. Subsequently, the glucoamylase was inactivated by boiling for 10 min, and the reaction mixtures were analyzed by HPAEC. 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