Increased amylosucrase activity and specificity, and identification of regions important for activity, specificity and stability through molecular evolution Bart A. van der Veen 1 , Lars K. Skov 2 , Gabrielle Potocki-Ve ´ rone ` se 1 , Michael Gajhede 2 , Pierre Monsan 1 and Magali Remaud-Simeon 1 1 Laboratoire Biotechnologie-Bioproce ´ de ´ s, UMR CNRS 5504, UMR INRA 792, Toulouse, France 2 Biostructural Research, Department of Medicinal Chemistry, The Danish University of Pharmaceutical Sciences, Copenhagen, Denmark Glucansucrases constitute a class of enzymes produ- cing glucose polymers using sucrose as the sole sub- strate and are usually members of glycoside hydrolase (GH) family 70 [1]. Amylosucrase (EC 2.4.1.4) is an exceptional glucansucrase, because it belongs to GH family 13, in which many polyglucan-degrading enzymes (e.g. a-amylase) are found. Furthermore, it produces a glucan consisting of only a-1,4-linked glu- cose residues [2,3], which has recently been shown to be identical to amylose [4]. Unlike amylosucrase, other enzymes responsible for the synthesis of such amylose- like polymers require the addition of expensive activated sugars such as ADP- or UDP-glucose [5]. Amylosucrase can also be used to modify the structure of polysaccharides such as glycogen by the addition of a-1,4-linked glucosyl units [6]. These properties make amylosucrase an interesting enzyme for industrial applications. This requires, however, improvement of its catalytic efficiency on sucrose alone (k cat ¼ 1Æs )1 ) and its stability (t ½ ¼ 21 h at 30 °C), and decrease of the catalysis of nondesired side reactions resulting in sucrose isomer formation, which limits the yield of polymer [6]. Amylosucrase from Neisseria polysaccharea was the first amylosucrase to be studied as a recombin- ant enzyme [2,6]. It is the only glucansucrase for which the structure has been determined [7], and the second Keywords amylosucrase; molecular evolution; polymerase; reaction specificity; sucrose- binding site Correspondence M. Remaud-Simeon, Laboratoire Biotechnologie-Bioproce ´ de ´ s, UMR CNRS 5504, UMR INRA 792, INSA, 135 avenue de Rangueil, 31077 Toulouse Cedex 4, France Fax: +33 561 55 94 00 Tel: +33 561 55 94 46 E-mail: remaud@insa-tlse.fr (Received 12 July 2005, revised 5 October 2005, accepted 24 November 2005) doi:10.1111/j.1742-4658.2005.05076.x Amylosucrase is a transglycosidase which belongs to family 13 of the glyco- side hydrolases and transglycosidases, and catalyses the formation of amy- lose from sucrose. Its potential use as an industrial tool for the synthesis or modification of polysaccharides is hampered by its low catalytic efficiency on sucrose alone, its low stability and the catalysis of side reactions result- ing in sucrose isomer formation. Therefore, combinatorial engineering of the enzyme through random mutagenesis, gene shuffling and selective screening (directed evolution) was applied, in order to generate more effi- cient variants of the enzyme. This resulted in isolation of the most active amylosucrase (Asn387Asp) characterized to date, with a 60% increase in activity and a highly efficient polymerase (Glu227Gly) that produces a lon- ger polymer than the wild-type enzyme. Furthermore, judged from the screening results, several variants are expected to be improved concerning activity and ⁄ or thermostability. Most of the amino acid substitutions observed in the totality of these improved variants are clustered around specific regions. The secondary sucrose-binding site and b strand 7, connec- ted to the important Asp393 residue, are found to be important for amylo- sucrase activity, whereas a specific loop in the B-domain is involved in amylosucrase specificity and stability. Abbreviations DNS, dinitrosalicylic acid; (EP-)PCR, (error prone-)polymerase chain reaction; GH, glycoside hydrolase; GST, glutathione-S-transferase; IPTG, isopropyl thio-b- D-galactoside; OB, oligosaccharide binding site; SB, sucrose-binding site. FEBS Journal 273 (2006) 673–681 ª 2006 The Authors Journal compilation ª 2006 FEBS 673 family 13 enzyme following CGTase [8] for which the structure of a covalent intermediate is available [9]. A- mylosucrase possesses the characteristic (b ⁄ a) 8 -barrel catalytic A domain, a B domain between b strand 3 and a helix 3, and a C-terminal domain consisting of a sandwich of two Greek key motifs. In addition to these common structural features, amylosucrase pos- sesses two unique domains: an a-helical N-terminal domain and a B¢ domain between b strand 7 and a helix 7 in the catalytic core, which has been sugges- ted to be involved in the polymerase activity of this enzyme. The B and B¢ domains contribute largely to the formation of an active site pocket, which is closed on one side by a salt bridge [7]. Several structures of amylosucrase complexed with substrate and products [10,11] have indicated the presence of various import- ant regions inside and outside the active site pocket characterized by sucrose and oligosaccharide-binding sites (SB and OB, respectively, Fig. 1). Combined with biochemical and mutagenic studies [12–15] this allowed elucidation of the enzyme’s features implicated in the amylosucrase reaction mechanism and specificity. Rational engineering based on these data resulted in the construction of a highly efficient polymerase [16]. Further rational improvement of catalytic efficiency or stability would benefit from comparisons of similar enzymes with different characteristics [17]. Such data are not available for amylosucrase, because the only other described amylosucrase, from Deinococcus radiodurans, has very similar catalytic properties and stability [18]. This study deals with optimization of the catalytic properties of amylosucrase to adapt it to industrial synthesis conditions using directed evolution tech- niques, describing the positive variants found by screening of a large variant library. Results and Discussion Library construction and screening for improved variants Genetic variation was introduced by error prone polymerase chain reactions (EP-PCR), followed by shuffling of the PCR products. Cloning and transfor- mation of the shuffling products to Escherichia coli TOP10 yielded % 50 000 clones, the plasmid DNA iso- lated from these clones constituting the variant library. Transformation of this library to E. coli JM109 cells yielded over 100 000 colonies, indicating that most of the 50 000 clones should be represented on the plates. Ninety clones showing amylase formation after one day of growth, and thus probably expressing the most active or efficient polymerases present in the library, were used for screening. Initial screening rounds for increased enzymatic activity or stability resulted in the selection of 39 possibly improved variants, which were transferred in duplicate to a new microtitre plate. Screening of these 39 positives was repeated using the same conditions, finally yielding seven clones improved for various characteristics, each the result of one or two amino acid substitutions (Table 1). Two of the improved clones (E9 and H4) showed significant amylose production after 3 h incubation with sucrose at 37 °C, whereas no amylose production by the wild-type was observed. Variant E9 also showed Fig. 1. Stereo representation of the structure of the Glu328Gln amylosucrase complexed with sucrose bound in the active site pocket (PDB code 1JGI). Surface sites binding sucrose (SB) and oligosaccharides (OB) were added from other structures (PDB 1MW3 and 1MW0, respectively). The central (b ⁄ a) 8 -barrel catalytic domain (A) is flanked by a helical N-terminal domain (N), and a C-terminal domain (C) consist- ing of b strands. Domains B and B¢ are extended loops 3 and 7, respectively, protruding from the A domain. The active site pocket repre- sents SB1 (or OB1); alternative sucrose-binding sites are found in the B¢ domain (SB2), in the N-terminal domain (SB3), and in the B domain (SB4); alternative oligosaccharide binding sites are found in the B¢ domain (OB2), and in the C domain (OB3). Bound sucrose molecules are shown in green, bound oligosaccharides are shown in cyan. All residues that were mutated in the various clones are marked and represen- ted as sticks. The figure was produced using MOLSCRIPT [28] and RENDER3D [29]. Molecular evolution to improve amylosucrase B. A. van der Veen et al. 674 FEBS Journal 273 (2006) 673–681 ª 2006 The Authors Journal compilation ª 2006 FEBS significantly increased activity under all conditions, including retention of activity after preincubation at 50 or 60 °C. These two variants were selected for more detailed characterization. They were cloned in pGEX- 6P-3 and the proteins purified to homogeneity as des- cribed, and verified by electrophoresis followed by silver staining (results not shown). Kinetic analysis of the improved variants The kinetic profile of amylosucrase action on sucrose does not present a classical Michaelian behaviour, but it can be modelled by two different Michaelis–Menten equations, resulting in a high affinity and low V max at low sucrose concentrations (V max1 and Km 1 ) and low affinity and high(er) V max at high sucrose concentra- tions (V max2 and Km 2 ) [13]. In Table 2 the kinetic data for the wild-type and the selected variants are shown. As expected from the screening results, variant E9 shows a general increase in activity and catalytic effi- ciency. Although activity did not show the threefold increase found during screening, this variant is the most active amylosucrase found to date. In contrast, variant H4, selected for improved polymer formation, showed a general decrease in catalytic efficiency. The improvement of this variant compared with the wild- type is found in the significantly increased polymeriza- tion activity at high sucrose concentrations, and the twofold increased ratio of polymerization over hydro- lysis at both low and high sucrose concentrations. Polymerase efficiency of the improved variants The results of the iodine staining of polymer formed by the variants are shown in Table 3. Contrary to the wild-type, variant H4 produces polymer from low con- centrations of sucrose (5 mm) and under all conditions this variant produces longer amylose chains than the wild-type, as judged by the increase in k max . These findings can be related to the increased ratio of poly- merization over hydrolysis activity. Thus, in this variant the different reactions (hydrolysis and poly- merization) are affected differently, in which case the Table 1. Screening and sequence results of the improved variants. Act., activity based on DNS response; Tstab, improved thermostability; Pol., improved polymerase. Characteristics screening Purified enzyme Mutations DNA Protein A9 Act. 1.5· C45T, A226G N76D A10 Tstab. C469G, G691T,C1441T P157A, D231Y D1 Tstab. C698T,C1239T,G1597A P234L, G554S D8 Act. 1.5· G184A, C1317T, G1516A E62K, D506N E9 Act. 3·⁄Tstab. Improved Act. ⁄ Tstab. G99A, G495A, A1159G N387D F9 Act. 1.5· G1221T, G1839T Q613H H4 Pol. + Improved Pol. A680G E227G D2 a Act. 2· Improved Act. ⁄ Pol. C123T, G1165C, A1509T V389L, N503I G1 a Act. 2· Improved Act. ⁄ Pol. C58T, G444T, T1793C R20C, F598S a Variants described previously [19]. Table 2. Kinetics of the action on sucrose of (variant) enzymes. Kin- etic values that reflect the improved properties suggested by the screening results [improved activity (E9) or enhanced polymer for- mation (H4)] are indicated in bold. Km 1 (mM) k cat1 (s )1 ) k cat1 ⁄ Km 1 (s )1 ÆmM )1 ) Km 2 (mM) k cat2 (s )1 ) k cat2 ⁄ Km 2 (s )1 ÆmM )1 ) Total activity Wild-type 4.0 0.74 0.19 71 1.4 0.020 H4 4.4 0.60 0.14 165 1.7 0.010 E9 4.2 1.19 0.28 82 2.2 0.026 Hydrolysis Wild-type 2.5 0.35 0.14 29 0.52 0.018 H4 0.8 0.18 0.23 48 0.43 0.009 E9 2.3 0.54 0.23 62 0.86 0.014 Polymerization Wild-type 8.1 0.43 (1.2) a 0.05 112 0.90 (1.7) 0.008 H4 9.6 0.36 (2.0) 0.04 300 1.43 (3.3) 0.005 E9 5.6 0.64 (1.2) 0.11 102 1.30 (1.5) 0.013 a Values between brackets indicate the ratio polymerization ⁄ hydro- lysis. Table 3. k max of the iodine-stained reaction products from different concentrations of sucrose after 24 h incubation at 30 °C with (vari- ant) enzymes. The average DP of the amylose products, calculated using the formula in the methods section, is shown between brack- ets. nd, not detectable. [Suc] (m M) 5 1020 50100200 Wild-type nd nd 560 (45) 575 (57) 570 (52) 555 (42) H4 580 (62) 595 (84) 605 (108) 600 (94) 605 (108) 585 (68) E9 nd nd 570 (52) 580 (62) 570 (52) 560 (45) B. A. van der Veen et al. Molecular evolution to improve amylosucrase FEBS Journal 273 (2006) 673–681 ª 2006 The Authors Journal compilation ª 2006 FEBS 675 nature of the produced polymer is affected. Similarly, a general increase in catalytic efficiency, as observed for variant E9, does not significantly affect polymer synthesis. Furthermore, polymer formation occurs in the later stages of the reaction (initially polymerization consists of oligosaccharide formation), and also depends on the affinity for the oligosaccharides pro- duced to be used as acceptors. This appears to be improved for variant H4, as has been shown previ- ously for mutant Arg226Ala [16]. Temperature dependency of (variant) amylosucrases Under screening conditions, variant E9 also showed some increased thermostability, hence the temperature dependency of amylosucrase activity was investigated (Fig. 2). The wild-type enzyme is very rapidly dena- tured at temperatures over 50 °C, thus no activity can be measured at these temperatures (manuscript in preparation). Compared with the wild-type, activity at elevated temperatures had increased drastically for variant E9, which indicates increased stability. In con- trast, variant H4, which was not selected for increased thermostability, appears to have a decreased stability and the temperature optimum is decreased compared with that of the wild-type. Structural analysis of the mutations The effects of the mutations on enzyme properties are given in Table 1, and the positions of the mutated resi- dues in the crystal structure of amylosucrase are shown in Fig. 1, which also shows the binding sites of sucrose [10] and oligosaccharides [11]. It is immediately obvi- ous that the mutations are grouped in certain regions of the structure. Although several mutations are found in the vicinity of the sucrose-binding site SB2, which is separated from the active site pocket by a salt bridge formed by residues Asp144 and Arg509 (Fig. 1), few mutations are found at the other binding sites, and none in the amylosucrase-specific B¢ domain, or at the substrate access channel. Regions involved in activity In each of the two variants described in more detail here, only one amino acid substitution was found. In the first variant, E9, which is the most active amylo- sucrase found to date, Asn387 in b strand 7 is replaced by an aspartate. A positive effect on activity by muta- tions in b strand 7 is also shown by variant D2 in which Val389 at the end of b strand 7 is replaced by leucine [19]. These mutations probably affect the first part of loop 7 (B¢ domain) and consequently the important Asp393 residue (Fig. 3), which is conserved in all GH family 13 enzymes, and plays an essential role in catalysis by stabilizing the glucose residue bound at subsite )1 in the various reaction stages [8]. Interestingly, a second mutation in variant D2, Asn503Ile, is situated in the group of mutations close to SB2 (Fig. 4). It is found in the part of loop 8 that also contains Arg509, and interacts with a sucrose bound at SB2 via the backbone nitrogen of Ser508. Another mutation found in this loop is Asp506Asn in variant D8, which also shows increased activity under screening conditions. Such mutations probably influ- ence the properties of loop 8 in this region, thus affect- ing SB2 and Arg509 forming the salt bridge, indicating that these specific amylosucrase features are involved in catalysis. Regions involved in reaction specificity A very interesting region containing mutations near SB2 is the loop in the B domain including residue Glu227 which has been mutated in variant H4 (Fig. 5). Variant Glu227Gly found in the shuffling library and site-directed mutant Arg226Ala [16] both result in highly efficient polymerases. Thus via this loop the B domain is very important for reaction specificity via 0 20 40 60 80 100 120 15 25 35 45 55 65 Temperature (°C) Relative activity (%) Fig. 2. Temperature optima of the variants. Wild-type (s), H4 (n), and E9 (d) amylosucrase activity was measured at different tem- peratures, and the values recalculated as the percentage of the maximal activity for the enzyme concerned. Molecular evolution to improve amylosucrase B. A. van der Veen et al. 676 FEBS Journal 273 (2006) 673–681 ª 2006 The Authors Journal compilation ª 2006 FEBS oligosaccharide binding in the active site (OB1; Fig. 5A), which is also observed in other family 13 enzymes such as cyclodextrin glycosyltransferase, in which several residues of the B domain are essential for the catalysis of the characteristic cyclization reac- tion [20,21]. Regions involved in thermostability Both variant enzymes Arg226Ala and Glu227Gly show reduced thermostability, indicating that this loop in the B domain is also involved in the thermostability of amylosucrase. In fact, two variants that were positive when screening for improved thermostability have amino acid substitutions in this loop. In variant A10 an Asp231Tyr mutation occurs, which is actually the only mutation that directly affects a sucrose-binding residue (Fig. 5B). Furthermore, Asp231 has been des- cribed as the most important ‘geometric lock’ respon- sible for a closed conformation of a highly flexible loop in the B¢ domain. Removal of the Asp231 side chain allowed simulation of large movements of this loop using geometric techniques [22]. The Asp231Tyr mutation probably improves interactions with hydro- phobic residues of this neighbouring loop in the B¢ domain, further stabilizing it. In variant A10, this mutation is combined with a Pro157Ala mutation in loop 2, a substitution which is not expected when looking for thermostability. In another variant, D1, such a contradictory mutation is Pro234Leu in the connection of the Glu227 loop to a b sheet in the B domain. However, in this variant a second mutation is Gly554Ser in the loop connecting the catalytic domain and the C domain, which may be another important area for protein stability. Mutations in the N-terminal domain Besides the remarkable cluster close to SB2, also sev- eral mutations are found in the N-terminal domain. In variant D8, containing the Asp506Asn mutation, a Glu62Lys mutation is found in an a helix in the N-ter- minal domain, which does not provide a logical explanation for the increase in activity. Also in variant G1 a mutation (Arg20Cys) is found in the N-terminal domain, however, in this case, the mutated residue (Arg20) participates in the SB3 site and may in this way affect the enzymatic activity. Another mutation in Fig. 4. Detail of the structure of amylosucrase complexed with sucrose (PDB code 1MW3), showing the positions of the mutated residues Asn503 and Asp506. In this structure a Tris molecule is bound at the catalytic site, indicated by the three catalytic residues (Asp286, Glu328 and Asp393), and sucrose (Suc) is bound at SB2, close to the salt bridge formed by Asp144 and Arg509. Mutated residues Asn503 and Asp506 are located in a flexible loop connect- ing two helical parts of loop 8 (purple). Besides Arg509 the second helix contains residues Ser508, hydrogen bonding to the sucrose with its backbone nitrogen. The central b-barrel is shown as solid strands depicted in yellow. The figure was produced using PYMOL (W. L. DeLano, DeLano Scientific, San Carlos, CA). Fig. 3. Detail of the structure of the Glu328Gln amylosucrase com- plexed with maltoheptaose (PDB code 1MW0), showing the posi- tions of the mutated residues Asn387 in b strand 7, and Val389 in the first part of loop 7 (purple). Only the two glucose residues (G2) around the cleavage site are shown and represented as sticks, as are the three catalytic residues (Asp286, Gln328, and Asp393), and the residues forming the salt bridge that closes the active site (Asp144 and Arg509). The central b-barrel is shown as solid strands depicted in yellow. The figure was produced using PYMOL (W. L. DeLano, DeLano Scientific, San Carlos, CA) B. A. van der Veen et al. Molecular evolution to improve amylosucrase FEBS Journal 273 (2006) 673–681 ª 2006 The Authors Journal compilation ª 2006 FEBS 677 the N-terminal domain that appears to have a positive effect on activity is found in variant A9. Here, the only substitution is Asn76Asp, situated in a bend connect- ing two a helices and no obvious structurally based reason for the improvement can be found. Mutations in the C-terminal domain A second mutation in variant G1 is Phe598Ser in the C-terminal domain, which may have some effect, because it replaces a solvent-exposed hydrophobic resi- due with a hydrophilic residue. In the C domain another mutation found is Gln613His, in variant F9, which shows a slight increase in activity under screen- ing conditions. Also for these substitutions no direct explanation for a positive effect on enzyme activity can be derived from the structure. In conclusion, screening and analysis of a large amy- losucrase variant library resulted in the isolation of a very efficient polymerase and the most active amylo- sucrase enzyme characterized to date, both resulting from mutations that would not be chosen rationally. Furthermore, regions could be identified in the enzyme that are clearly important for amylosucrase activity, as b strand 7, connecting to the important Asp393 resi- due, and the region close to the salt bridge and the secondary sucrose-binding site SB2. Other regions are involved in specificity and thermostability, as the loop containing Glu227 in the B domain. These findings provide new perspectives for engineering improved amylosucrase enzymes for industrial applications by site-directed or massive mutagenesis in the identified regions. Experimental procedures Bacterial strains and plasmids ⁄ growth conditions One ShotÒ E. coli TOP10 (Invitrogen, Carlsbad, CA) was used for transformation of ligation mixtures. E. coli JM109 (Promega, Madison, WI) was used to screen amylosucrase variants and large-scale production of the selected mutants. Plasmid pZErO-2 (Invitrogen) was used for subcloning of PCR products and screening, and plasmid pGEX-6P-3 (Amersham Pharmacia Biotech, Piscataway, NJ) was used for production of glutathione S-transferase (GST)–amylo- sucrase fusion proteins. Bacterial cells were grown on Luria–Bertani (agar) containing 50 lgÆmL )1 kanamycin (when harbouring plasmid pCEASE01S01F), or 100 lgÆmL )1 ampicillin (when harbouring a pGEX-6P-3- derived plasmid). To express amylosucrase in E. coli JM109 media were supplemented with isopropyl thio-b-d-galacto- side (IPTG; 1 mm). When appropriate, Luria–Bertani agar plates contained 50 gÆL )1 sucrose for visualization of amy- losucrase activity, by halos formed through formation of amylose in the agar. A B Fig. 5. The Glu227 loop in (A) the structure of the Glu328Gln amy- losucrase complexed with maltoheptaose (B) the structure of amy- losucrase complexed with sucrose. This flexible loop (purple) is situated between an a helix and a b strand in the B domain. Unlike Asp226, none of the mutated residues in this loop interact with maltotheptaose bound in the active site in (A). However, Asp231 has hydrogen bonding interactions with the sucrose bound at SB2 in (B). Further, highlighted are the three catalytic residues (Asp286, Gln ⁄ Glu328 and Asp393), the residues forming the salt bridge that closes the active site (Asp144 and Arg509), and the Tris molecule bound in the active site (B). The central b-barrel is shown as solid yellow strands. Figure produced using PYSMOL (W. L. DeLano, DeLano Scientific, San Carlos, CA). Molecular evolution to improve amylosucrase B. A. van der Veen et al. 678 FEBS Journal 273 (2006) 673–681 ª 2006 The Authors Journal compilation ª 2006 FEBS DNA manipulations Restriction endonucleases and DNA-modifying enzymes were purchased from New England Biolabs (Ipswich, MA) and used according to the manufacturer’s instructions. DNA purification was performed using QIAQuick (gel extraction) and QIASpin (miniprep; Qiagen, Valencia, CA). DNA sequencing was carried out using the di-deoxy chain- termination procedure [23] by MilleGen (Labe ` ge, France). Generation of variant libraries EP-PCR using two different enzymes, Mutazyme (Strata- gene, La Jolla, CA) and Taq DNA-polymerase (New Eng- land Biolabs), was applied to introduce random mutations and the PCR products shuffled as described previously [19]. The shuffling products were digested with HindIII and Xho I and ligated with pZErO digested with the same enzymes. The resulting constructs were transformed to E. coli TOP10 cells and plated on Luria–Bertani agar plates containing sucrose. The colonies were scraped from these plates for isolation of the plasmids, constituting the shuffling library. Selection of positive clones The shuffling gene library pCEASE01S01F was trans- formed to E. coli JM109 and plated on Luria–Bertani agar containing sucrose. From these plates, clones showing for- mation of amylose after one day of growth, thus expressing highly active or efficient polymerases, were identified visu- ally due to the precipitation of the polymer. These were selected and grown in microtitre plates containing 200 lL Luria–Bertani per well, supplemented with 1 mm IPTG and 50 lgÆmL )1 kanamycin. These mini-cultures were horizon- tally shaken at 250 r.p.m., for 15 h at 30 ° C. Screening for improved amylosucrases Because amylosucrase is produced intracellularly, lysozyme was added to a final concentration of 0.5 gÆL )1 and the cells were frozen at )20 °C. After thawing for 30 min at room temperature, several screening conditions were applied to select improved amylosucrases. The screen for increased enzymatic activity was carried out with sucrose alone as substrate, at a final concentration of 150 mm. Reactions were performed at combinations of temperature and incubation time that resulted in only slight product formation for the wild-type. Incubations at 30 °C for 6 h or 37 °C for 3 h were used in this study. Reducing- sugar production was measured by adding 50 lL of the reaction mixture to 50 lL of dinitrosalicylic acid (DNS) [24], incubating at 95 °C for 7 min, adding 60 lL of this mixture to 180 lLH 2 O, and measuring the absorbance at 540 nm. The formation of the amylose-type polymer was analysed by adding 10 lL of iodine solution (100 mm KI, 6 mm I 2 , 0.02 m HCl) to the remaining reaction mix- ture, the positive clones being revealed by development of a blue colour. Changes in ratios of these separate measure- ments are indicative of changes in polymerization efficiency [19]. Screening for thermostability was carried out by preincu- bation of the microtitre plates at elevated temperatures (20 min 50 °C, 10 min 60 °C), which inactivates the wild- type enzyme. After cooling, sucrose and glycogen (final concentrations 150 mm and 5 gÆl )1 , respectively) were added as substrate, glycogen being a strong activator of amylo- sucrase activity [25]. After overnight incubation at 30 °C, iodine staining was used to detect polymer formation by variants that remained active. Production and purification of improved variants Selected clones were grown in 4 mL Luria–Bertani cultures for plasmid isolation. After sequencing, the genes of the most promising variants were subcloned in vector pGEX- 6P-3, using the EcoRI and XhoI restriction sites, for GST fusion protein expression. Variant GST–amylosucrases were produced in 100 mL cultures using E. coli JM109 as host and the proteins were extracted as described previously [6]. Purification of the variant amylosucrases was carried out as described by the provider of plasmid pGEX-6P-3 (Amer- sham Pharmacia Biotech), using the on column cleavage protocol to elute GST-free enzyme. The purity of the enzymes was analysed by electrophoresis on the PHAST system (Amersham Pharmacia Biotech), using PhastGel tm gradient 8–25 (Amersham Pharmacia Biotech) under dena- turing conditions, followed by staining with 0.5% (w ⁄ v) AgNO 3 . Previously purified wild-type GST–amylosucrase [19] was used as reference in characterization of the vari- ants; the GST-fusion having been reported as not influen- cing the catalytic properties of the enzyme [16]. Protein concentration determination Protein concentrations were determined with the Bradford method [26] using the Bio-Rad reagent (Bio-Rad Laborat- ories, Hercules, CA) and bovine serum albumin as a standard. Kinetic analysis of the improved variants Kinetic parameters of the action on sucrose were deter- mined by incubating various substrate concentrations (5 mm)1 m) with % 0.1 mgÆmL )1 of pure enzyme at 30 °C. At regular time intervals (5 min) 20 lL samples were taken and the amylosucrase was immediately inactivated by heat- ing (3 min 90 °C). The formation of glucose and fructose was analysed using the d-glucose ⁄ d-fructose UV-method B. A. van der Veen et al. Molecular evolution to improve amylosucrase FEBS Journal 273 (2006) 673–681 ª 2006 The Authors Journal compilation ª 2006 FEBS 679 (Boehringer Mannheim ⁄ R-Biopharm, Mannheim, Germany) according to the manufacturer’s procedure, but scaled down to be used in microtitre plates. The glucose formation reflects the hydrolysing activity, because it can only be formed when water is used as acceptor. The fructose forma- tion reflects the total consumption of sucrose, and thus the total activity. The fructose formation minus glucose forma- tion then reflects the polymerization activity [19]. Polymerase efficiency of the improved variants Polymer formation was analysed by iodine staining of a sample taken after 24 h incubation; the comparative length of produced polymer was judged by the optimal wavelength (higher k max ¼ longer polymer). For shorter amylose chains (< 120 glucose residues) such as produced by amylosucrase [4] an increase in k max with increase in the average degree of polymerization is observed according to the following formula [27]: average degree of polymerization ¼1:025e À2 =ð1k À1 max À1:558e À3 Þ: Temperature dependency of (variant) amylosucrases The optimal reaction temperature was determined by meas- uring the standard activity at different temperatures. Stand- ard activity is determined by incubating the enzyme with sucrose and glycogen at final concentrations of 146 mm and 0.1 gÆL )1 , respectively [6], and measuring the fructose for- mation using the DNS method. All assays were performed in duplicate at least, and devi- ations were < 10%. 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