International Symposium on Extremophiles and Their Applications 2005

The concept of the !- family: a rational tool for interconverting glucanohydrolases/glucanotransferases, and their specificities

Takashi Kuriki

Biochemical Research Laboratory, Ezaki Glico Co., Ltd., 4-6-5 Utajima, Nishiyodogawa-ku, Osaka 555-8502, Japan Email: [email protected]

Abstract Neopullulanase catalyzes the hydrolysis of !-1,4- and !-1,6-glucosidic linkages, as well as transglycosylation to form !-1,4- and !-1,6-glucosidic linkages. Based on the series of experimental results using the neopullulanase, we pointed out the same catalytic machinery and the common catalytic mechanism of the that catalyze these four reactions, and thus, proposed and defined the concept of the !-amylase family. Mutational and structural analyses provided the conclusive proof that one active center of neopullulanase participate in all four reactions. We have been trying to interconvert glucanohydrolases/glucanotransferases, and their specificities and create tailor-made industrially useful enzymes based on the concept of the !-amylase family. We engineered Thermus amylomaltase to essentially erase hydrolytic activity and created perfect 4-!-glucanotransferase for the industrial production of cycloamylose.

Keywords; !-amylase family, neopullulanase, catalytic mechanism, catalytic machinery, protein engineering

1. Introduction

All enzymes known to date have individual EC number based on the recommendation of the International Union of Biochemistry and Molecular Biology (IUBMB). This classification is based on two factors, the reaction catalyzed and the substrate specificity. This is consistent with the “key-lock” hypothesis postulated by Emil Fischer in 1884. It has been estimated that more than 25,000 enzymes exist in nature. However, Chothia [1] speculated that the number of basic topological motifs of protein is most likely limited to around one thousand. Therefore, it is quite natural that similar folding pattern is often seen in different enzymes which have clearly distinct functions [2]. The concept of !-amylase family [3] was proposed independently from the classification of carbohydrate-active enzymes based on sequence similarities [4, for updated form http://afmb.cnrs-mrs.fr/CAZY/]. In 1989, we had already pointed out the common catalytic machinery among !-amylase (EC 3.2.1.1), cyclomaltodextrin glucanotransferase (EC 2.4.1.19), (EC 3.2.1.41), isoamylase (EC 3.2.1.68), and neopullulanase, and the existence of four highly conserved regions which contains all the catalytic residues and the substrate-binding residues that bind glucosyl residues adjacent to the scissile linkage in the substrate even though each enzyme has different function, based on the experimental results for neopullulanase [5] (Fig. 1).

50 International Symposium on Extremophiles and Their Applications 2005

Enzyme Origin Region1 Region2 Region3 Region4

!-A myl ase As per gil lus or yzae 11 7 DVVANH 20 2 GLRI DTVKH 23 0 EVLD 29 2 FVENH D CG Tas e Ba cil lus ma cer ans 13 5 DFAPNH 22 5 GIRF DAVKH 25 8 EWFL 32 4 FIDNH D Pu llu lan ase Kl ebs iel la aer ogen es 60 0 DVVYNH 67 1 GFRF DLMGY 70 4 EGWD 82 7 YVSKH D Is oam yla se Ps eud omo nas am ylod era mos a 29 2 DVVYNH 37 1 GFRF DLASV 43 5 EPWA 50 5 FIDVH D Br anc hin g e nzy me Es che ric hia co li 33 5 DWVPGH 40 1 ALRV DAVAS 45 8 EEST 52 1 LPLSH D Ne opu llu lan ase Ba cil lus st ear othe rmo phi lus 24 2 DAVFNH 32 4 GWRL DVANE 35 7 EIWH 41 9 LLGSH D Am ylo pul lul ana se Th erm oan aer oba cter et han oli cus 48 7 DGVFNH 59 3 GWRL DVANE 62 6 ELWG 69 8 LLGSH D !-G luc osi das e Sa cch aro myc es carl sbe rge nes is 10 6 DLVINH 21 0 GFRI DTAGL 27 6 EVAH 34 4 YIENH D Cy clo dex tri nas e Th erm oan aer oba cter et han oli cus 23 8 DAVFNH 32 1 GWRL DVANE 35 4 EVWH 41 6 LIGSH D Ol igo -1, 6-g luc osid ase Ba c il lus ce reu s 98 DLVVNH 19 5 GFRM DVINF 25 5 EMPG 32 4 YWNNH D De xtr an glu cos idas e St rep toc occ us muta ns 98 DLVVNH 19 0 GFRM DVIDM 23 6 ETWG 30 8 FWNNH D Am ylo mal tas e St rep toc occ us pneu mon iae 22 4 DMWAND 29 1 IVRI DHFRG 33 2 EELG 39 1 YTGTH D Gl yco gen de bra nchi ng en zym e Ho mo sap ien s 29 8 DVVYNH 50 4 GVRL DNCHS 53 4 ELFT 60 3 MDITH D Am ylo suc ras e Ne iss eri a p oly sacc har ea 19 0 DFIFNH 29 0 IL RM DAVAF 33 6 EAIV 39 6 YVRSH D Tr eha los e-6 -ph osph ate hy dro las e Es che ric hia co li 10 0 DMVFNH 19 6 GLRL DVVNL 25 1 EMSS 32 0 FWCNH D Ma lto oli gos ylt reha los e h ydr ola se Ar thr oba cto r sp . 20 2 DVVYNH 26 3 GLRL DAVHA 30 4 ESDL 39 5 CSQNH D Ma lto oli gos ylt reha los e s ynt has e Ar thr oba cto r sp . 87 DIVPNH 23 3 GLRI DHPDG 26 4 EKIL 47 8 TLSTH D Tr eha los e s ynt hase Pi mel oba cto r sp. 108 DFVMNH 20 6 GF RL DAVPY 25 2 EANQ 32 2 F L RNH D Fig. 1 Enzymes belonging to !-amylase family and four highly conserved regions. Invariable three catalytic sites are highlighted by inversion. Numbering of the amino acid sequences of the enzymes starts at the amino-terminal amino acid of each mature enzyme.

The study on neopullulanase was the key to open the door for the formulation of the concept of the !-amylase family [6]. We found a new enzyme, neopullulanase (EC 3.2.1.135) from Bacillus stearothermophilus [7], and showed that it catalyzes the hydrolysis of !-1,4- and !-1,6-glucosidic linkages [8], as well as transglycosylation to form !-1,4- and !-1,6- glucosidic linkages [3]. The replacement of several amino acid residues that constitute the active center of the neopullulanase showed that one active center of the enzyme participated in all four reactions described above [9]. Pointing out the same catalytic machinery and the common catalytic mechanism of the enzymes that catalyze these four reactions, we proposed and defined a general idea for the !-amylase family [3]. We describe here the concept of the !-amylase family focusing attention on our work. Other important contributions to establish the concept have been also described in excellent reviews [10, 11, 12]. We defined !-amylase family as one enzyme family that satisfy the following requirements: the !-amylase family enzymes; (i) act on !-glucosidic linkages; (ii) hydrolyze !-glucosidic linkages to produce !-anomeric mono- and oligo-saccharides or form !- glucosidic linkages by transglycosylations; (iii) have four highly conserved regions in their primary sequences which contain all the catalytic and most of the important substrate-binding sites; and (iv) have Asp, Glu, and Asp residues as catalytic sites corresponding to Asp206, Glu230, and Asp297 of Taka-amylase A [3]. We did not include the ("/!)8-barrel structure which is commonly seen in !-amylase family enzymes as one of our original definitions of !-amylase family. From the viewpoints of the convergent evolution of the ancestral proteins and the chemical reactions, we think that there should be many enzymes that catalyze the same reaction even though they have totally different structures [2]. Indeed, the xylanases from Cellulomonas fimi and Streptomyces lividans are composed of ("/!)8-barrel structure although the xylanase from Bacillus circulans is composed of "-jelly roll (http://afmb.cnrs- mrs.fr/CAZY/). We think that the importance may lie in the local geometrical arrangements of catalytic and substrate-binding residues, but not in the whole structure of the protein. Whole structure may reflect the stable topological motif of the protein in water and the evolutional relationship. In fact, recent study revealed the existence of !-,

51 International Symposium on Extremophiles and Their Applications 2005

amylopullulanases (EC 3.2.1.1/41), and 4-!-glucanotransrerases (2.4.1.25) in the proteins having ("/!)7-barrel structure [13]. By the classification of carbohydrate-active enzymes based on amino acid sequence similarities (http://afmb.cnrs-mrs.fr/CAZY/), these enzymes are classified into glycoside families 57 (GH-57), although the enzymes belonging to !-amylase family were in clan GH-H covering three families 13, 70, and 77 (GH-13, 70, and 77) [14]. This is also a fundamentally different point between the concept of the !-amylase family and the classification of carbohydrate-active enzymes based on amino acid sequence similarities (http://afmb.cnrs-mrs.fr/CAZY/). In order to explain the concept of the !-amylase family, we schematically represented the relationship of specificities for the target linkage and reaction of the enzymes typically belonging to the !-amylase family [2, 3, 6, 15] (Fig. 2). Hydrolysis of !-1,4-glicosidic linkage is typically catalyzed by !-amylase. Pullulanase and isoamylase hydrolyze !-1,6 linkage [16, 17]. Amylomaltase catalyzes transglycosylation to form !-1,4-glucosidic linkage. Branching enzyme catalyzes transglycosylation to form !-1,6 linkage [18-21]. These four reactions and classification of these enzymes have been clearly distinguished. Each of the four reactions is representatively catalyzed by one of four individual types of enzymes. However, some exceptional examples have been also reported. A type of !-amylase significantly catalyze !-1,4 transglycosylation in addition to the main reaction, !-1,4 hydrolysis [22]. Cyclomaltodextrin glucanotransferases (CGTase) feebly but significantly catalyze !-1,4 hydrolysis [23]. We may, therefore, reasonably conclude that the boundary between glucanohydrolases and glucanotransferases is not necessarily clear. A type of pullulanase [24] and an !-amylase [25] have been reported to hydrolyze not only !-1,6- but also !-1,4-glucosidic linkages. Therefore, we are aware of the existence of enzyme on the boundary between !-amylase and pullulanase or isoamylase. Neopullulanase catalyzes all four reactions, hence, the enzyme should be located in the center as shown in Fig. 2 [2, 3, 6, 15]. !-1,4 Specificity for glucosidic linkage !-1,6 Hydrolysis !-Amylase Amylopullulanase Pullulanase Fig. 2. Schematic representation of the T. vulgaris -amylase ! Isoamylase relationship of specificities for the target Bacterial linkage and reaction of the enzymes typically saccharifying !-amylase belong to the !-amylase family. CGTase, Fate of cyclomaltodextrin glucanotransferase; Reaction Neopullulanase T. vulgaris, Thermoactinomyces vulgaris.

CGTase Trans- Amylomaltase Branching enzyme glycosylation

2. Why the concept of the !-amylase family is important for us? Various !-amylases, , and isoamylases, have been industrially produced and used for various industrial purposes. CGTases have been also used for production of various cyclodextrins and glycosides [26-28]. We have first industrialized branching enzyme and developed a new material; Cluster DextrinTM [29]. We also have first industrialized amylomaltase and developed cyclic !-1,4 glucan with a degree of polymerization of 22 and higher [refers as cycloamylose hereafter; 29, 30]. Thus, we have been industrializing various !-amylase family enzymes [31-38]. We have also been trying to interconvert glucanohydrolases/glucanotransferases, and their specificity and create tailor-made

52 International Symposium on Extremophiles and Their Applications 2005

industrially useful enzymes based on the concept of the !-amylase family. We have controlled the specificity toward !-1,4- and !-1,6-glucosidic linkages and transglycosylation activity of neopullulanase [39]. We have changed the substrate specificities and product specificities of branching enzymes [40]. We have introduced raw-starch binding and hydrolyzing activities to an !-amylase by domain fusion with an CGTase [41-45]. The enzymes from hyperthermophile often show broad reaction/substrate specificities. The idea is now widely accepted that hyperthermophiles are most likely the ancestors of all existing life on earth. Therefore, it is reasonable to think that ancestral enzymes probably had broad specificities. Amylomaltase from hyperthermophile [46], Thermus aquaticus, and D- enzyme from potato [30] catalyze intramolecular transglycosylation of !-1,4 glucan to produce cycloamylose. The yield of cycloamylose using the D-enzyme was more than 90 % since the enzyme did not exhibit any hydrolytic activity [30], however, the enzyme was not thermostable enough to be used for industrial production of cycloamylose. Although the themostability was high enough to be used for the industrial scale, the yield of cycloamylose using Thermus auaticus (Taq) amylomaltase decreased to 60 % at the end point of reaction since the enzyme significantly exhibited hydrolytic activity [47, 48]. In such case, we have two ways to overcome the problems. One is enhancing the thermostability of potato D- enzyme, and another is erasing the hydrolytic activity of Taq amylomaltase. Indeed, we successfully enhanced thermostability of potato type L !-glucan phosphorylase [49] and Streptococcus sucrose phosphorylase [50]. We chose the latter strategy for Taq amylomaltase [47, 48]. The engineered amylomaltase with Y54G mutation exhibited 1.67-time higher cyclization activity than the wild-type enzyme and essentially no hydrolytic activity [47, 48]. The yield of cycloamylose using the engineered enzyme was remained at more than 90 % until the end of reaction, while the reducing power was not significantly increased [47, 48]. We are going further to improve enzymes based on the concept of the !-amylase family as a rational tool for designing and engineering the industrially useful enzymes.

3. The evidence that one active center of neopullulanase participates in all four reactions; hydrolysis of !-1,4- and !-1,6-glucosidic linkages and transglycosylation to form !-1,4- and !-1,6-glucosidic linkages by mutational analyses Neopullanase was the key enzyme to open the door for formulation of the concept of !-amylase family as described previously (Fig. 2). Neopullanase catalyzes !-1,4 and !-1,6 hydrolyses and !-1,4 and !-1,6 transglycosylations. If one active center of neopullanase definitely catalyzes the all four reactions, we can conclude that the catalytic mechanisms of the enzymes belong to !-amylase family are basically the same. That is an evidence that supports the concept of the !-amylase family; the same catalytic machinery and the common catalytic mechanism. If the same active center of neopullulanase participates in all four reactions, we should obtain the following results: (i) neopullulanase simultaneously loses all activities following the replacement of one of the catalytic residues, and (ii) the enzyme specificity toward each glucosidic linkage can be altered by the replacement of the residue involved in substrate recognition. Using the alignment of amino acid residues in the four highly conserved regions [5] (Fig. 1), a proposed substrate-biding model of Taka-amylase A [51], which was the only available three-dimensional structure of the !-amylase family enzyme at that time, was used for identification of amino acid residues constituting the active center of the neopullulanase [9] (Fig. 3). When one of the catalytic residues, Asp328, Glu357, and Asp424, was replaced by their amide form, Asn, Gln, and Asn, respectively,

53 International Symposium on Extremophiles and Their Applications 2005

neopullulanase lost all four activities [9] (Fig. 4). When one or two of the residues involved substrate recognition were changed, the mutated neopullulanases exhibited different specificities toward !-1,4- and !-1,6-glucosidic linkages [9] (Fig. 3). This was the first clear evidence showing one active center of an enzyme participates in hydrolysis of !-1,4 and !- 1,6-glucosidic linkages and transglycosylation to form !-1,4- and !-1,6-glucosidic linkages by mutational analysis. These observations were the key evidence to formulate the concept of the !-amylase family as described previously. Ser-Val

Fig. 3 Preliminary substrate-binding model of neopullulanase based on that of Taka-amylase His A [51], and introducing mutation at catalytic sites and the residues involved in substrate recognition of neopullulanase. Catalytic and Asn substrate-binding sites for Taka-amylase A are Glu indicated oblongs and rectangles, respectively. Corresponding amino acid residues of neopullulanase are shown in parentheses. Gln

Asn

Glu

4. Structural analyses provide the conclusive proof that one active center of neopullulanase participates in all four reactions; hydrolysis of !-1,4- and !-1,6- glucosidic linkages and transglycosylation to form !-1,4- and !-1,6-glucosidic linkages We recently determined crystal structure of the neopullulanase [52] and its complexes with panose, maltotetraose, and isopanose [53]. The active enzyme forms a dimer in the crystalline state and in solution. The monomer enzyme is composed of four domain, N, A, B, and C, and has a ("/!)8-barrel in domain A. The lies between domain A and domain N from the other monomer. Fig. 4 indicates the structure of the catalytic center of neopullulanase binding of panose, maltotetraose, and isopanose. Panose is the main product from by neopullulanase [7]. Neopullulanase mainly cleaves the middle position of the three !-1,4 linkages of maltotetraose and exclusively cleaves !-1,6 linkage of isopanose [8]. Since maltotetraose and isopanose are substrate of neopullualnase, deactivated mutant replacing the proton donor, Glu357, by Gln was used for complex crystallization with maltotetraose and isopanose. In the panose complex, the cleavage point is located at the reducing end. In the maltotetraose complex, the cleavage point is located at the central !-1,4 linkage. In the isopanose complex, the cleavage point is located at the !-1,6 linkage. Indeed, the electron densities of bound oligosaccharides were clearly identified at the same active center (Fig. 4). As mentioned previously, neopullulanase strongly catalyzes a transglycosylation reaction to form both !-1,4- and !-1,6-glucosidic linkages. A significant electron density was observed at the proximate position of bound maltotetraose and isopanose, but not at bound panose [53]. The important point to note is that neopullulanase produce by attacking the central !-1,4 linkage of maltotetraose and !-1,6 linkage of isopanose, as mentioned above (Fig. 4) . The enzyme cannot act on panose since it is one of the final products when pullulan is used as a substrate (Fig. 4). Although deactivated mutant enzyme was used in the crystals of maltotetraose- and isopanose-binding complexes, it is possible that maltose is produced by hydrolysis of maltotetraose or isopanose, as seen with

54 International Symposium on Extremophiles and Their Applications 2005

other !-amylase family enzymes [54, 55]. Therefore, we conclude that this electron density is maltose, which works as an acceptor for transglycosylation. This was also the first evidence showing one active center of an enzyme participates in hydrolysis of !-1,4 and !-1,6-glucosidic linkages and transglycosylation to form !-1,4- and !-1,6-glucosidic linkages on the structural basis.

Fig. 5 Fo-Fc electron density omit maps of neopullulanase with structure models of bound oligosaccharides.

5. Concluding remarks and future prospects The main focus of biocatalysis has been on distinguishing and classifying enzymes into different species with a history of discovering new enzymes. However, with the increasing amount of information on the structure of enzymes and their comparison may change this conventional tendency. This change may have been done by the prediction of Chothia [1]; “There are not as many basic protein folding patterns as there are number of enzymes”, the work of Henrissat [4]; “Sequence-based classification of carbohydrate-active enzymes”, and our proposal of the concept of the !-amylase family [3]; “Not only the same catalytic machinery but also the common catalytic mechanism lies in the significant number of glucanohydrolases and glucanotransferases”. These ideas have been individually proposed almost the same time, and therefore, point to the new horizon of rational designing of protein engineering of glycoenzymes.

References [1] Chothia, C. (1992) One thousand families for molecular biologist. Nature, 357, 543-544. [2] Kuriki, T. and Imanaka, T (1999) The concept of the !-amylase family: structural similarity and common catalytic mechanism. J. Biosci. Bioeng., 87, 557-565. [3] Takata, H., Kuriki, T., Okada, S., Takesada, Y., Iizuka, M., Minamiura, N. and Imanaka, T. (1992) Action of neopullulanase: neopullulanase catalyzes both hydrolysis and transglycosylation at !-(1!4)- and !-(1! 6)- glucosidic linkages. J. Biol. Chem. 267, 18447-18452. [4] Henrissat, B. (1991) A classification of glycosyl based on amino acid sequence similarities. Biochem. J. 280, 309-316. [5] Kuriki, T. and Imanaka, T. (1989) Nucleotide sequence of the neopullulanase gene from Bacillus stearothermohpilus. J. Gen. Microbiol. 135, 1521-1528. [6] Kuriki, T. (1999) The concept of !-amylase family. in Recent Advances in Carbohydrate Bioengineering (Gilbert, H.J., Davies, G.J., Henrissat, B., and Svensson, B., Eds) pp. 107-113, Royal Society of Chemistry, Cambridge. [7] Kuriki, T., Okada, S. and Imanaka, T. (1988) New type of pullulanase from Bacillus stearothermophilus and molecular cloning and expression of the gene in Bacillus subtilis. J. Bacteriol. 170: 1554-1559. [8] Imanaka, T. and Kuriki, T. (1989) Pattern of action of Bacillus stearothermophilus neopullualanse on pullulan. J. Bacteriol. 171, 369-374. [9] Kuriki, T., Takata, H., Okada, S. and Imanaka, T. (1991) Analysis of active center of Bacillus stearothermophilus neopullualanse. J. Bacteriol. 173, 6147-6152.

55 International Symposium on Extremophiles and Their Applications 2005

[10] Svensson, B. (1994) Protein engineering of !-amylase family. Plant Mol. Biol. 25, 141-157. [11] MacGregor, E.A., Janecek, S. and Svensson, B. (2001) Relationship of sequence and structure to specificity in the !-amylase family of enzymes. Biochim. Biophys. Acta 1546, 1-20. [12] Janecek, S. (2002) How many conserved sequence regions are there in the !-amylase family? Biologia, Blartislava 57/Suppl. 11, 29-41. [13] Zona, R., Chang-Pi-Hin, F., O’Donohue, M.J. and Janecek, S. (2004) Bioinformatics of the glycoside hydrolase family 57 and identification of catalytic residues in amylopullulanase from Thermococcus hydrothermalis. Eur. J. Biochem. 271, 2863-2872. [14] Bourne, Y. and Henrissat, B. (2001) Glycoside hydrolases and glycosyltransferases: families and functional modules. Curr. Opin. Struc. Biol. 11, 593-600. [15] Kuriki, T. (1992) Can protein engineering interconvert glucanohydrolases/glucanotransferases, and their specificities? Trend Glycosci. Glycotechnol. 4, 567-572. [16] Kuriki, T., Park, J.-H., Okada, S. and Imanaka, T. (1988) Purification and characterization of thermostable pullulanase from Bacillus stearothermophilus and molecular cloning and expression of the gene in Bacillus subtilis. Appl. Environ. Microbiol. 54, 2881-2883. [17] Kuriki, T., Park, J.-H. and Imanaka, T. (1990) Characteristics of thermostable pullulanase from Bacillus stearothermophilus and the nucleotide sequence of the gene. J. Ferment. Bioeng. 69, 204-210. [18] Takata, H., Takaha, T., Kuriki, T., Okada, S., Takagi, S. and Imanaka, T. (1994) Properties and active center of the thermostable branching enzyme from Bacillus stearothermophilus. Appl. Environ. Microbiol. 60, 3096-3104. [19] Guan, H., Kuriki, T., Sivak, M. and Preiss, J. (1995) Maize branching enzyme catalyzes synthesis of glycogen-like in glgB-deficient Escherichia coli. Proc. Natl. Aca. Sci. USA 92, 964-967. [20] Kuriki, T., Guan, H., Sivak, M. and Preiss, J. (1996) Analysis of the active center of branching enzyme II from maize endosperm. J. Prot. Chem. 15, 305-313. [21] Takata, H., Ohdan, K., Takaha, T., Kuriki, T. and Okada, S. (2003) Properties of branching enzyme from hyperthermophilic bacterium, Aquifex aeolicus, and its potential for production of highly-branched cyclic dextrin. J. Appl. Glycosci. 50, 15-20. [22] Hehre, E.J. and Genghof, D.G. (1971) The !-amylase as glycosylases, with wider catalytic capacities than envisioned or explained their representation as hydrolases. Arch. Biochem. Biophys. 142, 382-393. [23] Terada, Y., Sanbe, S., Takaha, T., Kitahata, S., Koizumi, K. and Okada, S. (2001) Comparative study of cyclization reactions of three bacterial cyclomaltodextrin glucanotransferases. Appl. Environ. Microbiol. 67, 1453-1460. [24] Mathupala, S.P., Lowe, S.E., Podkovyrov, S.M. and Zeikus, J.G. (1993) Sequenceing of amylopullulanase (apu) gene of Thermoanaerobactor ethanolicus 39E, and identification of the active site by site-directed mutagenesis. J. Biol. Chem. 268, 16332-16344. [25] Sakano, Y., Fujushima, J. and Kobayashi, T. (1983) Hydrolysis of !-1,4- and !-1,6-glucosidic linkages in trisaccharides by Theromactinomyces vulgaris !-amylase. Agric. Biol. Chem. 47, 2211-2216. [26] Sugimoto, K., Nishimura, T., Nomura, K., Sugimoto, K. and Kuriki, T. (2003) Synthesis of Arbutin-!- glicosides and a comaparison of their inhibitory effects with those of !-arbutin and arbutin on human tyrosinase. Chem. Pharm. Bull. 51, 798-801. [27] Sugimoto, K., Nishimura, T., Nomura, K., Sugimoto, K. and Kuriki, T. (2004) Inhibitory effrects of !- arbutin on melanin synthesis in cultured human melanoma cells and a three-dimentinal human skin model. Biol. Pharm. Bull. 27, 510-514. [28] Sugimoto, K., Nomura, K., Nishimura, T., Kiso, T., Sugimoto, K. and Kuriki, T. (2005) Synthesis of !- arbutin- !-glycosides and their inhibitory effects on human tyrosinase. J. Biosci. Bioeng. 99, 272-276. [29] Fujii, K., Takata, H., Yanase, M., Terada, Y., Ohdan, K., Takaha, T., Okada, S. and Kuriki, T. (2003) Bioengineering and application of novel polymers. Biocatal. Biotransform. 21: 167-172. [30] Takaha, T.,Yanase, M., Takata, H., Okada, S. and Smith, S.M. (1996) Potato D-enzyme catalyzes the cyclization of amylase to produce cycloamylose, a novel cyclic glucan. J. Biol. Chem. 271, 2902-2908. [31] Kuriki, T., Tsuda, M., and Imanaka, T. (1992) Continuous production of panose by immobilized neopullulanase. J. Fermant. Bioeng. 73, 198-202. [32] Kuriki, T., Yanase, M., Takata, H., Takesada, Y., Imanaka, T. and Okada, S. (1993) A new way of producing isomalto-oligosaccharides syrup by using the transglycosylation reaction of neopullulanase. Appl. Environ. Microbiol. 59, 953-959. [33] Kuriki, T., Yanase, M., Takata, H., Imanaka, T. and Okada, S. (1993) Highly branched oligosaccharides produced by the transglycosylation reaction of neopullulanase. J. Frement. Bioeng. 76, 184-190.

56 International Symposium on Extremophiles and Their Applications 2005

[34] Kuriki, T., Yanase, M., Takata, H. and Okada, S. (1997) Production of isomalto/branched-oligosaccharides by using immobilized neopullulanase and preliminary evaluation of the syrup as a food additive. J. Appl. Glycosci. 44, 15-22. [35] Kamasaka, H., Sugimoto, K., Takata, H., Nishimura, T. and Kuriki, T. (2002) Bacillus stearothermophilus neopullualanase selective hydrolysis of amylase to maltose in the presence of amylopectin. Appl. Environ. Mocrobiol. 68, 1659-1664. [36] Kamasaka, H., Sugimoto, K., Takata, H., Nishimura, T. and Kuriki, T. (2002) Macromolecule recognition of Bacillus stearothermophilus neopullualanase. Biologia, Bratislava 57/Suppl. 11:83-85. [37] Yanase, M., Takata, H., Takaha, T., Kuriki, T., Smith, S.M. and Okada, S. (2002) Cyclization reaction catalyzed by glycogen branching enzyme (EC 2.4.1.25/EC 3.2.1.33) and its potential for cycloamylose production. Appl. Environ. Microbiol. 68, 4233-4239. [38] Kamasaka, H., Sugimoto, K., Takata, H., Nishimura, T. and Kuriki, T. (2003) Neopullulanase exhibits distinct specificity toward amylase and amylopectin. J. Appl. Glycosci. 50, 273-275. [39] Kuriki, T., Kaneko, H., Yanase, M., Takata, H., Shimada, J., Takada, T., Umeyama, H. and Okada, S. (1996) Controlling substrate preference and transglycosylation activity of neopullulanase by manipulating steric constraint hydrophobicity in active center. J. Biol. Chem. 271: 17321-17329. [40] Kuriki, T., Stewart, D.C. and Preiss, J. (1997) Construction of chimeric enzymes out of maize endosperm branching enzymes I and II. J. Biol. Chem. 272: 28999-29004. [41] Ohdan, K., Kuriki, T., Kaneko, H., Simada, J., Takada, T., Fujimoto, Z., Mizuno, H. and Okada, S. (1999) Characteristics of two forms of !-amylases and structural implication. Appl. Environ. Microbiol. 65: 4652-4658. [42] Ohdan, K., Kuriki, T., Tatata, H. and Okada, S. (2000) Cloning of the cyclodextrin glucanotransferase gene from alkalophilic Bacillus sp. A2-5a and analysis of raw starch binding-domain. Appl. Microbiol. Biotechnol. 53: 430-434. [43] Ohdan, K., Kuriki, T., Tatata, H., Kaneko, H. and Okada, S. (2000) Introduction of raw starch-binding domains into Bacillus subtilis !-amylase by fusion with the starch-binding domain of Bacillus cyclomaltodextrin glucanotransferase. Appl. Environ. Microbiol. 66: 3058-3064. [44] Ohdan, K. and Kuriki, T. (2000) An approach for introducing a different function to an industrial enzyme. Trends Glycosci. Glyoctechnol. 12, 403-410. [45] Ohdan, K., Takata, H., Kuriki, T. and Okada, S. (2001) Flexibility with truncation of the amino- and carboxyl-termini of Bacillus subtilis X-23 !-amylase. J. Appl. Glycosci. 48, 37-44. [46] Terada, Y., Fujii, K., Takaha, T. and Okada, S. (1999) Thermus aquaticus ATCC 33923 amylomaltase gene cloning and expression and enzyme characterization: production of cycloamylose. Appl. Environ. Microbiol. 65: 910-915. [47] Fujii, K., Minagawa, H., Terada, Y., Takaha, T., Kuriki, T., Shimada, J. and Kaneko, H. (2005) Use of random and saturation mutageneses to improve the properties of Thermus aquaticus amylomaltase for efficient production of cycloamylose. Appl. Environ. Microbiol. 71: 5823-5827. [48] Fujii, K., Minagawa, H., Terada, Y., Takaha, T., Kuriki, T., Shimada, J. and Kaneko, H. (2005) Improvement of amylomaltase from Thermus aquaticus by random and saturation mutagenesis. J. Appl. Glycosci. 52: 137-143. [49] Yanase, M. Takata, H., Fujii, K., Takaha, T. and Kuriki, T. (2005) Cumulative effect of amino acid replacements results in enhanced thermostability of potato type L !-glucan phosphorylase. Appl. Environ. Microbiol. 71: 5433-5439. [50] Fujii, K., Iiboshi, M., Yanase, M., Takaha, T. and Kuriki, T. (2006) Enhancing the thermostability of sucrose phosphorylase from Streptococcus mutans by random mutagenesis. J. Appl. Glycosci. (in press) [51] Matsuura, Y., Kusunoki, M., Harada, Y. and Kakudo, M. Structure and possible catalytic residues of Taka- amylase A. J. Biochem. 95, 697-702. [52] Hondoh, H., Kuriki, T. and Matsuura, Y. (2002) Three-dimensional structure of Bacillus stearothermophilus neopullulanase. Biologia, Brastislava 57/Suppl. 11, 77-82. [53] Hondoh, T., Kuriki, T. and Matsuura, Y. (2003) Three-dimensional structure and substrate binding of Bacillus stearothermophilus neopullulanase. J. Mol. Biol. 326, 177-188. [54] Yoshikawa, Y., Hasegawa, K., Matsuura, Y., Katsube, Y. and Kubota, M. (1997) Crystal structure of a mutant maltotetraose-forming exo-amylase cocrystallized with maltopentaose. J. Mol. Biol. 277, 393-407. [55] Klein, C., Hollender, J., Bender, H. and Shulz, G,E. (1992) Catalytic center of cyclodextrin glycosyltransferase derived from X-ray structure analysis combined with site-directed mutagenesis. Biochemistry 31, 8740-8746.

57