アミロース製造に利用する酵素の開発と改良 Phosphorylase and Muscle Phosphorylase B

125

J. Appl. Glycosci., 54, 125―131 (2007) !C 2007 The Japanese Society of Applied Glycoscience Proceedings of the Symposium on Amylases and Related Enzymes, 2006 Developing and Engineering Enzymes for Manufacturing Amylose

(Received December 5, 2006; Accepted January 5, 2007)

Michiyo Yanase,1,＊ Takeshi Takaha1 and Takashi Kuriki1

1 Biochemical Research Laboratory, Ezaki Glico Co., Ltd. (4 ―6 ―5, Utajima, Nishiyodogawa-ku, Osaka 555-8502, Japan)

Abstract: Amylose is a functional biomaterial and is expected to be used for various industries. However at present, manufacturing of amylose is not done, since the purification of amylose from starch is very difficult. It has been known that amylose can be produced in vitro by using α-glucan phosphorylases. In order to obtain α-glucan phosphorylase suitable for manufacturing amylose, we isolated an α-glucan phosphorylase gene from Thermus aquaticus and expressed it in Escherichia coli. We also obtained thermostable α-glucan phosphorylase by introducing amino acid replacement onto potato enzyme. α-Glucan phosphorylase is suitable for the synthesis of amylose; the only problem is that it requires an expensive substrate, glucose 1-phosphate. We have avoided this problem by using α-glucan phosphorylase either with sucrose phosphorylase or cellobiose phosphorylase, where inexpensive raw material, sucrose or cellobiose, can be used instead. In these combined enzymatic systems, α-glucan phosphorylase is a key enzyme. This paper summarizes our work on engineering practical α-glucan phosphorylase for industrial processes and its use in the enzymatic synthesis of essentially linear amylose and other glucose polymers.

Key words: amylose, glucose polymer, α-glucan phosphorylase, glycogen debranching enzyme, protein engineering

α-1,4 glucan is the major form of energy reserve from Enzymes for amylose synthesis. microorganisms to animals, and mainly occurs in the form Amylose can be produced by the enzymes listed in Ta- of starch (amylose and amylopectin) or glycogen. Amy- ble 1. Starch (glycogen) synthase (EC 2.4.1.21) is the en- lose is a mostly linear polymer of α-1,4-linked glucose zyme employed for starch (glycogen) biosynthesis in vivo. with rare α-1,6 branched points and normally contains ap- The enzymes from plants and microorganisms use ADP- proximately 20% in starch granules.1,2) Amylopectin is the glucose as a substrate, whereas the enzymes from animals major component of starch (80%), in which short amylose use UDP-glucose.10,11) Since ADP-glucose and UDP-glu- chains are connected together with α-1,6 linkages to form cose are very expensive, these are not practical starting a characteristic cluster structure.2,3) Glycogen is also a materials for manufacturing amylose. Therefore, these re- branched glucan, but it differs from amylopectin in the actions are not suitable for industrial production of syn- number and organization of α-1,6 branched linkages and thetic amylose. absence of a cluster structure.4) Cyclodextrin or cycloamy- Isoamylase (EC 3.2.1.68) and pullulanase (EC 3.2.1.41) lose is another form of α-1,4 glucan. Cyclodextrins are catalyze the hydrolysis of α-1,6-glucosidic linkage in cyclic α-1,4 glucans with a degree of polymerization (DP) starch.12,13) These enzymes have been known to be used for from 6 to 8,5) which are produced by treating starch with the conversion of amylopectin into amylose. However, cyclomaltodextrin glucanotransferase (CGTase, EC 2.4.1. most of the amylose produced retains the original unit 19). Cycloamyloses are larger homologues of cyclodex- chains of amylopectin; therefore, the average DP of the trins with a DP larger than 17 and produced by an intra- amylose is around 20. From this reason, it is not possible molecular transglycosylation reaction of amylomaltases to produce amylose with controlled molecular size with (EC 2.4.1.25).6,7) These glucose polymers are different not these enzymes. only in their structure but also in their physical and Amylosucrase (EC 2.4.1.4) catalyzes the transfer of the chemical properties. Among the four glucose polymers glucose moiety from sucrose to the accepter molecule and described above, amylopectin and cyclodextrins (DP 6―8) produces amylose.14) Amylosucrase is very attractive since are available in pure form as industrial raw materials, but it produces amylose directly from sucrose, a less expen- neither amylose nor glycogen is currently available as an sive and renewable biomass. However, the molecular size industrial raw material. We have been studying enzymes of amylose produced by this enzyme is reported to be that can be used to produce many types of glucose poly- about 10 kDa or smaller.15) The amylosucrase system is mers with controlled molecular size and structures.8) Amy- not suitable to produce amylose either, since it cannot lose is one of most interesting targets, since it is expected produce amylose with the desired molecular size. to be used in various industries as a functional biomaterial Cyclomaltodextrin glucanotransferases catalyze the and also as starting material to produce cycloamylose and linearization of cyclodextrin with a so-called “coupling re- other glucose polymers.8,9) action” and produce amylose.16) Though the maximum yield of amylose was more than 90%, the DP of the prod- ＊ Corresponding author (Tel. +81―6―6477―8425, Fax. +81―6―6477― uct was reported to be 34―43 glucose units. This system 8362, E-mail: [email protected]). is also unable to produce amylose with the desired mo- 126 J. Appl. Glycosci., Vol. 54, No. 2 (2007)

Table 1. Various enzymes for the amylose synthesis.

Enzyme Substrate

(1) Starch synthase (EC 2.4.1.21) ADP-Glucose (Glycogen synthase) (UDP-Glucose) (2) α-Glucan phosphorylase (EC 2.4.1.1) Glucose 1-phosphate (3) Amylosucrase (EC 2.4.1.4) Sucrose (4) Pullulanase (EC 3.2.1.41) Starch Isoamylase (EC 3.2.1.68) (5) Cyclodextrin glucano- (EC 2.4.1.19) Cyclodextrin transferase (6) Glycogen debranching (EC 2.4.1.25／ Starch enzyme EC 3.2.1.33) lecular size. Fig. 1. Action of glycogen debranching enzyme. Amylose synthesis using glycogen debranching en- Glycogen debranching enzyme transfers maltosyl or maltotriosyl zyme. units from the shortened chain to a non-reducing end of another Glycogen debranching enzyme (EC 2.4.1.25／EC 3.2.1. chain with its 4-α-glucanotransferase activity (A), and leaves a single glucosyl residue attached. The glycosyl stub remaining at the 33) is a multifunctional enzyme and possesses 4 -α- branch point is next cleaved with its amylo-1,6-glucosidase activity glucanotransferase and amylo-1,6-glucosidase activities, in (B). When glycogen debranching enzyme was reacted with amy- the same polypeptide chain.17,18) Glycogen debranching en- lopectin, amylose was produced in the early stage of the reaction. zyme is distributed in mammals and yeasts and is in- Then this enzyme next catalyzed the cyclization reaction of amylose to produce cycloamylose (C). Open and closed circles indicate volved in the degradation of glycogen together with α- glucosyl residue connected with α-1,4-glucosidic linkage, and verti- glucan phosphorylase (EC 2.4.1.1) in vivo. α-Glucan cal lines indicate α-1,6-branched linkage. phosphorylase degrades glycogen from its non-reducing ends, leaving dextrin with shortened side chains. Glyco- origins differ in their modes of regulation and their sub- gen debranching enzyme then transfers maltosyl or malto- strate preferences.21,25) α-Glucan phosphorylases from po- triosyl units from the shortened chain to a non-reducing tato26,27) and rabbit muscle20) have been successfully em- end of another chain with its transferase activity, and ployed in the synthesis of amylose in vitro. The molecular leaves a single glucosyl residue attached. The glucosyl weight of the amylose thus produced has a narrow distri- stub remaining at the branch point is next cleaved with its bution (Mw／Mn<1.2) and can be controlled by the G-1-P／ amylo-1,6-glucosidase activity. We presumed relatively primer molar ratio.26) long amylose might be produced when glycogen debranching enzyme acts on amylopectin in vitro in the ab- α-Glucan phosphrylase from Thermus aquaticus. sence of α-glucan phosphorylase (Fig. 1). When glycogen Enzymes used in industrial processes are required to be debranching enzyme reacted with amylopectin, amylose stable, even at elevated temperatures or when stored. Be- was produced in the early stage of the reaction. However, cause amylose easily forms a precipitate at low tempera- this enzyme next catalyzes the cyclization reaction of tures and this precipitation can be significantly inhibited amylose to produce cycloamylose, which account for at elevated temperatures, α-glucan phosphorylase for amy- about 60% in the final stage of the reaction (Fig. 1). We lose production also expected to be thermostable. The α- discovered that glycogen debranching enzyme was not glucan phosphorylase gene from T. aquaticus was iso- suitable for amylose production but was a very attractive lated, and the properties of the enzyme expressed in Es- enzyme for producing cycloamylose from starch.19) cherichia coli were characterized.28) The optimum temperature for phosphorylation reaction of the enzyme was Amylose synthesis using α-glucan phosphorylase. 70°C and the optimum temperature for glucan synthetic α-Glucan phosphorylase catalyzes a transfer of the glu- reaction was 50°C, and the enzyme retained about 80% of cose moiety from glucose 1-phosphate (G-1-P) to malto- its activity even after incubation at 80°C for 30 min. The dextrin primer and can produce essentially linear amylose minimum primer for glucan synthesis reaction and the by repeating this activity. The molecular size of amylose minimum effective substrate for phosphorylation of the has been reported to be controlled by changing the G-1-P enzyme from T. aquaticus are maltotriose and maltotetra- ／primer molar ratio,20) so it is potentially a good system ose, respectively. These values corresponded well to those for manufacturing amylose. of the enzyme from Thermus thermophilus,29) but are one α-Glucan phosphorylase is found widely in animals, unit shorter than those of potato enzyme. plants and micro-organisms and catalyses the reversible We performed the amylose synthesizing reaction at ele- phosphorolysis of α-1,4-glucan. All known α-glucan vated temperatures (70°C) using the thermostable α- phosphorylases seem to share a similar catalytic mecha- glucan phosphorylase of T. aquaticus, which resulted in nism.21―24) Although all α-glucan phosphorylases belong to the synthesis of amylose with a very narrow distribution a large highly homologous group that includes glycogen (Mw／Mn<1.01), without producing any precipitate of amy- phosphorylase from bacteria, yeast and animals, starch lose throughout the reaction.8) phosphorylase from plants, and maltodextrin phosphorylases (MalP) from bacteria, these enzymes from distinct Developing and Engineering Enzymes for Manufacturing Amylose 127

Fig. 3. Thermostability of the wild-type and mutated α-glucan phosphorylases. The enzymes were incubated at 60°C(A)and65°C(B)in20mM sodium citrate buffer (pH 6.7), and the remaining activity was measured as described in Ref 34). The initial activity is denoted as 100%. The assays of α-glucan phosphorylase activity were performed in duplicate with less than 10% difference between the two readings. ○, wild-type; □, F39L; △, N135S; ◇, T706I; ■, F39L／N135S; ▲,F 39L／T706I; ◆, N135S／T706I; ●, F39L／N135S／T706I.

Fig. 2. Phylogenetic tree of α-glucan phosphorylases. phorylase from T. aquaticus at the beginning stage. How- The deduced amino acid sequences of various α-glucan phospho- ever, bacterial α-glucan phosphorylases appeared to have rylases were compared by using the CLUSTAL W 1.81 program higher Km values of maltooligosaccharides such as malto- (http:／／align.genome.jp／). The phylogenetic tree was constructed tetraose as the primer for glucan synthesis compared to by the neighbor joining method.44) The number of amino acid sub- - potato type L glucan phosphorylase. Furthermore T. stitutions per site is shown in the scale. α-Glucan phosphorylases - α- from T. aquaticus, T. thermophilus, T. litoralis, Potato type L and aquaticus α-glucan phosphorylase and potato-type L α- H, E. coli and rabbit muscle are indicated by dots. glucan phosphorylase were relegated to different subgroups by their primary structure and substrate specificity. Classification of α-glucan phosphorylases. The potato enzymes are not stable at elevated tempera- α-Glucan phosphorylase has been known to be present tures and readily degrade when stored. Therefore, we in- in several thermophilic organisms. Among those, only α- troduced random mutation into the gene for potato-type L glucan phosphorylases from Bacillus stearothermo- α-glucan phosphorylase to enhance the thermostability of philus,30) T. thermophilus, Thermococcus litoralis 25) and T. this enzyme.34) We obtained three single-residue mutations, aquaticus have been subjected to biochemical experi- Phe39→Leu (F39L), Asn135→Ser (N135S) and Thr706 ments; information on all the others is based on deduced →Ile (T706I), and each mutant enzyme was purified from amino acid sequences obtained from the genome sequenc- recombinant E. coli. The purified enzymes produced a ing project. We proposed to divide α-glucan phosphory- single clear band on SDS-PAGE with an apparent mo- lases into two subgroups which can be distinguished by lecular mass of 105 kDa. These mutant enzymes retained their primary structure and substrate specificity. One sub- their activity even after heat treatment at 60°C for 2h, group includes well-characterized α-glucan phosphory- while the wild-type enzyme was completely inactivated lases from rabbit muscle, potato (types H and L), E. coli (Fig. 3A). α-Glucan phosphorylase genes that contain two (MalP and GlgP) and B. stearothermophilus. These en- or three mutations were constructed by introducing the zymes are already known to use maltotetraose as the second or third mutation to genes containing a single mu- minimum primer and maltopentaose as the minimum ef- tation by site-directed mutagenesis. The simultaneous mu- fective substrate for phosphorylation. The second sub- tation of two (F39L／N135S, F39L／T706I and N135S／ group includes some α-glucan phosphorylases from thermo- T706I) or three (F39L／N135S／T706I) residues further in- philic organisms such as T. aquaticus, T. litoralis and creased the thermostability of the enzyme, indicating that Thermotoga maritime. 31) Although most of the enzymes the effect of the replacement of the residues is cumulative. within this new group are putative α-glucan phosphory- The triple-mutant enzyme retained more than 95% of its lases and their properties have not been studied, three activity after incubation at 60°C for 60 min (Fig. 3A); fur- characterized enzymes (T. aquaticus, T. thermophilus and thermore it retained 50% of its original activity after heat T. litoralis) within this group use maltotetraose as the treatment at 65°C for 20 min (Fig. 3B). The variant en- minimum primer and maltopentaose as the minimum ef- zymes thus obtained have increased stability against ther- fective substrate for phosphorylation. About 400 se- mal denaturation, but retain other properties (specific ac- quences of α-glucan phosphorylases have been accumu- tivity, pH stability, Km values, pyridoxal 5′-phosphate lated in databanks such as CAZy32) and Pfam.33) The num- contents and the secondary-structure contents).34) ber of enzymes belonging to this second group has been Three amino acids (Phe39, Asn135 and Thr706) of the increasing and has now reached more than 60 records potato-type L α-glucan phosphorylase were then replaced (Fig. 2). with 19 other amino acid residues. The residual activity of the wild-type and mutant enzymes after heat treatment at Enhancing thermostability of α-glucan phosphorylase 60°C for 10 min was measured. At position 39 and 706, from potato. the most thermostable variants achieved were F39L and To obtain α-glucan phosphorylase suitable for industrial T706I, respectively, which are the same mutations as applications, we focused on thermostable α-glucan phos- those obtained by random mutagenesis. At position 135, 128 J. Appl. Glycosci., Vol. 54, No. 2 (2007) on the other hand, N135G was the most thermostable that degradation occurred within the insertion sequence, as variant and N135S, obtained by random mutagenesis, was reported by Mori et al. 37) In this study, we found that α- the sixth most thermostable.35) glucan phosphorylase with a F39L or T706I mutation was Introduction of the F39L or T706I mutation not only not broken down under the same storage conditions and affected the thermal stability of potato-type L α-glucan retained a clear single band with a molecular mass of 105 phosphorylase, but also affected the stability of the en- kDa (Fig. 4). These results suggest that the introduction zyme upon storage. To investigate the stability of variant of a F39L or T706I mutation protects potato-type L α- enzymes against storage, variant enzymes were stored at 4 glucan phosphorylase from degradation, which is most °C for 5 months and then subjected to native PAGE and likely to occur in the insertion sequence. The position of SDS-PAGE. Wild-type and N135S enzymes produced two the insertion sequence (amino acid number 414 to 491) major bands (ca. 45 kDa), whereas all of the other mu- and its interaction with Phe39 or Thr706 is of great inter- tants produced a single band with a molecular mass of est. We could estimate the approximate position of the in- 105 kDa on SDS-PAGE, which corresponded well to the sertion sequence from the positions of nearby amino acids value for the α-glucan phosphorylase subunit. These and indicate that the insertion sequence is located on the analyses indicate that enzymes with a F39L and a T706I surface of this protein.34) Mori et al. 37) predicted that the mutation are resistant to possible proteolytic degradation, 78-residue insertion sequence located on the surface of the which readily occurs when the wild-type enzyme is stored molecule has a flexible structure. Therefore, it is possible at 4°C (Fig. 4). that Phe39 and Thr706 might be located at an interactive It has been reported that type L α-glucan phosphorylase distance to the insertion sequence in potato-type L α- from potato and sweet potato are easily degraded when glucan phosphorylase. they are stored. Brisson et al .36) indicated that the prote- These results suggest that mutations F39L and T706I olysis of potato-type L α-glucan phosphorylase occurs in probably cause a change in the conformation of potato- vivo and is not an artifact of the isolation procedure. Mori type L α-glucan phosphorylase that stabilizes the protein et al. 37) reported that purified potato-type L α-glucan against thermal denaturation as well as proteolytic degra- phosphorylase was digested within the 78 amino acid in- dation. sertion sequence (amino acid number 414 to 491) and We successfully enhanced the thermostability of potato- broken down to several fragments with a molecular mass type L α-glucan phosphorylase by random and site- of around 50 kDa. Chen et al. 38) also reported that the directed mutagenesis. Some variants were also stable presence of amino acid sequences in sweet potato α- against proteolytic degradation, which readily occurred glucan phosphorylase (110 kDa) which was recognized when the wild-type enzyme was stored at 4°C. These en- and cleaved by a site-specific protease into two polypep- hanced properties may be very important not only for the tides with a molecular mass of around 50 kDa. The se- use of this enzyme for industrial purposes, but also for the quence is located at an equivalent position to the insertion production and purification of this enzyme on an indus- sequence of potato enzyme. In our experiment, wild-type trial scale. α-glucan phosphorylase and the N135S variant were also broken down to several smaller polypeptides (data not Phosphorylase coupling for amylose synthesis. shown) and finally into two polypeptides with similar mo- As described previously, α-glucan phosphorylase is po- lecular masses of around 45 kDa (Fig. 4), which indicates tentially a good enzyme for manufacturing amylose. How- ever, its substrate, G-1-P, is expensive as a starting material, so we need to combine the system to provide G-1-P for this enzyme. Sucrose phosphorylase (EC 2.4.1.7) catalyzes the phosphorolysis of sucrose in the presence of inorganic phosphate (Pi) and produces G-1-P. Waldmann et al. 39) reported the combined use of sucrose phosphorylase and α- glucan phosphorylase in the production of amylose from sucrose. We have engineered sucrose phosphorylase from Streptococcus mutans to increase thermostability by random and site-directed mutagenesis40) andusedthisen- zyme, together with the triple-mutant of potato α-glucan phosphorylase (F39L／N135S／T706I), for the production of synthetic amylose from sucrose. By using these two Fig. 4. SDS-PAGE of purified potato-type L α-glucan phosphory- genetically engineered thermostable phosphorylases, we lases. succeeded in producing amylose from inexpensive sucrose 35) The protein samples (0.6 µg) were loaded onto a gel. After elec- with a very high yield. The molecular size of the amy- trophoresis, the gel was stained with Coomassie Brilliant Blue. lose thus produced is strictly controlled by the sucrose／ Lanes 1a and 1b are the wild-type enzyme samples just after being primer molar ratio, as in the case of amylose production purified and stored at 4°C for 5 months, respectively. Lanes 2―8are from G-1-P. the variants with F39L, N135S, T706I, F39L N135S, F39L T706I, ／／ We then examined another phosphorylase, cellobiose N135S／T706I and F39L／N135S／T706I mutations stored at 4°Cfor 5 months, respectively. Numbers on the left are the estimated mo- phosphorylase (EC 2.4.1.20), which catalyzes the phos- lecular masses of marker protein. phorolysis of cellobiose to produce G-1-P.41) We have Developing and Engineering Enzymes for Manufacturing Amylose 129 cloned and expressed the cellobiose phosphorylase gene tion of novel glucose polymers. Biocatal. Biotransform., 21, from the Clostridium thermocellum strain42) using E. coli 167―172 (2003). 9 ) M. Tanihara, J. Takahara and Y. Morihara: Enzymatic synthe- as a host. Partially purified cellobiose phosphorylase was sis of amylose and its medical application. BIO INDUSTRY , incubated with cellobiose and α-glucan phosphorylase in 22,58―66 (2005). the presence of Pi. Various sizes of amylose (4.2×104 to 10) T. Baba, M. Nishihara, K. Mizuno, T. Kawasaki, H. Shimada, 7.3×105) were synthesized, but the yield (38.6%) was not E. Kobayashi, K. Ohnishi, K. Tanaka and Y. Arai: Identifica- tion, cDNA cloning and gene expression of soluble starch syn- as high as that of the sucrose phosphorylase and α-glucan thase in rice (Oryza sativa L.) immature seeds. Plant Physiol., phosphorylase system. However, the yield of amylose was 103, 565―573 (1993). increased to 64.8% by adding mutarotase (EC 5.1.3.3) and 11) K. Furukawa, M. Tagaya, K. Tanizawa and T. Fukui: Identifi- glucose oxidase (EC 1.1.3.4) to the reaction mixture, cation of Lys277 at the active site of Escherichia coli glyco- which removed the glucose accumulated in the reaction gen synthase. Application of affinity labeling combined with site-directed mutagenesis. J. Biol. Chem., 269, 868―871 (1994). mixture. Amylose production from cellulose is extremely 12) T. Harada, A. Misaki, H. Akai, K. Yokobayashi and Y. Sugi- interesting, not only from the viewpoint of it being an ef- moto: Characterization of Pseudomonas isoamylase by its ac- fective use of biomass, but also as a one-step conversion tions on amylopectin and glycogen: comparison with Aerobac- of β-1,4 glucan to α-1,4 glucan by using multiple en- ter pullulanase. Biochem. Biophys. Acta, 268, 497―505 (1972). zymes.41) 13) H. Akai, K. Yokobayashi, A. Misaki and T. Harada: Complete hydrolysis of branching linkages in glycogen by Pseudomonas isoamylase: distribution of linear chains. Biochem. Biophys. Conclusion and future prospects. Acta, 237, 422―429 (1971). As described in this article, we have engineered ther- 14) C. Albenne, L.K. Skov, O. Mirza, M. Gajhede, G. Feller, S. mostable α-glucan phosphorylase and established the sys- D’Amico, G. Andre´, G. Potocki-Veronease, B.A. van der Veen, P. Monsan and M. Remaud Simeon: Molecular basis of tems to produce amylose from sucrose or cellobiose. - the amylose-like polymer formation catalyzed by Neisseria Combined use of phosphorylases is very attractive from polysaccharea amylosucrase. J. Biol. Chem., 279, 726―734 the point of industrial application, since it enables us to (2004). utilize inexpensive substrates. It should be noted that 15) G. Potocki-Veronease, J.L. Putaux, D. Dupeyre, C. Albenne, these systems are not only useful for the production of M. Remaud-Simeon, P. Monsan and A. Buleon: Amylose syn- synthetic amylose, but also for the production of other thesized in vitro by amylosucrase: morphology, structure, and 35) properties. Biomacromolecules, 6, 1000―1011 (2005). glucose polymers. We have already reported the produc- 16) T. Shibuya, T. Yamauchi, H. Chaen, M. Nakano, S. Sakai and tion of glucose polymers with a branched or cyclic struc- M. Kurimoto: The formation of amylose-granules from cyclo- ture from G-1-P, by the combined use of a branching en- maltodextrin glucanotransferase by α-cyclodextrin. Denpun zyme43) (EC 2.4.1.18) or 4-α-glucanotransferase (EC 2.4.1. Kagaku, 40, 375―381 (1993). 25) with glucan phosphorylase. The tailor made synthe- 17) B.K. Gillard and T.E. Nelson: Amylo-1,6-glucosidase／4-α- α- - glucanotransferase: use of reversible substrate model inhibitors sis of glucose polymers with the desired molecular struc- to study the binding and active sites of rabbit muscle de- ture is already in the practical stage. branching enzyme. Biochemistry, 16, 3978―3987 (1977). 18) A. Nakayama, K. Yamamoto and S. Tabata: Identification of This research was supported by Research and Development Pro- the catalytic residues of bifunctional glycogen debranching en- gram for New Bio-industry Initiatives (2005―2008) of Bio-oriented zyme. J. Biol. Chem., 276, 28824―28828 (2001). Technology Research Advancement Institution (BRAIN), Japan. 19) M. Yanase, H. Takata, T. Takaha, T. Kuriki, S.M. Smith and S. Okada: Cyclization reaction catalyzed by glycogen debranching enzyme (EC 2.4.1.25／EC 3.2.1.33) and its potential REFERENCES for cycloamylose production. Appl. Environ. Microbiol., 68, 4233―4239 (2002). 1 ) S. Hizukuri, Y. Takeda and M. Yasuda: Multi-branched nature 20) S. Kitamura: Starch polymers, natural and synthetic. in The of amylose and the action of debranching enzymes. Carbo- Polymeric Materials Encyclopedia, Synthesis, Properties and hydr. Res., 94, 205―213 (1981). Applications, J.C. Salamone, ed., CRC Press, Boca Raton, pp. 2 ) J.C. Shannon and D.L. Garwood: Genetics and physiology of 7915―7922 (1996). starch development. in Starch, Chemistry and Technology,R. 21) D. Palm, R. Goerl and K.J. Burger: Evolution of catalytic and L. Whister, J.N. Bemiller and E.F. Paschall, eds., 2nd ed., regulatory sites in phosphorylases. Nature, 313, 500―502 Academic Press, Orlando, pp. 25―86 (1984). (1985). 3 ) S. Hizukuri: Starch, analytical aspects. in Carbohydrates in 22) T. Fukui, S. Shimomura and K. Nakano: Potato and rabbit Food , A.C. Eliasson, ed., Marcel Dekker Inc., New York, pp. muscle phosphorylases: comparative studies on the structure, 347―429 (1996). function and regulation of regulatory and nonregulatory en- 4 ) D.J. Manners: Recent developments in our understanding of zymes. Mol. Cell. Biochem., 42, 129―144 (1982). glycogen structure. Carbohydr. Polym., 16,37―82 (1991). 23) J.W. Hudson, G.B. Golding and M.M. Crerar: Evolution of al- 5 ) S. Kitamura: Cyclic oligosaccharides and polysaccharides. in losteric control in glycogen phosphorylase. J. Mol. Biol., 234, Cyclic Polymers, J.A. Semlyen, ed., 2nd ed., Kluwer Aca- 700―721 (1993). demic Publishers, Dordrecht, pp. 125―160 (2000). 24) R. Schinzel and N. Nidetzky: Bacterial α-glucan phosphory- 6 ) T. Takaha, M. Yanase, H. Takata, S. Okada and S.M. Smith: lases. FEMS Microbiol. Lett., 171,73―79 (1999). Potato D-enzyme catalyzes the cyclization of amylose to pro- 25) K.B. Xavier, R. Peist, M. Kossmann, W. Boos and H. Santos: duce cycloamylose, a novel cyclic glucan. J. Biol. Chem., 271, Maltose metabolism in the hyperthermophilic archaeon Ther- 2902―2908 (1996). mococcus litoralis: Purification and characterization of key en- 7 ) Y. Terada, K. Fujii, T. Takaha and S. Okada: Thermus aquati- zymes. J. Bacteriol., 181, 3358―3367 (1999). cus ATCC 33923 amylomaltase gene cloning and expression 26) M.J. Gidley and P.V. Bulpin: Aggregation of amylose in aque- and enzyme characterization: production of cycloamylose. ous systems: The effect of chain length phase behavior and ag- Appl. Environ. Microbiol., 65, 910―915 (1999). gregation kinetics. Macromolecules, 22, 341―346 (1989). 8 ) K. Fujii, H. Takata, M. Yanase, Y. Terada, K. Ohdan, T. 27) C. Niemann, W. Sanger, B. Pfannemu¨ller, W.D. Eigner and A. Takaha, S. Okada and T. Kuriki: Bioengineering and applica- Huber: Phospholytic synthesis of low-molecular-weight amy- 130 J. Appl. Glycosci., Vol. 54, No. 2 (2007)

loses with modified terminal groups; Comparison of potato アミロース製造に利用する酵素の開発と改良 phosphorylase and muscle phosphorylase b. in ACS Symposium 1 1 1 Series, M.J. Comstock, ed., ACS Books Advisory Board, 柳瀬美千代，鷹羽武史，栗木隆 Washington, DC, pp. 189―204 (1991). 1 江崎グリコ株式会社生物化学研究所 28) T. Takaha, M. Yanase, H. Takata and S. Okada: Structure and properties of Thermus aquaticus α-glucan phosphorylase ex- (555―8502 大阪市西淀川区歌島 4―6―5) pressed in Escherichia coli. J. Appl. Glycosci., 48,71―78 アミロースは，グルコースが α-1,4 結合により直鎖状に (2001). 29) B. Boeck and R. Schinzel: Purification and characterization of 連なったポリマーであり，シクロアミロース，グリコー an α-glucan phosphorylase from the thermophilic bacterium ゲンなど種々のグルコースポリマーの原料となる．また， Thermus thermophilus. Eur. J. Biochem., 239, 150―155 (1996). 30) H. Takata, T. Takaha, S. Okada, M. Takagi and T. Imanaka: 機能材料として，種々の産業分野におけるアミロースの Purification and characterization of α-glucan phosphorylase 利用が期待されている．アミロースは，天然のデンプン from Bacillus stearothermophilus. J. Ferment. Bioeng., 85, 156―161 (1998). の成分として自然界に大量に存在しているが，デンプン 31) M. Bibel, C. Brettl, U. Gosslar, G. Kriegshauser and W. Liebl: の主成分であるアミロペクチンとの分離が難しいため， Isolation and analysis of genes for amylolytic enzymes of the hyperthermophilic bacterium Thermotoga maritima. FEMS Mi- 天然アミロースの工業的な生産は行われていなかった． crobiol. Lett., 158,9―15 (1998). 一方，アミロースは酵素を利用して合成できることが知 32) P.M. Coutinho and B. Henrissat: CAZy Family GT35, Carbohy- drate-Active Enzymes server, http:／／afmb.cnrs-mrs.fr／CAZY／られている．中でも α-グルカンホスホリラーゼは，合成 [accessed 20 July 2006]. するアミロースの分子量を制御することが可能であり， 33) Pfam 17.0 (7868 families), http:／／pfam.wustl.edu／index.html [accessed 11 May 2005]. アミロース合成において優れた酵素である．そこで，私 34) M. Yanase, H. Takata, K. Fujii, T. Takaha and T. Kuriki: Cu- たちは，産業化利用を目的として，熱安定性に優れた α- mulative effect of amino acid replacements results in enhanced thermostability of potato-type L α-glucan phosphorylase. Appl. グルカンホスホリラーゼの開発を行った．まず，高度好 Environ. Microbiol., 71, 5433―5439 (2005). 熱菌 Thermus aquaticus 由来の α-グルカンホスホリラーゼ 35) M. Yanase, T. Takaha and T. Kuriki: α-Glucan phosphorylase and its use in carbohydrate engineering. J. Sci. Food Agric., 遺伝子を取得し，大腸菌で発現させ耐熱性酵素を得た． 86, 1631―1635 (2006). 次に，タンパク質工学的手法を用いて，馬鈴薯由来の α- 36) N. Brisson, H. Giroux, M. Zollinger, A. Camirad and C. Si- mard: Maturation and subcellular compartmentation of potato グルカンホスホリラーゼの耐熱化を行った．また，α-グ starch phosphorylase. Plant Cell., 21, 559―566 (1989). ルカンホスホリラーゼは高価なグルコース 1-リン酸を原 37) H. Mori, K. Tanizawa and T. Fukui: A chimeric α-glucan phosphorylase of plant type L and H isozymes: functional role 料とするため，私たちは，スクロースやセルロースから of 78-residue insertion in type L isozyme. J. Biol. Chem., 263, グルコース 1-リン酸を合成できるスクロースホスホリ 5574―5581 (1993). 38) H.M. Chen, S.C. Chung, C.C. Wu, T.S. Cuo, J.S. Wu and R. ラーゼやセロビオースホスホリラーゼを α-グルカンホス H. Juang: Regulation of the catalytic behavior of L-form starch ホリラーゼと組み合わせて反応させることにより，安価 phosphorylase from sweet potato roots by proteolysis. Physiol. Plant., 114, 506―515 (2002). な原料からアミロースを量産化する方法を開発した．さ 39) H. Waldmann, D. Gygax, M.D. Bednarski, R. Shangraw and らにこの反応に，グルカン転移酵素を組み合わせること G.M. Whitesides: The enzymic utilization of sucrose in the synthesis of amylose and derivatives of amylose, using phos- により，種々のグルコースポリマーの合成も可能となり， phorylases. Carbohydr. Res., 157,c4―c7 (1986). α-グルカンホスホリラーゼはこれらグルコースポリマー 40) K. Fujii, M. Iiboshi, M. Yanase, T. Takaha and T. Kuriki: En- hancing the thermal stability of sucrose phosphorylase from 合成の鍵となる酵素である．ここでは，産業的利用を目 Streptococcus mutans by random mutagenesis. J. Appl. Gly- 的とした α-グルカンホスホリラーゼの開発と改良につい cosci., 53,91―97 (2006). 41) K. Ohdan, K. Fujii, M. Yanase, T. Takaha and T. Kuriki: En- て，さらに本酵素を利用したアミロース合成について報 zymatic synthesis of amylose. Biocatal. Biotransform., 24,77― 告する． 81 (2006) 42) Y.K. Kim, M. Kitaoka, M. Krishnareddy, Y. Mori and K. Hayashi: Kinetic studies of a recombinant cellobiose phospho- ＊＊＊＊＊ rylase (CBP) of the Clostridium thermocellum YM4strainex- pressed in Escherichia coli. J. Biochem., 132, 197―203 (2002). 〔質問〕食総研徳安 43) H. Takata, K. Ohdan, T. Takaha, T. Kuriki and S. Okada: 耐熱性を上げる目的として，酵素反応温度を高くして Properties of branching enzyme from hyperthermophilic bacterium, Aquifex aeolicus, and its potential for production of 反応効率を上げることが考えられるが，得られた変異酵 highly-branched cyclic dextrin. J. Appl. Glycosci., 50,15―20 素を高温で用いた際のメリットはどのようなものがあっ (2003). 44) N. Saitou and M. Nei: The neighbor-joining method: a new たのか． method for reconstructing phylogenetic trees. Mol. Biol. Evol., 〔答〕 4, 406―425 (1987). 今回のアミノ酸置換により得た酵素は，熱安定性を向上させることはできましたが，至適温度は変化しませんでした．そのため，反応温度を上げることにより，反応効率を上昇させる効果は期待できませんでした．しかし，酵素反応を高温で行うことにより，アミロースの老化を抑制することができます．また，酵素の熱安定性を向上 Developing and Engineering Enzymes for Manufacturing Amylose 131

させたことにより，夾雑タンパク質を熱処理 (60°C 以上) グループに属しています．二つのグループの酵素は，構により除去することができ，酵素精製工程において大き造だけでなくグルカン合成反応の最小のプライマーが異なメリットがありました．なるなど，性質も大きく異なることが報告されています． T. aquaticus 由来の酵素の属するグループには，耐熱性菌〔質問〕石川県立大谷口由来の酵素が多く存在しますが，馬鈴薯由来の酵素のグ 1) Thermus aquaticus の耐熱性酵素が存在する中で，ループには，これまでに耐熱性菌由来の酵素は見出され敢えて potato 酵素の耐熱化を続けた理由は？ておりません．産業化利用を目的として，それぞれのグ 2) 得られた耐熱性変異酵素とT. aquaticus 由来のループから耐熱性の高い酵素を得るため，このグループ amylose 合成における優劣はどうか？に属する馬鈴薯由来の酵素の耐熱化を試みました．〔答〕 2) アミロース合成においては，それぞれの酵素の特 1) α-グルカンホスホリラーゼは，アミノ酸配列の相徴を活かし，目的に応じて選択していきたいと考えてい同性から二つのグループに分かれると考えられ，T. ます． aquaticus 由来の酵素と馬鈴薯由来の酵素はそれぞれ別の