Shigetaka Okada

Potato D-enzyme Catalyzes the Cyclization of Amylose to Produce Cycloamylose, a Novel Cyclic Glucan

Potato D-enzyme was purified from recombinant Escherichia coli, and its action on synthetic amylose (average M of 320,000) was analyzed. D-enzyme treatment resulted in a decrease in the ability of the amylose to form a blue complex with iodine. Analysis of the products indicated that the enzyme catalyzes an intramolecular transglycosylation reaction on amylose to produce cyclic α-1,4-glucan (cycloamylose). Confirmation of the cyclic structure was achieved by demonstrating the absence of reducing and nonreducing ends, resistance to hydrolysis by glucoamylase (an exoamylase), and by “time of flight” mass spectrometry. The degree of polymerization of cycloamylose products was determined by time of flight mass spectrometry analysis and by high-performance anion-exchange chromatography following partial acid hydrolysis of purified cycloamylose molecules and was found to range from 17 to several hundred. The yield of cycloamylose increased with time and reached >95%. D-enzyme did not act upon purified cycloamylose, but if glucose was added as an acceptor molecule, smaller cyclic and linear molecules were produced. The mechanism of the cyclization reaction, the possible role of the enzyme in starch metabolism, and the potential applications for cycloamylose are discussed.

Cyclodextrins are not the major cyclic alpha-1,4-glucans produced by the initial action of cyclodextrin glucanotransferase on amylose.

The initial action of cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) from an alkalophilicBacillus sp. A2-5a on amylose was investigated. Synthetic amylose was incubated with purified CGTase then terminated in the very early stage of the enzyme reaction. When the reaction mixture was treated with glucoamylase and the resulting glucoamylase-resistant glucans were analyzed with high performance anion exchange chromatography, cyclic α-1,4-glucans, with degree of polymerization ranging from 9 to more than 60, in addition to well known α-, β-, and γ-cyclodextrin (CD), were detected. The time-course analysis revealed that larger cyclic α-1,4-glucans were preferentially produced in the initial stage of the cyclization reaction and were subsequently converted into smaller cyclic α-1,4-glucans and into the final major product, β-CD. CGTase from Bacillus maceransalso produced large cyclic α-1,4-glucans except that the final major product was α-CD. Based on these results, a new model for the action of CGTase on amylose was proposed, which may contradict the widely held view of the cyclization reaction of CGTase.

Bioengineering and Application of Novel Glucose Polymers

Abstract Glucan phosphorylase, branching enzyme, and 4-α-glucanotransferase were employed to produce glucose polymers with controlled molecular size and structures. Linear or branched glucan was produced from glucose-1-phosphate by glucan phosphorylase alone or together with bracnhing enzyme, where the molecular weight of linear glucan was strictly controlled by the glucose-1-phosphate/primer molar ratio, and the branching pattern by the relative branching enzyme/glucan phosphorylase activity ratio. Cyclic glucans were produced by the cyclization reaction of 5-αglucanotransferases and branching enzyme on amylose and amylopectin. Molecular size and structure of cyclic glucan was controlled by the type of enyzyme and substrate chosen and by the reaction conditions. This in vitro approach can be used to manufacture novel glucose polymers with applicable value.

Controlling Substrate Preference and Transglycosylation Activity of Neopullulanase by Manipulating Steric Constraint and Hydrophobicity in Active Center

The substrate specificity and the transglycosylation activity of neopullulanase was altered by site-directed mutagenesis on the basis of information from a three-dimensional structure predicted by computer-aided molecular modeling. According to the predicted three-dimensional structure of the enzyme-substrate complex, it was most likely that Ile-358 affected the substrate preference of the enzyme. Replacing Ile-358 with Trp, which has a bulky side chain, reduced the acceptability of α-(1→6)-branched oligo- and polysaccharides as substrates. The characteristics of the I358W-mutated enzyme were quite different from those of wild-type neopullulanase and rather similar to those of typical starch-saccharifying α-amylase. In contrast, replacing Ile-358 with Val, which has a smaller side chain, increased the preference for α-(1→6)-branched oligosaccharides and pullulan as substrates. The transglycosylation activity of neopullulanase appeared to be controlled by manipulating the hydrophobicity around the attacking water molecule, which is most likely used to cleave the glucosidic linkage in the hydrolysis reaction. We predicted three residues, Tyr-377, Met-375, and Ser-422, which were located on the entrance path of the water molecule might be involved. The transglycosylation activity of neopullulanase was increased by replacing one of the three residues with more hydrophobic amino acid residues; Y377F, M375L, and S422V. In contrast, the transglycosylation activity of the enzyme was decreased by replacing Tyr-377 with hydrophilic amino acid residues, Asp or Ser.

Structure of the cyclic glucan produced from amylopectin by Bacillus stearothermophilus branching enzyme

The thermostable branching enzyme (BE, EC 2.4.1.18) from Bacillus stearothermophilus TRBE14 produces large cyclic glucans from waxy rice amylopectin similar to those obtained from amylose as described elsewhere [H. Takata, T. Takaha, S. Okada. M. Takagi, and T. Imanaka, J. Bacteriol., 178 (1996) 1600-1606]. The structure of the product (P-1) from the late-stage reaction was analyzed in detail. The weight-average degree of polymerization (dpw) of P-1 was 900. Its chain-length distribution was not significantly changed compared with that of amylopectin, although the amount of long chains (dp > 38) was slightly decreased. The cyclic component of P-1, which was isolated by the extensive action of glucoamylase, had dpw of 49. Three point five alpha-1,6 linkages were directly involved in the formation of the ring structure with several non-cyclic side chains linked to the ring. Based on these results, the action and new roles of BE are discussed.

Purification and Some Properties of α-Amylase from Bacillus subtilis X-23 That Glucosylates Phenolic Compounds Such as Hydroquinone

Abstract Six hundred strains of soil microorganisms were screened for the production of hydroquinone glucosylating enzyme (HGE). One of these strains, Bacillus subtilis strain X-23, produced an enzyme that glucosylated hydroquinone in the culture filtrate. The HGE was successively purified by ammonium sulfate fractionation, and Q-Sepharose, Phenyl-Toyopearl, and Superose 12 column chromatographies. The molecular weight was estimated as 65 kDa by SDS-polyacrylamide gel electrophoresis, and 54 kDa by gel filtration. Based on an analysis of the hydrolytic products from soluble starch, HGE was considered to be a kind of α-amylase. The structure of the hydroquinone glucoside produced by HGE was identified as 4- hydroxyphenyl -O-α- d -glucopyranoside by α- and β-glucosidase treatments, and 1H-NMR and 13C-NMR.

Introduction of raw starch-binding domains into Bacillus subtilis α-amylase by fusion with the starch-binding domain of Bacillus cyclomaltodextrin glucanotransferase

ABSTRACT We constructed two types of chimeric enzymes, Ch1 Amy and Ch2 Amy. Ch1 Amy consisted of a catalytic domain of Bacillus subtilis X-23 α-amylase (Ba-S) and the raw starch-binding domain (domain E) of Bacillus A2-5a cyclomaltodextrin glucanotransferase (A2-5a CGT). Ch2 Amy consisted of Ba-S and D (function unknown) plus E domains of A2-5a CGT. Ch1 Amy acquired raw starch-binding and -digesting abilities which were not present in the catalytic part (Ba-S). Furthermore, the specific activity of Ch1 Amy was almost identical when enzyme activity was evaluated on a molar basis. Although Ch2 Amy exhibited even higher raw starch-binding and -digesting abilities than Ch1 Amy, the specific activity was lower than that of Ba-S. We did not detect any differences in other enzymatic characteristics (amylolytic pattern, transglycosylation ability, effects of pH, and temperature on stability and activity) among Ba-S, Ch1 Amy, and Ch2 Amy.

Normal starch content and composition in tubers of antisense potato plants lacking D-enzyme (4-α-glucanotransferase)

Abstract. Transgenic potato (Solanum tuberosum L.) plants were created with sense and antisense copies of the potato D-enzyme (disproportionating enzyme; EC␣2.4.1.25) cDNA linked to patatin and cauliflower mosaic virus 35 S promoters, and screened for D-enzyme activity in tubers. Transformants with sense constructs mostly had wild type D-enzyme activity but two plants had only about 1% wild-type activity. Transformants with antisense constructs had activity ranging from 90% to about 1% of wild type. Three 35 S antisense plants with very low activity were analysed in detail. Western blot analysis showed that D-enzyme was present in greatly reduced amounts in tubers and in leaves, whereas plastidic starch phosphorylase (EC 2.4.1.1) was unaffected. The lack of D-enzyme resulted in slow plant growth but development was otherwise apparently normal. Furthermore, the starch content of tubers was not appreciably altered in amount, proportion of amylose, molecular weight of debranched amylopectin, or branch chain length, despite the lack of D-enzyme. These results do not indicate a direct requirement for D-enzyme in the synthesis and accumulation of storage starch in tubers. The results are discussed in terms of the known reactions catalysed by D-enzyme and possible involvement of D-enzyme in starch metabolism.

Complex formation of large-ring cyclodextrins with iodine in aqueous solution as revealed by isothermal titration calorimetry

Complex formation of large cyclodextrins(CDs) having DP 21–32 with iodine in aqueous KI solution was studied by isothermal titration calorimetry. The curves obtained for the titration of the CDs with iodine cannot be analyzed by a model based on a single set of identical sites, but, rather, by a model assuming 1 : 2 complex formation with identical interacting sites. For the two identical interacting sites, the binding constants K1 and K2 (K1 < K2), defined relative to the progress of saturation, lie in the range 0.7 to 7.3×103 M–1 and 3.0 to 62.6×103 M–1, respectively. The values of ΔH2 and T ΔS2 lie in the range –34.9 to –136.4 kJ·mol–1 and –15.5 to –112.8 kJ·mol–1, respectively. The largest values of –T ΔS2 obtained for a CD of DP 26 can, in part, be attributed to a large decrease in conformational flexibility of the CD which occurs during complex formation.

Comparative Study of the Cyclization Reactions of Three Bacterial Cyclomaltodextrin Glucanotransferases

ABSTRACT The actions of cyclomaltodextrin glucanotransferases (CGTase; EC2.4.1.19 ) from alkalophilic Bacillus sp. strain A2-5a (A2-5a CGTase), Bacillus macerans (BmacCGTase), and Bacillus stearothermophilus (BsteCGTase) on amylose were investigated. All three enzymes produced large cyclic α-1,4-glucans (cycloamyloses) at the early stage of the reaction, but these were subsequently converted into smaller cycloamyloses. However, the rates of this conversion differed among the three enzymes. The product specificity of each CGTase in the cyclization reaction was determined by measuring the amount of each cycloamylose from CD6 to CD31 (CDn, a cycloamylose with a degree of polymerization of n). A2-5a CGTase produced 10 times more CD7, while Bmac CGTase produced 34 times more CD6 than other cycloamyloses. Bste CGTase produced 12 and 3 times more CD6 and CD7 than other cycloamyloses, respectively. The substrate specificities of the linearization reactions of CD6, CD7, CD8, and larger cycloamyloses (a mixture of CD22 to CD50) were investigated, and we found that CD7 and CD8 are extremely poor substrates for both hydrolytic and transglycosidic linearization (coupling) reactions while larger cycloamyloses are linearized at a much higher rate. By repeating these cyclization and linearization reactions, the larger cycloamyloses initially produced are converted into smaller cycloamyloses and finally into mainly CD6, CD7, and CD8. These three enzymes also differ in their hydrolytic activities, which seem to accelerate the conversion of larger cycloamyloses into smaller cycloamyloses.