Water mixtures of normal or waxy corn starch were treated with a high hydrostatic pressure (HHP) of 600 MPa at 40°C for 1 h, and the effect of starch content (10-70% (w/w)) on the physical properties of HHP-treated starch was evaluated by differential scanning calorimetry (DSC), X-ray diffraction, water holding capacity, cold water solubility and optical microscopy. With decreased starch content, HHP-treated starches showed loss in gelatinizaion enthalpy, less sharp X-ray diffraction patterns, and reduced water holding capacity and cold water solubility. More enhanced reductions in water holding capacity and cold water solubility were observed with normal corn starch than with waxy corn starch. The morphology of HHP-treated starch differed between normal and waxy corn starches. When completely pressure-gelatinized, the granular shape of HHP-treated normal corn starch was retained, while that of HHP-treated waxy corn starch was disintegrated.
We have investigated the enzymatic characterization of recombinant β-fructofuranosidase from Bifidobacterium longum JCM1217. This recombinant protein was expressed in Escherichia coli, and showed high activity of hydrolysis on fructo-oligosaccharides with a low degree of polymerization. The molecular mass of the purified recombinant protein was estimated to be about 64,000 by SDS-PAGE and 59,600 by MALDI TOF-MS. The optimum pH and pH stability of the enzyme were 5.7 and 5.0-7.9, respectively. The temperature stability of the enzyme was indicated up to 50°C. The Km (mM), Vmax (μmol/min/mg of protein), k0 (s-1) and k0/Km (mM-1 s-1) for 1-kestose, sucrose, neokestose, nystose, fructosylnystose and inulin were 1.2, 97, 96.4 and 80.3, 38, 64, 63.6 and 1.7, 2.1, 109, 105.3 and 50.1, 4.2, 52, 51.7 and 12.3, 3.1, 66, 65.6 and 21.2, 16.5, 72, 71.6 and 4.3, respectively. This recombinant protein had a high affinity for fructo-oligosaccharides.
Regular Papers (in Japanese with English Abstract)
A Furunori-like polysaccharide was prepared from the paste of wheat starch for 2 weeks by a new method combining α-amylase treatment of the paste and the washing of the paste to remove low molecular-weight materials during retrogradation of the paste. Structural properties of the Furunori-like polysaccharide and Furunori samples were investigated by comparing their iodine affinity, average molecular-weight, and distributions of the unit chain-length and whole chain-length. The degree of retrogradation was also compared using the β-amylase-pullulanase method and the X-ray diffractometer. All results of these experiments showed similarities between the Furunori-like polysaccharide and Furunori samples. Their similarity of structure and degree of retrogradation appears greatly to provide the Furunori-like polysaccharide with an adhesiveness and smoothness similar to that of the Furunori samples.
Rakkasei-tofu (peanut-tofu) is a kind of a processed food which is made from peanut milk squeezed from ground peanut flour with water, starch and water. It is usually prepared using sweet potato starch in the Okinawa region. We made the Rakkasei-tofu from seven kinds of starches: kuzu starch, sweet potato starch, a low temperature gelatinization sweet potato starch called “minazuki”, corn starch, tapioca starch and modified tapioca starches called “PB108” and “PB2000”. The texture (hardness, cohesiveness and adhesiveness), degree of gelatinization and sensory evaluation were measured for each of these preparations. The physicochemical properties (particle size distribution, swelling power, solubility, X-ray diffraction, thermal properties using a differential scanning calorimetry; DSC and pasting properties using a Rapid Visco Analyzer; RVA) of the starches were also examined. It was demonstrated that Minazuki gelatinized at a low temperature similar to PB108 and PB2000. As the storage period increased, the cohesiveness and the gelatinization degree of the Rakkasei-tofu made from kuzu and sweet potato decreased, but those made from minazuki and PB2000 retained higher cohesiveness and a greater degree of gelatinization. These results suggest that the Rakkasei-tofu made from starches showing low temperature gelatinization had good keeping qualities.
Thermosetting bio-plastic produced from a slurry mixture of saccharide and polyisocyanate by heating includes electric parts demanding heat-resistance as phenol resin. It is generally called “prepolymer”, and is unstable and self-curing at room temperature. The purpose of this study is to understand the self-curing mechanisms of the prepolymerization using tools such as infrared and X-ray spectroscopy. The experimental results suggested that the self-curing process was divided into 4 steps: polyisocyanate hydration, amine group production, urea group production and viewlet group production. They were caused by absorptive water of saccharide, and the cross-linkage formation of the viewlet group provided the cure. It was also possible that the urea group was produced from the proton originating from saccharide and polyisocyanate. We also found that fructose, maltose monohydrate and glucose-supplemented water had long curing characteristics, which was different from the affinity for adsorptive water. But crystal water and the crystallinity of saccharide did not affect the cure. Hence, the absorptive water was very important, since the viewlet group was not produced and the cure was delayed during the urea group production using it. Consequently, this study could be a very important step for stable prepolymer production in industrial processes.
Proceedings of the Symposium on Amylases and Related Enzymes, 2009
A new enzymatic process for glycogen production was developed. In this process, short-chain amylose is used as the substrate for branching enzymes (BE, EC 220.127.116.11). The molecular weight of the enzymatically synthesized glycogen (ESG) depends on the type of BE used and the molecular weight and the concentration of the substrate amylose. Although a plant BE (kidney bean) and Bacillus cereus BE could not synthesize high molecular weight glucan, BEs from 6 other bacterial sources produced ESG. The BE from Aquifex aeolicus was the most suitable for the production of glycogen with a weight-average molecular weight (Mw) of 3000 kDa to 30,000 kDa. Furthermore, the addition of amylomaltase (AM, EC 18.104.22.168) significantly enhanced the efficiency of this process, and the yield of ESG reached approximately 65%. Typical preparations of ESG obtained by this method were subjected to structural analyses. The average chain length, interior chain length, and exterior chain length of the ESGs were 8.2-11.6, 2.0-3.3 and 4.2-7.6, respectively. Transmission electron microscopy showed that the ESG molecules formed spherical particles. Viscometric analyses also supported a spherical nature for the product. Unlike starch, the ESGs were barely degraded by pullulanase. Solutions of ESG were opalescent (milky-white and slightly bluish), and gave a reddish-brown color on the addition of iodine. These analyses revealed that ESG shares similar molecular shapes and solution properties with natural source glycogen (NSG).
Cellobiose (528-50-7) was synthesized from starch using two phosphorylases. α-Glucan phosphorylase (EC 22.214.171.124) converted 39.6% of glucose residues in the starch molecule into glucose 1-phosphate in the presence of 1 M inorganic phosphate. Inorganic phosphate was selectively dialyzed out from the resultant reaction mixture by electrodialysis equipped with an ion exchange membrane having a molecular weight cut-off of 100. Thus glucose1-phosphate solution free from inorganic phosphate was obtained with 82.1% yield. Cellobiose phosphorylase (EC 126.96.36.199) converted 89.2% of added glucose 1-phosphate into cellobiose when the above G1P solution was incubated with a roughly equimolar amount of glucose in the presence of magnesium acetate under alkaline conditions (pH 8.0). As a result of three successive steps using two phosphorylases, cellobiose was produced from starch with a 29.0% yield. Cellobiose was reduced under a sponge nickel catalyst (Raney nickel) and the obtained cellobiitol was crystallized with an 86.7% yield. The 1H- and 13C-NMR spectra of this compound agreed well with those expected from 4-O-β-glucosyl-D-glucitol (cellobiitol, 535-94-4). The X-ray crystallographic analysis of the obtained cellobiitol crystal revealed it to be a monoclinic space group P21 (4), with the following unit-cell parameters: a = 1.0007, b = 0.8683, c = 2.3137 nm, β = 127.27°. From a thermo gravimetric-differential scanning calorimetric analysis, the cellobiitol crystal was found to have a melting point of 103.6°C and to be in the form of a monohydrate. Cellobiitol showed a relative sweetness of around 20, being less sweet than other sugar alcohols such as glucitol (sorbitol) and maltitol. Cellobiitol was found to be much less hygroscopic than sorbitol and maltitol. The median lethal dose of cellobiitol in rats was determined to be more than 5000 mg/kg body weight. All these properties of cellobiitol indicate it to be a promising compound as a food ingredient.
The alkaliphilic soil bacterium Bacillus clarkii 7364 was found to produce cyclodextrin glucanotransferase (CGTase), an enzyme which converts starch into γ-cyclodextrin (γ-CD) with high specificity. The bacterium also intracellularly produced cyclodextrinase (CDase). The gene encoding the CDase (Cda) was located about 200 bases downstream of the gene encoding the CGTase (Cgt). Comparison of the amino acid sequence of Cgt with those of other CGTases revealed that several amino acids which contribute to substrate binding are absent or different at subsites +3, +2, -3 and -7 in Cgt. The replacement of Ala223 at subsite +2 by three basic amino acids, histidine, lysine and arginine, strongly enhanced γ-CD-forming activity in the neutral pH range, although the optimum pH for the activity (pH 10.0) and CD production specificity remained the same. The interaction between the protonated amino group in the side chain of these basic amino acids and the linear substrate is thought to play an important role in the enhancement of the activity. Cda had extremely high hydrolytic specificity for γ-CD. Cda also displayed a transglycosylation activity, where a maltotriose moiety could be transferred, unlike other CD-degrading enzymes. In Cda, the N-domain normally found in other CD-degrading enzymes which functions in the dimerization, thus contributing to the preference for CDs, was deleted and instead, a long extra sequence was present in the C-terminus. Despite the lack of the N-domain, Cda showed a dodecameric structure. These unique features indicate that Cda is a novel CD-degrading enzyme.
Glycosyltransferases are of growing importance in in vitro synthesis of oligosaccharides and modification of glycoproteins, and several expression systems for recombinant glycosyltransferases have been investigated. We have created gene libraries of human glycosyltransferases using the Gateway® system, and each gene encoding the catalytic domain of the glycosyltransferase was expressed as a soluble enzyme using human embryonic kidney (HEK) 293T cells or yeast expression systems. In the case of the mammalian expression system, HEK293T transfectant cells successfully expressed most of the human glycosyltransferases. On the other hand, 23 glycosyltransferases were secreted into the culture media as active forms among 53 genes tested in the methylotrophic yeast Ogataea minuta. In a further study involving the optimization of the yeast expression system, we found that several factors, such as cultural conditions, chaperone activity in the host cells and truncation of the glycosyltransferase gene to be expressed were critical for high-level production. In the case of ST3Gal-I, optimization of each parameter caused a greater than 300-fold increase in the activity in the culture supernatant. Finally, the library of glycans or glycopeptides having various structures was synthesized by combination with glycosyltransferases from HEK293T cells. We also demonstrated the modification of various pyridylaminated N-glycan structures by sequential reactions with the recombinant enzymes from the yeast system. Our expression system for human glycosyltransferases may be applicable to the preparation of glycan arrays and the production of therapeutic glycoproteins with homogeneous glycans.
Highly water-soluble, artificial glycopolypeptides with a γ-polyglutamic acid (γ-PGA) backbone derived from Bacillus subtilis and multivalent sialyloligosaccharide units have been chemoenzymatically synthesized as potential polymeric inhibitors of infection by influenza virus. 5-Trifluoroacetamidopentyl β-N-acetyllactosaminide (1) was enzymatically synthesized from N-acetyllactosamine (LacNAc: Galβ1-4GlcNAc) by cellulase-mediated condensation with 5-trifluoroacetamido-1-pentanol. Next, enzymatic sugar elongation of the LacNAc unit to 1 was carried out by consecutive use of β1,3-N-acetylglucosaminyltransferase II (β3GnTII) and β1,4-galactosyltransferase I (β4GalTI) to produce tetra- and hexasaccharide glycosides (2 and 3) with tandem and triplet LacNAc repeats. After deacetylation, the resulting 5-aminopentyl di-, tetra- and hexasaccharide glycosides (4-6) were coupled to the α-carboxy groups of the γ-PGA side chains. Next, in order to synthesize an artificial sialoglycopolypeptide, we developed a large-scale production of rat α2,6-sialyltransferases (α2,6-SiaT). The α2,6-SiaT was expressed in fifth instar silkworm larval hemolymph using recombinant both cysteine protease- and chitinase-deficient Bombyx mori nucleopolyhedrovirus (BmNPV-CP--Chi-) bacmid. The expressed α2,6-SiaT and commercially available α2,3-SiaT were used for sialylation of asialoglycopolypeptides (7-9). The structure-activity relationship of the resulting α2,3/6-sialoglycopolypeptides (10-15) with different glycans in the array has been investigated by focus-forming and solid-phase binding assays. The avian viruses specifically bound to glycopolypeptides carrying a short sialo-glycan with higher affinity than to a long glycan. In contrast, human viruses preferentially bound to long α2,3/6 sialylated glycan with LacNAc repeats in the receptors. Our strategy provides a facile way to design strong polymeric inhibitors of infection by avian and human influenza viruses.
Xylanase J (XynJ) from alkaliphilic Bacillus sp. strain 41M-1 is a multi-domain enzyme consisting of a glycoside hydrolase (GH) family 11 catalytic domain and a carbohydrate-binding module family 36 xylan-binding domain (XBD). Structural comparison of the GH family 11 catalytic domains indicated that there were several specific salt bridges in the catalytic cleft of XynJ. Mutant enzymes were prepared by substituting several amino acid residues responsible for the characteristic salt bridges. Elimination of the salt bridges caused an acidophilic shift in optimum pH, suggesting that the characteristic salt bridges contributed to the alkaliphily of XynJ. On the other hand, reinforcing one of the characteristic salt bridges in the catalytic domain shifted the optimum pH of XynJ from 8.5 to 9.0. Furthermore, introducing excess Arg residues on the protein surface was also effective to improve the alkaliphily of XynJ. Xylan-binding properties of various XBD mutants were investigated as fusion proteins with glutathione-S-transferase. Furthermore, the same substitutions were introduced into the XBD region of XynJ and insoluble xylan-hydrolyzing activity was measured. The results indicated that some Asp, Trp and Tyr residues were important for xylan-binding activity of the XBD, and that xylan-hydrolyzing activity of XynJ was closely correlated to xylan-binding activity of the XBD region.
The pro-form (Pro-EndoPG I) of Stereum purpureum endopolygalacturonase I has a unique C-terminal region (pro-sequence) that is lacking in PGs of other origins. Mature EndoPG I purified from the culture filtrate of this fungus does not have the 44-amino-acid pro-sequence present in Pro-EndoPG I. We expressed Pro-EndoPG I in Escherichia coli and examined its activity. It was found that Pro-EndoPG I had no PG activity initially but some was acquired after the degradation of a portion of the pro-sequence with V8 protease. These results suggest that the pro-sequence inactivates auto-PG activity. No similar characteristic has been reported for any glycoside hydrolase. We then constructed C-terminal deletion mutants of Pro-EndoPG I and showed that 31 or 32 residues of the 44 amino acid residues in the pro-sequence were needed for the inactivation. Furthermore, we identified two Glu residues, E364 and E366, that were also related to the auto-inactivation. A test involving injection of the enzyme into apple trees showed that Pro-EndoPG I induced the same silver-leaf symptoms as mature EndoPG I. It is assumed that the Pro-EndoPG I was activated with plant proteases.
Eun-Seong Seo, Joakim M. Andersen, Morten M. Nielsen, Malene B. Vester-Christensen, Camilla Christiansen, Johanne M. Jensen, Janos A. Mótyán, Mikkel A. Glaring, Andreas Blennow, Lili Kandra, Gyöngyi Gyémánt, Štefan Janecek, Richard Haser, Nushin Aghajari, Maher Abou Hachem, Birte Svensson
Two carbohydrate binding surface sites (SBSs) on barley α-amylase 1 (AMY1) of glycoside hydrolase family 13 (GH13) displayed synergy in interactions with starch granules, thus being pivotal for hydrolysis of supramolecular substrates. Mutational analysis showed that SBS1 is more critical for the conversion of starch granules, while SBS2 has higher affinity than SBS1 for β-cyclodextrin (β-CD). Noticeably, the binding preference of β-CD to SBS2 differed distinctly from that of maltooligosaccharides to the catalytic nucleophile mutant D180A AMY1. Binding energy maps at subsites -8 through +4 of the active site indicated remarkably elevated affinity due to the Y380A mutation at SBS2. The high-yield AMY2 expression variant A42P, made it possible to show that Tyr378—corresponding to Tyr380 in AMY1—has a role in interactions with starch granules, but not in β-CD binding. Besides SBSs, dedicated starch binding domains (SBDs) mediate binding to starch granules. SBDs are currently categorised into 9 carbohydrate binding module (CBM) families. A novel CBM20 subfamily encountered in regulatory enzymes possesses characteristically low affinity for β-CD. Although α-amylase is essential for starch mobilisation in germinating barley seeds, efficient degradation requires the concerted action of α-amylase, β-amylase, limit dextrinase (LD) and possibly α-glucosidase. Limit dextrinase (LD) is encoded by a single gene and represents the sole debranching activity during germination. Recent expression of functional LD in Pichia pastoris makes biochemical and biophysical characterisation of this GH13 enzyme possible. An endogenous limit dextrinase inhibitor was cloned and produced recombinantly and demonstrated to have sub-nanomolar affinity for LD.
The Editor-In-Chief apologize for the mistake in “Article Type” on Page 287 of Volume 56(4), 2009. The term “Regular Paper (in Japanese with English Abstract) ” was erroneously changed to “Note (in Japanese with English Abstract) ”.