Evaluation of the distribution and orientation of remineralized enamel crystallites in subsurface lesions by X-ray diffraction.

Remineralization is the process by which hydroxyapatite (HAp) is restored in enamel subsurface lesions, and transversal microradiography (TMR) has been used to analyze remineralization in terms of the recovery of mineral content. In this study, we directly detected the distribution and orientation of longitudinal HAp crystallite at the remineralized zone in enamel subsurface lesions by using an X-ray microbeam (6-mum diameter) diffraction method. This method was demonstrated and involves the simultaneous detection of wide-angle X-ray diffraction (WAXRD) and small-angle X-ray scattering (SAXS). WAXRD reflects the amount of HAp crystallites, and SAXS reflects that of voids in crystallites. The polished surface of a bovine enamel block was divided into three zones of sound, demineralized, and remineralized zones. Thin sections of approximately 150 mum thickness were then cut perpendicular to the surface, and subjected to WAXRD and SAXS following TMR. The increase in the amount of HAp crystallites and the decrease in voids in the crystallites at the remineralized zone were detected by WAXRD and SAXS, respectively, which was consistent with the result of TMR. This study indicates that both the spatial distribution and orientation of the restored HAp crystals in the remineralization process at the subsurface lesion can be simultaneously analyzed by the X-ray diffraction methods.

Evaluation of enamel crystallites in subsurface lesion by microbeam X-ray diffraction.

Early caries lesion is a demineralization process that takes place in the top 0.1 mm layer of tooth enamel. In this study, X-ray microbeam diffraction was used to evaluate the hydroxyapatite crystallites in the subsurface lesion of a bovine enamel section and the results are compared with those obtained by transversal microradiography, a method commonly used for evaluation of tooth mineral. Synchrotron radiation from SPring-8 was used to obtain a microbeam with a diameter of 6 microm. Wide-angle X-ray diffraction reports the amount of hydroxyapatite crystals, and small-angle X-ray scattering reports that of voids in crystallites. All three methods showed a marked decrease in the enamel density in the subsurface region after demineralization. As these diffraction methods provide structural information in the nanometre range, they are useful for investigating the mechanism of the mineral loss in early caries lesion at a nanometre level.

Fluids containing a highly branched cyclic dextrin influence the gastric emptying rate.

The rates of gastric emptying for highly branched cyclic dextrin (HBCD) and other carbohydrate (CHO) solutions were examined using ultrasonograph techniques. Ten healthy volunteers ingested water, physiological saline, or solutions containing various CHO, such as HBCD, glucose, maltose, sucrose, and commercially available dextrin. After a subject drank one of the solutions, the relaxed cross-sectional area of the pylorus antrum was measured at rest by real-time ultrasonography. The time required for gastric emptying was correlated with the relaxed cross-sectional area of the pylorus antrum. Among all of the solutions tested, physiological saline was transferred fastest from the stomach to the small intestine. For solutions of the same CHO, 5 % solution was transferred faster than 10 % solution. For CHO solutions other than HBCD, a low osmotic pressure was associated with rapid transfer from the stomach. The gastric emptying time (GET) of HBCD solution increased with an increase in its concentration. A shorter GET was observed for the CHO solutions at 59 to 160 mOsm regardless of their concentration. A sports drink based on 10 % HBCD adjusted to 150 mOsm by the addition of various minerals, vitamins, and organic acids was evacuated significantly (p < 0.05) faster than a 10 % HBCD solution or a sports drink based on 10 % commercially available dextrin (DE16), which has a higher osmotic pressure (269 mOsm). Our results suggest that a shorter GET could be achieved with CHO solutions with osmotic pressures of 59 - 160 mOsm. Therefore, a sports drink based on 10 % HBCD adjusted to 150 mOsm by the addition of minerals, vitamins, and organic acids could supply adequate quantities of CHO, fluid, and minerals simultaneously in a short time, without increasing GET.

Substrate selectivity in Aspergillus niger KU-8 acid phosphatase II using phosphoryl oligosaccharides.

The intracellular acid phosphatase II (ACPase II) produced by Aspergillus niger KU-8 preferentially dephosphorylates C-6 phosphate groups rather than C-3 phosphate groups of phosphoryl oligosaccharides. In this study, the kinetic parameters of ACPase II were measured. 3(2)-phosphoryl maltotriose and 6(2)-phosphoryl maltotriose, which differ only in the binding position of the phosphate group, were prepared and used as the substrates. The Km for both substrates were similar. However, the k(cat) value for the 6(2)-phosphoryl maltotriose was about three-fold of that for the 3(2)-phosphoryl maltotriose.

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

We constructed two types of chimeric enzymes, Ch1 Amy and Ch2 Amy. Ch1 Amy consisted of a catalytic domain of Bacillus subtilis X-23 alpha-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.

Cloning of the cyclodextrin glucanotransferase gene from alkalophilic Bacillus sp. A2-5a and analysis of the raw starch-binding domain.

The cyclodextrin glucanotransferase (CGTase) gene of alkalophilic Bacillus sp. A2-5a was cloned and expressed in Bacillus subtilis ANA-1 as a host. The DNA region included an open reading frame encoding a 704-amino-acid polypeptide with a typical raw starch-binding motif in its C-terminal region. The CGTase purified from Bacillus sp. A2-5a bound to raw starch as strongly as porcine pancreas alpha-amylase, as expected from the sequence motif. A chromosomal region (a DNA fragment of about 14.1 kbp) including the CGTase gene was also cloned and the nucleotide sequence was determined. Possible cyclodextrinase and putative cyclodextrin-binding protein genes were found in the flanking region of the CGTase gene, which implied that the novel starch-degradation pathway postulated for a gram-negative bacterium [Klebsiella oxytoca; Fiedler et al. (1996) J Mol Biol 256: 279-291] also exists in a gram-positive bacterium i.e. Bacillus.

Characteristics of two forms of alpha-amylases and structural implication.

Complete (Ba-L) and truncated (Ba-S) forms of alpha-amylases from Bacillus subtilis X-23 were purified, and the amino- and carboxyl-terminal amino acid sequences of Ba-L and Ba-S were determined. The amino acid sequence deduced from the nucleotide sequence of the alpha-amylase gene indicated that Ba-S was produced from Ba-L by truncation of the 186 amino acid residues at the carboxyl-terminal region. The results of genomic Southern analysis and Western analysis suggested that the two enzymes originated from the same alpha-amylase gene and that truncation of Ba-L to Ba-S occurred during the cultivation of B. subtilis X-23 cells. Although the primary structure of Ba-S was approximately 28% shorter than that of Ba-L, the two enzyme forms had the same enzymatic characteristics (molar catalytic activity, amylolytic pattern, transglycosylation ability, effect of pH on stability and activity, optimum temperature, and raw starch-binding ability), except that the thermal stability of Ba-S was higher than that of Ba-L. An analysis of the secondary structure as well as the predicted three-dimensional structure of Ba-S showed that Ba-S retained all of the necessary domains (domains A, B, and C) which were most likely to be required for functionality as alpha-amylase.

The concept of the alpha-amylase family: structural similarity and common catalytic mechanism.

This review reconsiders the concept of the alpha-amylase family in the light of the recent wealth of information on the structures, the catalytic mechanisms, and the classification of amylases. We proposed a general concept for an enzyme family, the alpha-amylase family including most of the amylases and related enzymes in 1992, based on the structural similarity and the common catalytic mechanisms. The study on neopullulanase was the key to open the door for the formulation of the concept. We discovered a new enzyme, neopullulanase, and proved that the enzyme catalyzes both hydrolysis and transglycosylation at alpha-1,4- and alpha-1,6-glucosidic linkages by one active center. Results from a series of experiments using neopullulanase indicated that the four reactions mentioned above could be catalyzed in the same mechanism. Progress in X-ray crystallographic analysis has allowed researchers to observe the structural similarities among alpha-amylases, cyclodextrin glucanotransferases, and an isoamylase. The primary structural analyses and the secondary structural predictions also suggest a close relationship among enzymes with three-dimensional structures which catalyze one of the four reactions. They possess a catalytic (beta/alpha)8-barrel as observed in the crystal structure of alpha-amylases, cyclodextrin glucanotransferases, and an isoamylase. Two crucial points, the common catalytic mechanisms and the structural similarities among the enzymes which catalyze the four reactions, led us to propose the concept of the alpha-amylase family. We would like to point out the significance and problems of the sequence-based classification of glycosyl hydrolases. The possible catalytic mechanism of the alpha-amylase family enzyme is also described for the rational design of tailor-made artificial enzymes.

Construction of chimeric enzymes out of maize endosperm branching enzymes I and II: activity and properties.

Branching enzyme I and II isoforms from maize endosperm (mBE I and mBE II, respectively) have quite different properties, and to elucidate the domain(s) that determines the differences, chimeric genes consisting of part mBE I and part mBE II were constructed. When expressed under the control of the T7 promoter in Escherichia coli, several of the chimeric enzymes were inactive. The only fully active chimeric enzyme was mBE II-I BspHI, in which the carboxyl-terminal part of mBE II was exchanged for that of mBE I at a BspHI restriction site and was purified to homogeneity and characterized. Another chimeric enzyme, mBE I-II HindIII, in which the amino-terminal end of mBE II was replaced with that of mBE I, had very little activity and was only partially characterized. The purified mBE II-I BspHI exhibited higher activity than wild-type mBE I and mBE II when assayed by the phosphorylase a stimulation assay. mBE II-I BspHI had substrate specificity (preference for amylose rather than amylopectin) and catalytic capacity similar to mBE I, despite the fact that only the carboxyl terminus was from mBE I, suggesting that the carboxyl terminus may be involved in determining substrate specificity and catalytic capacity. In chain transfer experiments, mBE II-I BspHI transferred more short chains (with a degree of polymerization of around 6) in a fashion similar to mBE II. In contrast, mBE I-II HindIII transferred more long chains (with a degree of polymerization of around 11-12), similar to mBE I, suggesting that the amino terminus of mBEs may play a role in the size of oligosaccharide chain transferred. This study challenges the notion that the catalytic centers for branching enzymes are exclusively located in the central portion of the enzyme; it suggests instead that the amino and carboxyl termini may also be involved in determining substrate preference, catalytic capacity, and chain length transfer.

A novel acid phosphatase from Aspergillus niger KU-8 that specifically hydrolyzes C-6 phosphate groups of phosphoryl oligosaccharides.

We had analyzed the detailed structures of the phosphoryl oligosaccharide-1 (PO-1) fraction that was the main component of phosphoryl oligosaccharides (POs) prepared from a potato starch hydrolysate. PO-1 fraction was made up of 3-phosphoryl oligosaccharides and 6-phosphoryl oligosaccharides. Aspergillus niger strain KU-8 produced two types of intracellular acid phosphatase (EC 3.1.3.2, ACPase); ACPase I and II. ACPase II preferentially dephosphorylated 6-phosphoryl oligosaccharides rather than 3-phosphoryl oligosaccharides. The molecular weight of the enzyme was estimated as 66 kDa by SDS-polyacrylamide gel electrophoresis and about 260 kDa by gel filtration, implying the active form to be a tetramer. The optimum pH and temperature of the enzyme were 2.0-2.5 and 60 degrees C, respectively. ACPase II was stable below 50 degrees C for 30 min and pH 2.0-10.0 for 60 min. In spite of the strict specificity toward 6-phosphoryl oligosaccharides in the PO-1 fraction, ACPase II was able to hydrolyze Fru-1,6-di-P, ATP, pyrophosphate, and polyphosphate as well as pNPP and Glc-6-P, a broad substrate specificity.

Improved separation and determination of phospholipids in animal tissues employing solid phase extraction.

Separation of five glycerophospholipids having different polar groups, phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylglycerol (PG) and cardiolipin (CL), was investigated by means of solid-phase extraction (SPE) cartridges. First, the phospholipids were retained in an aminopropyl-bonded phase (NH2) cartridge and subsequently eluted as neutral (PC and PE) and acidic (PS, PG and CL) glycerophospholipid fractions. Secondly, a combination of silica gel (SI) cartridge and NH2 cartridge was employed to separate five glycerophospholipids. The polarity of the eluent was responsible for neutral glycerophospholipid separation. Concerning acidic glycerophospholipids, the separation of PG and CL from PS depended mainly on the pH of the eluents, and the separation of PG and CL was affected by the solvent, depending on eluent polarities. Favorable recovery (not less than 95%, for five authentic phospholipids, 10-100 micrograms each) and repeatability (sigma = 2.3 for 10 micrograms ranges) were attained by the present method. This method of separation was applicable to the analysis of phospholipids in biological samples.

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 alpha-(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 alpha-amylase. In contrast, replacing Ile-358 with Val, which has a smaller side chain, increased the preference for alpha-(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.

Analysis of the active center of branching enzyme II from maize endosperm.

Analysis of the primary structure of mBEII, with those of other branching and amylolytic enzymes as reference, identifies four highly conserved regions which may be involved in substrate binding and in catalysis. When one of the amino acid residues corresponding to the putative catalytic sites of mBEII, i.e., Asp-386, Glu-441, and Asp-509, was replaced, activity disappeared. These putative catalytic residues are located in three different regions (regions 2-4) of the four highly conserved regions (regions 1-4) which exist in the primary structure of most starch hydrolases and related enzymes, including branching enzymes. Region 3, which contains Glu-441 as one of the putative catalytic residues, was located downstream of the carboxyl-terminal position previously reported. The importance of the carboxyl amino acid residues was also demonstrated by chemical modification of the branching enzyme protein using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.

Maize branching enzyme catalyzes synthesis of glycogen-like polysaccharide in glgB-deficient Escherichia coli.

The structure of alpha-glucan, isolated from wild-type Escherichia coli B, a glycogen branching enzyme (BE)-deficient E. coli AC71 (glgB-), or from AC71 transformed with genes coding for maize BEI and BEII individually as well as with both genes, was analyzed by high-performance anion-exchange chromatography (HPAEC) with pulsed amperometric detection. Transformation of the maize BE gene(s) in AC71 (glgB-) showed complementation in branching activity. Analysis by HPAEC revealed different structures between glycogen of E. coli B and alpha-glucan of AC71 transformed with a different maize BE gene(s). The individual chains of the alpha-glucan debranched with isoamylase were distributed between chain length (CL) 3 and > 30 and the chain with CL 6 was the most abundant. In comparison with the glycogen of E. coli B, the alpha-glucan of AC71 transformed with the maize BE gene(s) consisted of a lesser amount of chains with CL 7-9 and a larger amount of chains with CL > 14. It also showed a broad peak with chains of CL 9-12 as in maize amylopectin. This study provides in vivo evidence that glycogen BE and maize BE isozymes may have different specificities in the length of chain transferred. Furthermore, this study suggests that the specificity of glycogen synthase and starch synthase and their concerted action with BE play an important role in determining the structure of the polysaccharide synthesized.

Properties and active center of the thermostable branching enzyme from Bacillus stearothermophilus.

Although the branching enzyme (EC 2.4.1.18) is a member of the alpha-amylase family, the characteristics are not understood. The thermostable branching enzyme gene from Bacillus stearothermophilus TRBE14 was cloned and expressed in Escherichia coli. The branching enzyme was purified to homogeneity, and various enzymatic properties were analyzed by our improved assay method. About 80% of activity was retained when the enzyme was heated at 60 degrees C for 30 min, and the optimum temperature for activity was around 50 degrees C. The enzyme was stable in the range of pH 7.5 to 9.5, and the optimum pH was 7.5. The nucleotide sequence of the gene was determined, and the active center of the enzyme was analyzed by means of site-directed mutagenesis. The catalytic residues were tentatively identified as two Asp residues and a Glu residue by comparison of the amino acid sequences of various branching enzymes from different sources and enzymes of the alpha-amylase family. When the Asp residues and Glu were replaced by Asn and Gln, respectively, the branching enzyme activities disappeared. The results suggested that these three residues are the catalytic residues and that the catalytic mechanism of the branching enzyme is basically identical to that of alpha-amylase. On the basis of these results, four conserved regions including catalytic residues and most of the substrate-binding residues of various branching enzymes are proposed.

Two-dimensional electrophoresis as a complementary method of isolating peptide fragments of cleaved proteins for internal sequencing.

To determine the primary structure of proteins, usually proteolytic enzyme digests are separated by reversed-phase high-performance liquid chromatography (HPLC) and each fraction is collected and sequenced. The results obtained by different cleavages are combined to reveal the entire sequence. However, there are many N-terminal-blocked proteins and/or intact proteins or their particular fragments that are not eluted from HPLC columns. Internal fragments of such proteins were successfully isolated by the use of two-dimensional electrophoresis, after digestion. Electroblotted spots were easily sequenced to identify those difficult fragments which could not be obtained using HPLC.

The primary structure of branched-chain alpha-oxo acid dehydrogenase from Bacillus subtilis and its similarity to other alpha-oxo acid dehydrogenases.

The bfmB mutant of Bacillus subtilis requires branched short-chain carboxylic acids for growth because the organism is known to be defective in branched-chain alpha-oxo acid dehydrogenase. The DNA in the region of bfmB has now been cloned and sequenced, and the gene has been analyzed. The results show that there are three open reading frames in the area, each of which is preceded by a putative ribosome binding site, and the last of which is followed by a putative transcription termination site with inverted repeats. The amino acid sequences deduced by analysis of the reading frames are highly similar (with 32-49% identity) to the E1 alpha, El beta and E2 components of pyruvate, 2-oxoglutarate and branched-chain alpha-oxo acid dehydrogenases from different sources. The thiamin diphosphate binding, putative subunit interaction and phosphorylation sites of the E1 alpha of four reported branched-chain alpha-oxo acid dehydrogenases from different sources are very similar to those of the first open reading frame (E1 alpha) of bfmB. A similar result is also obtained with the lipoyl-binding site (lysine) and its domain of the E2 component of alpha-oxo acid dehydrogenases from different sources. The present data, along with the reported biochemical data, lead to the conclusion that bfmB encodes a branched-chain alpha-oxo acid dehydrogenase, which is composed of E1 alpha, E1 beta and E2 genes. This organization is identical to that of the 2-oxoglutarate dehydrogenase in B. subtilis.

A new way of producing isomalto-oligosaccharide syrup by using the transglycosylation reaction of neopullulanase.

A new way of producing isomalto-oligosaccharide syrup from starch was developed. Isomalto-oligosaccharides contain one or more alpha-(1-->6)-glucosidic linkages with or without alpha-(1-->4)-glucosidic linkages. The isomalto-oligosaccharide syrups are receiving increased attention as food additives because it is thought that they help prevent dental caries and improve human intestinal microflora, acting as a growth factor for bifidobacteria. The new system for production of isomalto-oligosaccharide syrup is based on the strong alpha-(1-->6)-transglycosylation reaction of neopullulanase. Bacillus subtilis saccharifying alpha-amylase was simultaneously used with neopullulanase to improve the yield of isomalto-oligosaccharides. The yield of isomalto-oligosaccharides was increased to more than 60%, compared with a yield of 45.0% obtained by the conventional system. To reduce the costs, the use of immobilized neopullulanase was investigated. Almost the same yield of isomalto-oligosaccharides was obtained by using immobilized neopullulanase.

Determination of felodipine enantiomers using chiral stationary phase liquid chromatography and gas chromatography/mass spectrometry, and the study of their pharmacokinetic profiles in human and dog.

A stereoselective and sensitive method for the determination of the enantiomers of felodipine, a dihydropyridine calcium antagonist, has been developed and the pharmacokinetic profiles of the enantiomers comparatively studied after oral administration to dogs and humans. D6-Felodipine, the internal standard, was added to the plasma, extracted with a solvent and then optically resolved into S(-) and R(+) enantiomers on a high performance liquid chromatographic Chiralcel OJ column. Each enantiomer in the effluent was analysed by capillary column gas chromatography/positive ion electron impact mass spectrometry. After oral administration of the felodipine racemate, the Tmax and t1/2 values hardly differed between the two enantiomers in dogs and humans. The Cmax and AUC0-24 h values of the S(-) enantiomer were slightly higher than those of the R(+) enantiomer in humans but the difference between the enantiomers was not significant. These results suggested that there is no large difference in the absorption, distribution and elimination of felodipine enantiomers after oral administration of the racemate in either dog or human.

Separation and determination of phospholipids in plasma employing thin-layer chromatographic plate with concentration zone or solid phase extraction.

Two efficient extraction methods for phospholipids have been established employing a TLC plate with a concentration zone or a solid-phase extraction cartridge. First, small amounts of five authentic phospholipids, 10 to 100 micrograms each, were separated on a TLC plate with a concentration zone. These phospholipids were not adsorbed to the silica gel of this TLC plate and the substrates were recovered in good yields. Second, a solid-phase extraction cartridge containing NH2-modified silica gel was able to retain all of the authentic phospholipids among C18-, diol-, and CN-modified silica gel and silica gel beds. Elution of neutral and acidic phospholipids from the NH2-cartridge was carried out with a chloroform-methanol mixture and a chloroform-methanol-28% ammonium mixture containing 0.05 M ammonium acetate, respectively. Recovery tests on the above two methods were carried out by conventional capillary gas chromatography. Thus, not less than 95% of each phospholipid was recovered from the TLC plate or NH2-cartridge in a 10 to 100 micrograms sample range. Also, the standard deviation of repeatability of recovery for phosphatidylcholine by solid phase extraction at a 10 micrograms sample range was favorable at sigma = 2.8. This method of separation was applied to determination of the fatty acid compositions of phospholipids in small amounts of plasma.