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ABSTRACT: The recently cloned β-galactosidase from Bacillus circulans ATCC 31382, designated BgaD, contains a multiple domain architecture including a F5/8 type C domain or a discoidin (DS) domain in the C-terminal peptide region. Here we report that the DS domain plays an essential role in repressing the production of galactooligosaccharides (GOSs). We prepared deletion mutants and point-mutated forms of rBgaD-A (deletion of the BgaD signal peptide) to compare their reaction behaviors. The yields of GOS for all of the point-mutated forms as well as the deletion mutants of rBgaD-As increased as compared to rBgaD-A. In particular, W1540A mutant BgaD-A (rBgaD-A_W1540A) produced much more GOSs than rBgaD-A. Surface plasmon resonance experiments indicated that both the wild-type and the W1540A mutant DS domain showed high affinity for galactosyllactose. rBgaD-A, which has a wild-type DS domain, showed high hydrolytic activity toward galactosyllactose, while the hydrolytic activities of rBgaD-D, without a DS domain, and rBgaD-A_W1540A, with a mutant DS domain were extremely low. The findings obtained in this study indicate that the wild-type DS domain of rBgaD-A has a function that aids galactosyllactose molecules to be properly oriented within the active site, so that they can be hydrolyzed efficiently to produce galactose/glucose by inhibiting the accumulation of GOS.
Bioscience Biotechnology and Biochemistry 01/2013; · 1.28 Impact Factor
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ABSTRACT: An indole compound with a strong purple-red color was produced by boiling a solution of indican under acidic conditions and purified by chromatographies on DEAE-650S Toyopearl TSK-gel and silica-gel columns. The purple-red compound purified was identified as indoxyl red, on the basis of FAB Mass, (13)C NMR, (1)H NMR, UV-visible spectra, and IR spectra. Although indoxyl red was first synthesized by Seidel(9) 70years ago, very little information has been available on its characteristics. We repot here that the compound was purple-red colored at acidic pH and green at pH 13, and showed antiproliferative and cytotoxic activities to the mouse B cell lymphoma cell line NSF202.
Bioorganic & medicinal chemistry letters 12/2012; · 2.65 Impact Factor
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ABSTRACT: An amorphous matrix, comprised of sugar molecules, is frequently used in the pharmaceutical industry. An amorphous sugar matrix exhibits high hygroscopicity, and it has been established that the sorbed water lowers the glass transition temperature T(g) of the amorphous sugar matrix. It is naturally expected that the random allocation and configuration of sugar molecules would result in heterogeneity of states for sorbed water. However, most analyses of the behavior of water, when sorbed to an amorphous sugar matrix, have implicitly assumed that all of the sorbed water molecules are in a single state. In this study, the states of water molecules sorbed in an amorphous sugar matrix were analyzed by Fourier-transform IR spectroscopy and a Fourier self-deconvolution technique. When sorbed water molecules were classified into five states, according to the extent to which they are restricted, three of the states resulted in a lowering of T(g) of an amorphous sugar matrix, while the other two were independent of the plasticization of the matrix. This finding provides an explanation for the paradoxical fact that compression at several hundreds of MPa significantly decreases the equilibrium water content at a given RH, while the T(g) remains unchanged.
Carbohydrate research 04/2012; 351:108-13. · 2.03 Impact Factor
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ABSTRACT: A gene of β-galactosidase from Bacillus circulans ATCC 31382 was cloned and sequenced on the basis of N-terminal and internal peptide sequences isolated from a commercial enzyme preparation, Biolacta(®). Using the cloned gene, recombinant β-galactosidase and its deletion mutants were overexpressed as His-tagged proteins in Escherichia coli cells and the enzymes expressed were characterized.
Bioscience Biotechnology and Biochemistry 06/2011; 75(6):1194-7. · 1.28 Impact Factor
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ABSTRACT: The presence of multiple types of β-galactosidases in a commercial enzyme preparation from Bacillus circulans ATCC 31382 and differences in their transgalactosylation activity were investigated. Four β-galactosidases, β-Gal-A, β-Gal-B, β-Gal-C, and β-Gal-D, which were immunologically homologous, were isolated and characterized. The N-terminal amino acid sequences of all of the enzymes were identical and biochemical characteristics were similar, except for galactooligosaccharide production. β-Gal-B, β-Gal-C, and β-Gal-D produced mainly tri- and tetra saccharides at maximum yields of 20-30 and 9-12%, while β-Gal-A produced trisaccharide with 7% with 5% lactose as substrate. The Lineweaver-Burk plots for all of the enzymes, except for β-Gal-A, showed biphasic behavior. β-Gal-A was truncated to yield multiple β-galactosidases by treatment with protease isolated from the culture broth of B. circulans. Treatment of β-Gal-A with trypsin yielded an active 91-kDa protein composed of 21-kDa and 70-kDa proteins with characteristics similar to those for β-Gal-D.
Bioscience Biotechnology and Biochemistry 02/2011; 75(2):268-78. · 1.28 Impact Factor
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ABSTRACT: An amorphous matrix comprised of sugar molecules are frequently used in the pharmaceutical industry. The compression of the amorphous sugar matrix improves the handling. Herein, the influence of compression on the water sorption of an amorphous sugar matrix was investigated. Amorphous sugar samples were prepared by freeze-drying, using several types of sugars, and compressed at 0-443 MPa. The compressed amorphous sugar samples as well as uncompressed samples were rehumidified at given RHs, and the equilibrium water content and glass transition temperature (T(g)) were then measured. Compression resulted in a decrease in the equilibrium water content of the matrix, the magnitude of which was more significant for smaller sized sugars. Diffusivity of water vapor in the sample was also decreased to one-hundredth by the compression. The T(g) value for a given RH remained unchanged, irrespective of the compression. Accordingly, the decrease in T(g) with increasing water content increased as the result of compression. The structural relaxation of the amorphous sugar matrices were also examined and found to be accelerated to the level of a non-porous amorphous sugar matrix as the result of the compression. The findings indicate that pores contained in freeze-dried sugar samples interfere with the propagation of structural relaxation.
International journal of pharmaceutics 02/2011; 408(1-2):76-83. · 2.96 Impact Factor
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ABSTRACT: The effects of various additives on the physical properties of an amorphous sugar matrix were compared. Amorphous, sugar-additive mixtures were prepared by freeze-drying and then rehumidified at given RHs. Sucrose and eighteen types of substances were used as the sugar and the additive, respectively, and water sorption, glass-to-rubber transition, and protein stabilization during freeze-drying for the various sucrose-additive mixtures were examined. The additives were categorized into two groups according to their effects on T(g) and water sorption. Presence of polysaccharides, cyclodextrins, and polymers (large-sized additives) resulted in a decrease in equilibrium water content from the ideal value calculated from individual water contents for sucrose and additive, and in contrast, low MW substances containing ionizable groups (small-ionized additives) resulted in an increase. The increase in T(g) by the addition of large-sized additives was significant at the additive contents >50 wt.% whereas the T(g) was markedly increased in the lower additive content by the addition of small-ionized additives. The addition of small-ionized additives enhanced the decrease in T(g) with increasing water content. The protein stabilizing effect was decreased with increasing additive content in the cases of the both groups of the additives.
Journal of Pharmaceutical Sciences 11/2010; 99(11):4669-77. · 3.06 Impact Factor
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ABSTRACT: A highly efficient method for dyeing textiles with indigo is described. In this method, the substrate, indican is first hydrolyzed at an acidic pH of 3 using an immobilized beta-glucosidase to produce indoxyl, under which conditions indigo formation is substantially repressed. The textile sample is then dipped in the prepared indoxyl solution and the textile is finally exposed to ammonia vapor for a short time, resulting in rapid indigo dyeing. As an enzyme, we selected a beta-glucosidase from Aspergillus niger, which shows a high hydrolytic activity towards indican and was thermally stable at temperatures up to 50-60 degrees C, in an acidic pH region. The A. niger beta-glucosidase, when immobilized on Chitopearl BCW-3001 by treatment with glutaraldehyde, showed an optimum reaction pH similar to that of the free enzyme with a slightly higher thermal stability. The kinetics for the hydrolysis of indican at pH 3, using the purified free and immobilized enzymes was found to follow Michaelis-Menten type kinetics with weak competitive inhibition by glucose. Using the immobilized enzyme, we successfully carried out repeated-batch and continuous hydrolyses of indican at pH 3 when nitrogen gas was continuously supplied to the substrate solution. Various types of model textiles were dyed using the proposed method although the color yield varied, depending on the type of textile used.
Journal of Bioscience and Bioengineering 09/2010; 110(3):281-7. · 1.79 Impact Factor
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ABSTRACT: We cultivated a filamentous fungus, Aspergillus oryzae IAM 2706 by three different cultivation methods, i.e., shaking-flask culture (SFC), agar-plate culture (APC), and membrane-surface liquid culture (MSLC), to elucidate the differences of its behaviors by different cultivation methods under the same media, by measuring the growth, secretion of proteases and alpha-amylase, secreted protein level, and gene transcriptional profile by the DNA microarray analysis. The protease activities detected by MSLC and APC were much higher than that by SFC, using both modified Czapek-Dox (mCD) and dextrin-peptone-yeast extract (DPY) media. The alpha-amylase activity was detected in MSLC and APC in a much larger extent than that in SFC when DPY medium was used. On the basis of SDS-PAGE analyses and N-terminal amino acid sequences, 6 proteins were identified in the supernatants of the culture broths using DPY medium, among which oryzin (alkaline protease) and alpha-amylase were detected at a much higher extent for APC and MSLC than those for SFC while only oryzin was detected in mCD medium, in accordance with the activity measurements. A microarray analysis for the fungi cultivated by SFC, APC, and MSLC using mCD medium was carried out to elucidate the differences in the gene transcriptional profile by the cultivation methods. The gene transcriptional profile obtained for the MSLC sample showed a similar tendency to the APC sample while it was quite different from that for the SFC sample. Most of the genes specifically transcribed in the MSLC sample versus those in the SFC sample with a 10-fold up-regulation or higher were unknown or predicted proteins. However, transcription of oryzin gene was only slightly up-regulated in the MSLC sample and that of alpha-amylase gene, slightly down-regulated.
Journal of Bioscience and Bioengineering 03/2010; 109(3):267-73. · 1.79 Impact Factor
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ABSTRACT: "H(2)O(2)-electrolysis" treatment is an alternative method for removing proteinaceous materials that are adsorbed to metal surfaces. The method is based on the generation of hydroxyl radicals by electrolysis of hydrogen peroxide and the subsequent decomposition of organic substances adhering to the metal surface. We herein investigated the influence of some parameters on the kinetics of protein removal by H(2)O(2)-electrolysis. These parameters included the properties of proteins and the type of metal surface. Sixteen types of proteins and nine types of metal surfaces were used. The removal of adsorbed protein from a metal surface during the treatment was monitored by ellipsometry. Apparent first-order rate constants for removal, k(c)(l), for various adsorption and treatment conditions were determined. The k(c)(l) value varied markedly with the type of protein and was also influenced by the pH used in the adsorption. The isoelectric point (pI) of protein used was found to be a major factor. The amount of adsorbed protein removed by a unit amount of ()OH was larger for a metal surface with a lower pI. The impact of the properties of the protein and metal surface on the removal kinetics are discussed, focusing on relationships with the adsorption characteristics of the protein.
Journal of Colloid and Interface Science 02/2010; 345(2):474-80. · 3.07 Impact Factor
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ABSTRACT: We report here on the purification, characterization, molecular cloning, and expression of a new aminoacylase, initially isolated from the supernatant of Streptomyces mobaraensis (Sm-AA). Purified wild-type Sm-AA was found to be a monomeric protein with a molecular mass of 55 kDa. The cloned gene of Sm-AA contained an ORF of 1,383 bp, encoding a polypeptide of 460 amino acids. A BLAST search revealed that Sm-AA belongs to the peptidase M20 family, with identities to a hypothetical protein from Streptomyces pristinaespiralis, a putative peptidase from Streptomyces avermitilis, peptidase M20 from Frankia sp., succinyl-diaminopimelate desuccinylase from Hemophilus influenzae, and aminoacylase-1 from porcine kidney at 89, 88, 67, 29, and 25% respectively. The Sm-AA gene was subcloned into an expression vector, pSH19, and was expressed in Streptomyces lividans TK24. The amount of the recombinant Sm-AA expressed in the S. lividans cells was approximately 42-fold higher than that of Sm-AA found in the supernatant of S. mobaraensis. Sm-AA showed high hydrolytic activity towards various N-acetyl-L-amino acids and N-(middle/long)-chain-fatty-acyl-L-amino acids, with a preference for the acyl derivatives of L-Met, L-Ala, L-Cys, etc. with an optimum pH and temperature for reaction of about 7.5 and 50 degrees Celsius (at pH 7.5).
Bioscience Biotechnology and Biochemistry 10/2009; 73(9):1940-7. · 1.28 Impact Factor
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ABSTRACT: An amorphous matrix comprised of sugar molecules is used as excipient and stabilizing agent for labile ingredients in the pharmaceutical industry. The amorphous sugar matrix is often compressed into a tablet form to reduce the volume and improve handling. Herein, the effect of compression on the crystallization behavior of an amorphous sucrose matrix was investigated. Amorphous sucrose samples were prepared by freeze-drying and compressed under different conditions, followed by analyses by differential scanning calorimetry, isothermal crystallization tests, X-ray powder diffractometry, Fourier transform infrared spectroscopy (FTIR), and gas pycnometry. The compressed sample had a lower crystallization temperature and a shorter induction period for isothermal crystallization, indicating that compression facilitates the formation of the critical nucleus of a sucrose crystal. Based on FTIR and molecular dynamics simulation results, the conformational distortion of sucrose molecules due to the compression appears to contribute to the increase in the free energy of the system, which leads to the facilitation of critical nucleus formation. An isothermal crystallization test indicated an increase in the growth rate of sucrose crystals by the compression. This can be attributed to the transformation of the microstructure from porous to nonporous, as the result of compression.
Journal of Pharmaceutical Sciences 09/2009; 99(3):1452-63. · 3.06 Impact Factor
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ABSTRACT: epsilon-Lysine acylase from Streptomyces mobaraensis (Sm-ELA), which specifically catalyzes hydrolysis of the epsilon-amide bond in various Nepsilon-acyl-L-lysines, was cloned and sequenced. The Sm-ELA gene consists of a 1617-bp open reading frame that encodes a 538-amino acid protein with a molecular mass of 55,816Da. An NCBI protein-protein BLAST search revealed that the enzyme belongs to the YtcJ-like metal-dependent amidohydrolase family, which is further characterized as the metallo-dependent hydrolase superfamily. The Sm-ELA gene was ligated into a pUC702 vector for expression in Streptomyces lividans TK24. Expression of recombinant Sm-ELA in S. lividans was approximately 300-fold higher than that in wild-type S. mobaraensis. The recombinant Sm-ELAs from the cell-free extract and culture supernatant were purified to homogeneity. The specific activities of the purified Sm-ELAs were 2500-2800U/mg, which were similar to that obtained for the wild-type Sm-ELA. Using the cell-free extract of the recombinant S. lividans cells, Nepsilon-lauroyl-L-lysine was synthesized from 500mM L-lysine hydrochloride and 50, 100, or 250mM lauric acid in an aqueous buffer solution at 37 degrees C. The yields were close to 100% after 6 and 9h of reaction for 50 and 100mM lauric acid, respectively, and 90% after 24h for 250mM lauric acid.
Journal of biotechnology 06/2009; 141(3-4):160-5. · 2.88 Impact Factor
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ABSTRACT: Adsorption characteristics of 18 proteins, with different sizes and isoelectric points, to a titanium oxide surface were studied. The adsorption isotherms were categorized based on protein type and pH: type 1, irreversible adsorption; type 2, Langmuir-type reversible adsorption; and type 3, reversible and irreversible adsorption. Most of the proteins tested were irreversibly adsorbed in the pH range of 3-8, whereas most adsorbed reversibly at pH 8.5-9.4. Protamine, with a pI value of 12, adsorbed reversibly in the pH range of 3-9. pH values that gave maximal sums of irreversibly and reversibly adsorbed proteins were in the pH range of 3-8 and tended to increase slightly with the pI value of the corresponding protein. pH values that gave maximal quantities of irreversibly adsorbed protein ranged between 4-6 and were nearly independent of pI.
Journal of Bioscience and Bioengineering 10/2008; 106(3):273-8. · 1.79 Impact Factor
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Spectroscopy Letters 09/2008; 41(6):305-312. · 0.72 Impact Factor
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ABSTRACT: True density of an amorphous matrix represents the state of molecular packing in the matrix, which is closely related to the physical/chemical properties of the material. Dry gas pycnometry is one possible technique for measuring the true density of an amorphous sugar matrix prepared by freeze-drying. We herein report on the influence of conditions used for pycnometry on the measured density value and propose a protocol for obtaining the true density. The technique is sufficiently accurate to permit values for matrices comprised of different types of sugar to be compared. Using the protocol, the true densities of several amorphous sugar samples containing different types of sugar, freeze-drying conditions (temperature and sugar concentration at the time of freezing of an aqueous sugar solution), pretreatment (compaction and grind) were determined and the results were compared. A model for simulating an amorphous matrix of sugar (trehalose) was constructed using molecular dynamics/mechanics calculations, and the true density of the simulated sugar matrix was found to agree with the value experimentally determined using the proposed protocol. The relationship among the true density, the states of intermolecular interactions, and strain of sugar molecules in the matrix are discussed using the simulated amorphous sugar matrix.
Journal of Pharmaceutical Sciences 08/2008; 97(7):2789-97. · 3.06 Impact Factor
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ABSTRACT: The characteristics of hydrogen bond formation between trehalose and polyvinylpyrrolidone (PVP) in amorphous mixtures at different hydration states were quantitatively investigated. Amorphous trehalose-PVP mixtures were prepared by freeze-drying and equilibrated at different relative humidities (RH). Infrared (IR) spectra of the trehalose-PVP mixtures were obtained by Fourier transform IR spectroscopy,(FTIR) and the IR band corresponding to C=O groups of PVP was deconvolved into the component bands responsible for C=O groups that were free and restricted by hydrogen bonds, to estimate the degree of the trehalose-PVP interactions. The FTIR analysis indicated that approximately 80% of the C=O groups of PVP formed hydrogen bonds with trehalose in the presence of more than 3 g of trehalose per gramme of PVP, independent of the RH. IR analysis of the O--H stretching vibration of the sugar demonstrated that the presence of PVP lead to an increase in the free hydroxyl groups of trehalose that did not form hydrogen bonds at RH 0%. On the other hand, the water sorption behavior of the trehalose-PVP mixtures suggested that rehumidification diminished the effect of PVP on increasing the free OH groups. Thus a peculiar relationship may exist between Tg, RH and the composition of the mixture: The presence of PVP increased Tg at RHs 0 and above 23% but decreased Tg at 11%.
Journal of Pharmaceutical Sciences 04/2008; 97(3):1301-12. · 3.06 Impact Factor
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ABSTRACT: The impact of a polymer additive (polyvinylpyrrolidone, PVP) on hydrogen bonding in amorphous sugar matrices as well as on the glass transition temperature, T(g), were examined by temperature scanning Fourier transform infrared spectroscopy (TS-FTIR). An amorphous sugar matrix containing PVP was prepared by air-drying an aqueous solution of a sugar-PVP mixture. The hydrogen bonds in the sugar-PVP mixture (sugar-PVP and sugar-sugar hydrogen bonds) were analyzed from the IR peak positions corresponding to the stretching vibration of C==O groups of PVP and O--H groups of the sugar and the temperature dependence of the peak position of the O--H stretching vibration band. The addition of PVP to amorphous mono and disaccharides significantly lowered the extent of hydrogen bond formation while interactions between sugars and the PVP tended to prevent the disruption of hydrogen bonds due to increasing temperature, the magnitude of which was larger for larger oligomers. The T(g) value for the amorphous sugar was increased by the addition of PVP in many cases. As the size of sugar molecule became larger, the relative magnitude of the increased T(g) by PVP to the difference between the T(g) values for sugar alone and PVP alone became larger and then reached a certain level; it was slight in the case of glucose. Collectively, these results demonstrate that the magnitude of the impact of PVP on an amorphous sugar matrix strongly vary and are dependent on the types of sugar.
Journal of Pharmaceutical Sciences 02/2008; 97(1):519-28. · 3.06 Impact Factor
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ABSTRACT: Angiotensin I converting enzyme (ACE)-inhibitory peptides were screened from a random peptide-displayed phage library using ACE-coupled liposomes. Among four kinds of inhibitory peptides selected by biopanning with two different elution strategies, a peptide (LSTLRSFCA) showed the highest inhibitory activity with an IC(50) value of 3microM. By measuring inhibitory activities of fragments of the peptide, it was found that the RSFCA region was a functional site to inhibit strongly the ACE catalytic activity, and particularly both Arg and Cys residues were essential for the strong inhibitory activity. The inhibitory activity of RRFCA was slightly increased, while that of the RSFRA, in which the Cys residue was replaced by Arg, was decreased to greater extent in comparison with the inhibitory activity of RSFCA. Taking into account the results obtained from the SPOT analysis, it was suggested that the Arg and Phe residues in RSFCA were important for a specific interaction with ACE, and the Cys residue inhibited the ACE activity. The cystein-based ACE-inhibitory peptides have not been isolated from processed food materials. These findings suggested that the biopanning method utilizing protein-coupled liposomes and random peptide libraries might have a possibility to screen new functional peptides that are not found in processed food materials.
Journal of Biotechnology 09/2007; 131(2):144-9. · 3.05 Impact Factor
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ABSTRACT: A Ser/Thr phosphatase gene cloned from Aspergillus oryzae, aoppt, revealed that the tetratricopeptide repeat (TPR) and catalytic domains of the full-length AoPPT are located at the N- and C-terminal regions, respectively, similar to those of human Ser/Thr phosphatase 5 (PP5) and yeast Ppt1. Four different regions of AoPPT, namely, a full-length polypeptide, the catalytic domain, the catalytic domain plus C-terminal 15 amino-acid residues and the TPR domain were expressed in Escherichia coli and their roles in dephosphorylation activity were examined, using p-nitrophenyl phosphate as the substrate. The full-length AoPPT showed the highest dephosphorylation activity while the catalytic domain had the lowest activity. The activity of the catalytic domain was not inhibited by the presence of the TPR domain and arachidonic acid did not increase the activity of the full-length enzyme. These findings suggest that the integrity of the entire enzyme would be necessary for its full activity to be expressed.
International Journal of Biological Macromolecules 09/2007; 41(3):281-5. · 2.45 Impact Factor
