The hydrophobicity of an amino acid residue in a flexible loop of KP-43 protease alters activity toward a macromolecule substrate.

KP-43, a 43-kDa alkaline serine protease, is resistant to chemical oxidants and surfactants, making it suitable for use in laundry detergents. An amino acid residue at position 195, in a unique flexible loop that binds a Ca2+ ion, dramatically affects the proteolytic activity and thermal stability of KP-43. In the present study, we obtained 20 variants with substitutions at position 195 and investigated how these residues affect hydrolytic activity toward a macromolecular substrate (casein) and a synthetic tetra-peptide (AAPL). At pH 10, the variant with the highest caseinolytic activity, Tyr195Gln, exhibited 4.4-fold higher activity than the variant with the lowest caseinolytic activity, Tyr195Trp. A significant negative correlation was observed between the hydrophobicity of the residue at position 195 and caseinolytic activity at pH 8-10. At pH 7, the correlation became weak; at pH 6, the correlation reversed to positive. Unlike casein, in the case of hydrolysis of AAPL, no correlation was observed at pH 10 or pH 6. Because the amino acid residue at position 195 is located on the protein surface and considered sufficiently far from the active cleft, the variation in caseinolytic activity between the 20 variants was attributed to changes in interaction efficiency with different states of casein at different pH values. To improve the enzymatic activity, we propose substituting amino acid residues on the protein surface to change the efficiency of interaction with the macromolecular substrates. KEY POINTS: • A single amino acid residue on the protein surface markedly changed enzyme activity. • The hydrophobicity of the amino acid residue and enzyme activity had a correlation. • The key amino acid residue for substrate recognition exists on the protein surface.

Mechanism for the antibacterial action of epigallocatechin gallate (EGCg) on Bacillus subtilis.

Catechins are a class of polyphenols and have high anti-bacterial activity against various microorganisms. Here, we report the mechanism for antibacterial activity of epigallocatechin gallate (EGCg) against Gram-positive bacteria Bacillus subtilis, which is highly sensitive to EGCg. Transmission electron microscope analysis revealed that deposits containing EGCg were found throughout the cell envelope from the outermost surface to the outer surface of cytoplasmic membrane. Aggregating forms of proteins and EGCg were identified as spots that disappeared or showed markedly decreased intensity after the treatment with EGCg compared to the control by two-dimensional electrophoresis. Among the identified proteins included 4 cell surface proteins, such as oligopeptide ABC transporter binding lipoprotein, glucose phosphotransferase system transporter protein, phosphate ABC transporter substrate-binding protein, and penicillin-binding protein 5. Observations of glucose uptake of cells and cell shape B. subtilis after the treatment with EGCg suggested that EGCg inhibits the major functions of these proteins, leading to growth inhibition of B. subtilis.

A single mutation within a Ca(2+) binding loop increases proteolytic activity, thermal stability, and surfactant stability.

We improved the enzymatic properties of the oxidatively stable alkaline serine protease KP-43 through protein engineering to make it more suitable for use in laundry detergents. To enhance proteolytic activity, the gene encoding KP-43 was mutagenized by error-prone PCR. Screening identified a Tyr195Cys mutant enzyme that exhibited increased specific activity toward casein between pH 7 and 11. At pH 10, the mutant displayed 1.3-fold higher specific activity for casein compared to the wild-type enzyme, but the activity of the mutant was essentially unchanged toward several synthetic peptides. Furthermore, the Tyr195Cys mutation significantly increased thermal stability and surfactant stability of the enzyme under oxidizing conditions. Examination of the crystal structure of KP-43 revealed that Tyr195 is a solvent exposed residue that forms part of a flexible loop that binds a Ca(2+) ion. This residue lies 15-20Å away from the residues comprising the catalytic triad of the enzyme. These results suggest that the substitution at position 195 does not alter the structure of the active center, but instead may affect a substrate-enzyme interaction. We propose that the Tyr195Cys mutation enhances the interaction with Ca(2+) and affects the packing of the Ca(2+) binding loop, consequently increasing protein stability. The simultaneously increased proteolytic activity, thermal stability, and surfactant stability of the Tyr195Cys mutant enzyme make the protein an ideal candidate for laundry detergent application.

New microbial mannan catabolic pathway that involves a novel mannosylglucose phosphorylase.

The consecutive genes BF0771-BF0774 in the genome of Bacteroides fragilis NCTC 9343 were found to constitute an operon. The functional analysis of BF0772 showed that the gene encoded a novel enzyme, mannosylglucose phosphorylase that catalyzes the reaction, 4-O-ß-d-mannopyranosyl-d-glucose+Pi→mannose-1-phosphate+glucose. Here we propose a new mannan catabolic pathway in the anaerobe, which involves 1,4-ß-mannanase (BF0771), a mannobiose and/or sugar transporter (BF0773), mannobiose 2-epimerase (BF0774), and mannosylglucose phosphorylase (BF0772), finally progressing to glycolysis. This pathway is distributed in microbes such as Bacteroides, Parabacteroides, Flavobacterium, and Cellvibrio.

Combined effect of improved cell yield and increased specific productivity enhances recombinant enzyme production in genome-reduced Bacillus subtilis strain MGB874.

Genome reduction strategies to create genetically improved cellular biosynthesis machineries for proteins and other products have been pursued by use of a wide range of bacteria. We reported previously that the novel Bacillus subtilis strain MGB874, which was derived from strain 168 and has a total genomic deletion of 874 kb (20.7%), exhibits enhanced production of recombinant enzymes. However, it was not clear how the genomic reduction resulted in elevated enzyme production. Here we report that deletion of the rocDEF-rocR region, which is involved in arginine degradation, contributes to enhanced enzyme production in strain MGB874. Deletion of the rocDEF-rocR region caused drastic changes in glutamate metabolism, leading to improved cell yields with maintenance of enzyme productivity. Notably, the specific enzyme productivity was higher in the reduced-genome strain, with or without the rocDEF-rocR region, than in wild-type strain 168. The high specific productivity in strain MGB874 is likely attributable to the higher expression levels of the target gene resulting from an increased promoter activity and plasmid copy number. Thus, the combined effects of the improved cell yield by deletion of the rocDEF-rocR region and the increased specific productivity by deletion of another gene(s) or the genomic reduction itself enhanced the production of recombinant enzymes in MGB874. Our findings represent a good starting point for the further improvement of B. subtilis reduced-genome strains as cell factories for the production of heterologous enzymes.

Effects of repeated morphine treatment on the antinociceptive effects, intestinal absorption, and efflux from intestinal epithelial cells of morphine.

The present study was conducted to investigate the effects of repeated treatment with morphine on the drug's antinociceptive effects, intestinal absorption, and transepithelial transport. The antinociceptive effects of morphine in rats were markedly decreased after repeated oral administration of the drug for 5 d, indicating the development of tolerance. In the morphine-tolerant rats, intestinal absorption of morphine was determined using the in situ loop method. Absorption of morphine from the jejunum was significantly decreased after repeated administration. The permeability of human intestinal epithelial Caco-2 cells was increased in the efflux direction after repeated treatment. The repeated administration of morphine also reduced the cellular accumulation and efflux of P-glycoprotein substrates ([(3)H]vincristine and rhodamine123) from Caco-2 cells, suggesting that it enhances P-glycoprotein-mediated efflux in Caco-2 cells. These results suggest that repeated use enhances the efflux of morphine in the epithelial cells of the small intestine, subsequently decreasing its intestinal absorption.

Site-directed mutagenesis of possible catalytic residues of cellobiose 2-epimerase from Ruminococcus albus.

The cellobiose 2-epimerase from Ruminococcus albus (RaCE) catalyzes the epimerization of cellobiose and lactose to 4-O-beta-D-glucopyranosyl-D-mannose and 4-O-beta-D-galactopyranosyl-D-mannose (epilactose). Based on the sequence alignment with N-acetyl-D-glucosamine 2-epimerases of known structure and on a homology-modeled structure of RaCE, we performed site-directed mutagenesis of possible catalytic residues in the enzyme, and the mutants were expressed in Escherichia coli cells. We found that R52, H243, E246, W249, W304, E308, and H374 were absolutely required for the activity of RaCE. F114 and W303 also contributed to catalysis. These residues protruded into the active-site cleft in the model (alpha/alpha)(6) core barrel structure.

Baicalein Inhibits Stx1 and 2 of EHE: Effects of Baicalein on the Cytotoxicity, Production, and Secretion of Shiga Toxins of Enterohaemorrhagic Escherichia coli.

Shiga toxin-producing enterohaemorrhagic Escherichia coli (EHEC) O157:H7 is an important foodborne pathogen. Baicalein (5,6,7-trihydroxylflavone), a flavone isolated from the roots of Scutellaria baicalensis, is considered as a potential antibacterial agent to control foodborne pathogens. Among seven compounds selected by in silico screening of the natural compound database, baicalein inhibited the cytotoxicity of both Shiga toxins 1 and 2 (Stx1 and Stx2) against Vero cells after pretreatment at 0.13 mmol/L. In addition, baicalein reduced the susceptibility of Vero cells to both Stx1 and Stx2. Real-time qPCR showed that baicalein increased transcription of stx1 but not of stx2. However, baicalein had no effects on production or secretion of Stx1 or Stx2. Docking models suggested that baicalein formed a stable structure with StxB pentamer with low intramolecular energy. The results demonstrate that inhibitory activity of baicalein against the cytotoxicity of both Stx1 and Stx2 might be due to of the formation of a binding structure inside the pocket of the Stx1B and Stx2B pentamers.

Ancestral sequence evolutionary trace and crystal structure analyses of alkaline alpha-amylase from Bacillus sp. KSM-1378 to clarify the alkaline adaptation process of proteins.

The crystal structure of alkaline liquefying alpha-amylase (AmyK) from the alkaliphilic Bacillus sp. KSM-1378 was determined at 2.1 A resolution. The AmyK structure belongs to the GH13 glycoside hydrolase family, which consists of three domains, and bound three calcium and one sodium ions. The alkaline adaptation mechanism of AmyK was investigated by the ancestral sequence evolutionary trace method and by extensive comparisons between alkaline and nonalkaline enzyme structures, including three other protein families: protease, cellulase, and phosphoserine aminotransferase. The consensus change for the alkaline adaptation process was a decrease in the Lys content. The loss of a Lys residue is associated with ion pair remodeling, which mainly consists of the loss of Lys-Asp/Glu ion pairs and the acquisition of Arg ion pairs, preferably Arg-Glu. The predicted replacements of the positively charged amino acids were often, although not always, used for ion pair remodeling.

Development of novel monoclonal antibodies directed against catechins for investigation of antibacterial mechanism of catechins.

Catechins are major polyphenolic compounds of green tea. To investigate mechanism for antibacterial action of catechins, 11 monoclonal antibodies (MAbs) were raised against a 3-succinyl-epicatechin (EC)-keyhole limpet hemocyanin (KLH) conjugate. Amino acid sequences of variable regions determined for MAbs b-1058, b-1565, and b-2106 confirmed their innovative character. MAb b-1058 strongly interacted with its target substances in the following order of magnitude: theaflavin-3,3'-di-O-gallate (TFDG)>theaflavin-3-O-gallate (TF3G)≥theaflavin-3'-O-gallate (TF3'G)>gallocatechin gallate (GCg)>penta-O-galloyl-ß-d-glucose (PGG)>epigallocatechin gallate (EGCg), as determined using surface plasmon resonance (SPR) on MAb-immobilized sensor chips. The affinity profiles of MAbs b-1058 and b-2106 to the various polyphenols tested suggested that flavan skeletons with both carbonyl oxygen and hydroxyl groups are important for this interaction to take place. S. aureus cells treated with EGCg showed green fluorescence around the cells after incubation with FITC-labeled MAb b-1058. The fluorescence intensity increased with increasing concentrations of EGCg. These MAbs are effective to investigate antibacterial mechanism of catechins and theaflavins.

Generation of fluorocarbon and hydrocarbon hybrid gemini surfactants controlled by micellar miscibility.

Hybrid surfactants were generated through the simple mixing of fluorocarbon and hydrocarbon gemini surfactants in aqueous solutions at 25°C. Disulfide exchange between the disulfide in the spacer chain occurred in the mixed micelles. However, the generation of hybrid gemini surfactants was particularly inhibited by the addition of salt. The suppression of the electrostatic repulsion between the hydrophilic headgroups led to the close packing of the hydrophobic chains in the micelles, resulting in the increased immiscibility of the fluorocarbon and hydrocarbon surfactants. On the other hand, when fluorocarbon-fluorocarbon or hydrocarbon-hydrocarbon surfactants were mixed, equilibrium with a 1:2 ratio of symmetric and dissymmetric gemini surfactants was attained after incubation for 24 h.

An endogenous factor enhances ferulic acid decarboxylation catalyzed by phenolic acid decarboxylase from Candida guilliermondii.

The gene for a eukaryotic phenolic acid decarboxylase of Candida guilliermondii was cloned, sequenced, and expressed in Escherichia coli for the first time. The structural gene contained an open reading frame of 504 bp, corresponding to 168 amino acids with a calculated molecular mass of 19,828 Da. The deduced amino sequence exhibited low similarity to those of functional phenolic acid decarboxylases previously reported from bacteria with 25-39% identity and to those of PAD1 and FDC1 proteins from Saccharomyces cerevisiae with less than 14% identity. The C. guilliermondii phenolic acid decarboxylase converted the main substrates ferulic acid and p-coumaric acid to the respective corresponding products. Surprisingly, the ultrafiltrate (Mr 10,000-cut-off) of the cell-free extract of C. guilliermondii remarkably activated the ferulic acid decarboxylation by the purified enzyme, whereas it was almost without effect on the p-coumaric acid decarboxylation. Gel-filtration chromatography of the ultrafiltrate suggested that an endogenous amino thiol-like compound with a molecular weight greater than Mr 1,400 was responsible for the activation.

Aqueous solution properties of disulfide-linked fluorinated gemini and cleaved monomeric thiol surfactants.

We prepared a fluorinated gemini surfactant containing a disulfide bond in its spacer chain, [C8F17(CH2)3N(CH3)2CH2CH2SSCH2CH2N(CH3)2(CH2)3C8F17]2Cl, and its analogue with a hexamethylene spacer. Monomeric thiol surfactant, [C8F17(CH2)3N(CH3)2CH2CH2SH]Cl, was readily produced by the cleavage of the gemini surfactant using dithiothreitol in water. The critical micelle concentration was determined using surface tension, conductivity, and fluorescence probe methods. The critical micelle concentration of the monomeric surfactant was significantly larger than that of the gemini surfactant. The surface tension of aqueous solution for the cleaved monomeric thiol surfactant returned gradually to the original value through the formation of the disulfide bond via air oxidation.

Dissymmetric gemini surfactants generated by disulfide exchange in mixed micelles.

We found the generation of dissymmetric gemini surfactants from symmetric ones in aqueous micelle solutions of gemini surfactants with a spacer containing disulfide linkage. The equilibrium was attained during 5 h incubation at 25 degrees C with 1:2 ratio of symmetric to dissymmetric ones when the total concentrations were sufficiently high compared with monomer concentrations. The generation of dissymmetric form is attributed to the disulfide exchange between the spacer chains, which occurs in the mixed micelles of gemini surfactants with different hydrocarbon chains. The pseudo-phase separation model for micellization was successfully used to predict not only the cmc of binary and ternary mixtures composed of symmetric and dissymmetric gemini surfactants but also the monomer concentrations after the equilibrium. Reversibility of the disulfide exchange was ascertained by the generation of symmetric gemini surfactants from dissymmetric ones.

Aqueous solution properties of disulfide linked gemini and cleaved monomeric thiol surfactants.

Monomeric thiol surfactants, [C(n)H(2n+1)N(CH(3))(2)CH(2)CH(2)SH]Br, were produced by the cleavage of gemini surfactant containing a disulfide bond in the spacer chain, [C(n)H(2n+1)N(CH(3))(2)CH(2)CH(2)SSCH(2)CH(2)N(CH(3))(2)C(n)H(2n+1)]2Br. The disulfide bond was completely reduced by the addition of four times moles of dithiothreitol in water at room temperature. The critical micelle concentrations of monomeric surfactants were significantly increased in comparison with original gemini surfactants. The monomeric thiol surfactants were stable in the presence of dithiothreitol, whereas they returned gradually to their original gemini surfactants within several days due to air oxidation in water without dithiothreitol. The micelle formation induced by the disulfide linkage formation was suggested by the fluorescence intensity ratio of pyrene. The time course of decrease in thiol concentration associated with the recovery of gemini surfactants was confirmed by the absorption spectra utilizing the reactions with 4,4'-dithiopyridine.

Enhancement by grapefruit juice of morphine antinociception.

The aim of this study was to investigate the effect of grapefruit juice intake on the antinociception of morphine in rats. The antinociception of morphine (30 mg/kg, per os (p.o.)) was significantly enhanced by the oral administration of grapefruit juice (2 ml/rat). Further, the effect of grapefruit juice was examined in morphine-tolerant rats. The repeated administration of morphine (100 mg/kg p.o.) for 5 d caused a marked decrease in the antinociception, indicating the development of morphine-tolerance. In the morphine-tolerant rats, oral administration of grapefruit juice potentiated significantly the antinociceptive effect of morphine. To examine the pharmacokinetics of morphine after the repeated treatment with morphine for 5 d, microdialysis probes were implanted into the jugular vein and spinal intrathecal space in rats. The morphine concentrations in the blood and intrathecal cerebrospinal fluid (CSF) were gradually decreased by the repeated treatment with morphine. The grapefruit juice treatment significantly increased the blood concentration of morphine in morphine-tolerant rats. These results suggest that oral administration of grapefruit juice enhances the morphine antinociception by increasing the intestinal absorption of this agent.

Enhanced recombinant protein productivity by genome reduction in Bacillus subtilis.

The emerging field of synthetic genomics is expected to facilitate the generation of microorganisms with the potential to achieve a sustainable society. One approach towards this goal is the reduction of microbial genomes by rationally designed deletions to create simplified cells with predictable behavior that act as a platform to build in various genetic systems for specific purposes. We report a novel Bacillus subtilis strain, MBG874, depleted of 874 kb (20%) of the genomic sequence. When compared with wild-type cells, the regulatory network of gene expression of the mutant strain is reorganized after entry into the transition state due to the synergistic effect of multiple deletions, and productivity of extracellular cellulase and protease from transformed plasmids harboring the corresponding genes is remarkably enhanced. To our knowledge, this is the first report demonstrating that genome reduction actually contributes to the creation of bacterial cells with a practical application in industry. Further systematic analysis of changes in the transcriptional regulatory network of MGB874 cells in relation to protein productivity should facilitate the generation of improved B. subtilis cells as hosts of industrial protein production.

New fluorescent probes applicable to aggregates of fluorocarbon surfactants.

We developed new benzofurazan (NBD) labeled probes for fluorocarbon surfactant systems. The fluorescence behavior depended on the solubilization site of the fluorescent probes in the surfactant aggregates. The NBD-labeled probes suffered virtually complete reduction in the presence of Na(2)S(2)O(4) owing to the solubilization at the surface of 2-hydroxy-1,1,2,3,3-pentahydroperfluoroundecyldiethylammonium bromide (FC(8)DAB) aggregates. On the other hand, N-(3-sulfopropyl)acridinium (SPA) in FC(8)DAB aggregates showed residual fluorescence in spite of NaBH(4) addition. The large vesicles of FC(8)DAB were confirmed by DLS measurements. These facts suggest that SPA is solubilized in an inner water phase of the vesicles. The NBD labeled fluorescence probe is quite effective for the study of the aggregation behavior of fluorocarbon surfactants.

Cloning and sequencing of the cellobiose 2-epimerase gene from an obligatory anaerobe, Ruminococcus albus.

Cellobiose 2-epimerase (EC 5.1.3.11) was first identified in 1967 as an extracellular enzyme that catalyzes the reversible epimerization between cellobiose and 4-O-beta-D-glucopyranosyl-D-mannose in a culture broth of Ruminococcus albus 7 (ATCC 27210(T)). Here, for the first time, we describe the purification of cellobiose 2-epimerase from R. albus NE1. The enzyme was found to 2-epimerize the reducing terminal glucose moieties of cellotriose and cellotetraose in addition to cellobiose. The gene encoding cellobiose 2-epimerase comprises 1170 bp (389 amino acids) and is present as a single copy in the genome. The deduced amino acid sequence of the mature enzyme contains the possible catalytic residues Arg52, His243, Glu246, and His374. Sequence analysis shows the gene shares a very low level of homology with N-acetyl-D-glucosamine 2-epimerases (EC 5.1.3.8), but no significant homology to any other epimerases reported to date.