Influence of pH, concentration of sodium lactate as an additive and ultrasonic treatment on synthesis of zinc phosphate white pigments.

Zinc oxide that has the photocatalytic activity is used as a white pigment for cosmetics. A certain degree of sebum on the skin is decomposed by the ultraviolet radiation in sunlight. In this work, zinc phosphates were prepared from zinc nitrate and phosphoric acid at pH 5 and 7 with and without the addition of sodium lactate and ultrasonic treatment as a novel white pigment for use in cosmetics. The chemical composition, powder properties, photocatalytic activity, colour phase, moisture retention and smoothness of the zinc phosphates were studied. The obtained materials had a Zn/P ratio of about 1.5, which corresponds to zinc orthophosphate Zn3 (PO4 )2 . Samples prepared with ultrasonic treatment indicated the high ratios of large particles in scanning electron microscopy images and particle-size distributions. The photocatalytic activity of these zinc phosphate particles was too less to protect the sebum on the skin. The materials obtained and their thermal products at 100°C showed a high reflectance within the range of visible light. The slipping resistance and roughness of the powder were enough low for use in cosmetics.

Strong nucleic acid binding to the Escherichia coli RNase HI mutant with two arginine residues at the active site.

The biochemical properties of the mutant protein D10R/E48R of Escherichia coli RNase HI, in which Asp(10) and Glu(48) are both replaced by Arg, were characterized. This mutant protein has been reported to have metal-independent RNase H activity at acidic pH [Casareno et al. (1995) J. Am. Chem. Soc. 117, 11011-11012]. The far- and near-UV CD spectra of this mutant protein were similar to those of the wild-type protein, suggesting that the protein conformation is not markedly changed by these mutations. Nevertheless, we found that this mutant protein did not show any RNase H activity in vitro. Instead, it showed high-nucleic-acid-binding affinity. Protein footprinting analyses suggest that DNA/RNA hybrid binds to or around the presumed substrate-binding site of the protein. In addition, this mutant protein did not complement the temperature-sensitive growth phenotype of the rnhA mutant strain, E. coli MIC3001, even at pH 6.0, suggesting that it does not show RNase H activity in vivo as well. These results are consistent with a current model for the catalytic mechanism of the enzyme, in which Glu(48) is not responsible for Mg(2+) binding but is involved in the catalytic function.

Overproduction in Escherichia coli, purification and characterization of a family I.3 lipase from Pseudomonas sp. MIS38.

Determination of the nucleotide sequence of the gene encoding a lipase from Pseudomonas sp. MIS38 (PML) revealed that PML is a member of the lipase family I.3 and is composed of 617 amino acid residues with a calculated molecular weight of 64510. Recombinant PML (rPML) was overproduced in Escherichia coli in an insoluble form, solubilized in the presence of 8 M urea, purified in a urea-denatured form and refolded by removing urea in the presence of the Ca(2+) ion. Gel filtration chromatography suggests that this refolded protein is monomeric. rPML showed relatively broad substrate specificities and hydrolyzed glyceryl tributyrate and olive oil with comparable efficiencies. rPML was active only in the form of a holo-enzyme, in which at least 12 Ca(2+) ions bound. These Ca(2+) ions bound too tightly to be removed from the protein upon dialysis, but were removed from it upon EDTA treatment. The resultant apo-enzyme was fully active in the presence of 10 mM CaCl(2), but was inactive in the absence of the Ca(2+) ion. PML has a GXSXG motif, which is conserved in lipases/esterases and generally contains the active-site serine. The mutation of Ser(207) within this motif to Ala completely inactivated PML, suggesting that Ser(207) is the active-site serine of PML.

Catalytic center of an archaeal type 2 ribonuclease H as revealed by X-ray crystallographic and mutational analyses.

The catalytic center of an archaeal Type 2 RNase H has been identified by a combination of X-ray crystallographic and mutational analyses. The crystal structure of the Type 2 RNase H from Thermococcus kodakaraensis KOD1 has revealed that the N-terminal major domain adopts the RNase H fold, despite the poor sequence similarity to the Type 1 RNase H. Mutational analyses showed that the catalytic reaction requires four acidic residues, which are well conserved in the Type 1 RNase H and the members of the polynucleotidyl transferase family. Thus, the Type 1 and Type 2 RNases H seem to share a common catalytic mechanism, except for the requirement of histidine as a general base in the former enzyme. Combined with the results from deletion mutant analyses, the structure suggests that the C-terminal domain of the Type 2 RNase H is involved in the interaction with the DNA/RNA hybrid.

Conformational changes of aminoacyl-tRNA and uncharged tRNA upon complex formation with polypeptide chain elongation factor Tu.

The conformation change of Thermus thermophilus tRNA(1Ile) upon complex formation with T. thermophilus elongation factor Tu (EF-Tu) was studied by analysis of the circular dichroism (CD) bands at 315 nm (due to the 2-thioribothymidine residue in the T-loop) and at 295 nm (due to the core structure of tRNA). Formation of the ternary complex of isoleucyl-tRNA(1Ile) and EF-Tu.GTP increased the intensities of these CD bands, indicating stabilization of the association between the T-loop and the D-loop and also a significant conformation change of the core region. Upon complex formation of EF-Tu.GTP and uncharged tRNA, however, the conformation of the core region is not changed, while the association of the two loops is still stabilized. On the other hand, the binding with EF-Tu.GDP does not appreciably affect the conformation of isoleucyl-tRNA or uncharged tRNA. These indicate the importance of the gamma-phosphate group of GTP and the aminoacyl group in the formation of the active complex of aminoacyl-tRNA and EF-Tu.GTP.

Ca(2+)-induced folding of a family I.3 lipase with repetitive Ca(2+) binding motifs at the C-terminus.

In order to understand a role of the Ca(2+) ion on the structure and function of a Ca(2+)-dependent family I.3 lipase from Pseudomonas sp. MIS38, apo-PML, holo-PML, holo-PML*, and the N-terminal domain alone (N-fragment) were prepared and biochemically characterized. Apo-PML and holo-PML represent refolded proteins in the absence and presence of the Ca(2+) ion, respectively. Holo-PML* represents a holo-PML dialyzed against 20 mM Tris-HCl (pH 7.5). The results suggest that the C-terminal domain of PML is almost fully unfolded in the apo-form and its folding is induced by Ca(2+) binding. The folding of this C-terminal domain may be required to make a conformation of the N-terminal catalytic domain functional.

Identification of the histidine and aspartic acid residues essential for enzymatic activity of a family I.3 lipase by site-directed mutagenesis.

A lipase from Pseudomonas sp. MIS38 (PML) is a member of the lipase family I.3. We analyzed the roles of the five histidine residues (His(30), His(274), His(291), His(313), and His(365)) and five acidic amino acid residues (Glu(253), Asp(255), Asp(262), Asp(275), and Asp(290)), which are fully conserved in the amino acid sequences of family I.3 lipases, by site-directed mutagenesis. We showed that the mutation of His(313) or Asp(255) to Ala almost fully inactivated the enzyme, whereas the mutations of other residues to Ala did not seriously affect the enzymatic activity. Measurement of the far- and near-UV circular dichroism spectra suggests that inactivation by the mutation of His(313) or Asp(255) is not due to marked changes in the tertiary structure. We propose that His(313) and Asp(255), together with Ser(207), form a catalytic triad in PML.

Isolation of TBP-interacting protein (TIP) from a hyperthermophilic archaeon that inhibits the binding of TBP to TATA-DNA.

We have isolated TBP (TATA-binding protein)-interacting protein (TIP) from cell lysates of a hyperthermophilic archaeon, Pyrococcus kodakaraensis KOD1, by affinity chromatography with TBP-agarose. Based on the internal amino acid sequence information, PCR primers were synthesized and used to amplify the gene encoding this protein (Pk-TIP). Determination of the nucleotide sequence and characterization of the recombinant protein revealed that Pk-TIP is composed of 224 amino acid residues (molecular weight of 25,558) and exists in a dimeric form. BIAcore analyses for the interaction between recombinant Pk-TIP and recombinant Pk-TBP indicated that they interact with each other with an equilibrium dissociation constant, KD, of 1.24-1.46 microM. A gel mobility shift assay indicated that Pk-TIP inhibited the interaction between Pk-TBP and a TATA-DNA. Pk-TIP may be one of the archaeal factors which negatively regulate transcription.

Identification of catalytically essential residues in Escherichia coli esterase by site-directed mutagenesis.

Escherichia coli esterase (EcE) is a member of the hormone-sensitive lipase family. We have analyzed the roles of the conserved residues in this enzyme (His103, Glu128, Gly163, Asp164, Ser165, Gly167, Asp262, Asp266 and His292) by site-directed mutagenesis. Among them, Gly163, Asp164, Ser165, and Gly167 are the components of a G-D/E-S-A-G motif. We showed that Ser165, Asp262, and His292 are the active-site residues of the enzyme. We also showed that none of the other residues, except for Asp164, is critical for the enzymatic activity. The mutation of Asp164 to Ala dramatically reduced the catalytic efficiency of the enzyme by the factor of 10(4) without seriously affecting the substrate binding. This residue is probably structurally important to make the conformation of the active-site functional.

Unusual enzyme characteristics of aspartyl-tRNA synthetase from hyperthermophilic archaeon Pyrococcus sp. KOD1.

The aspA gene, encoding the aspartyl-tRNA synthetase (AspRS) from the hyperthermophilic archaeon Pyrococcus sp. KOD1, was expressed in Escherichia coli. The KOD1 AspRS, which was purified to homogeneity and was shown to be functional in dimeric form, aminoacylated tRNA from KOD1. The optimum temperature for this activity was 65 degrees C, which was lower than that for the cell growth of KOD1 (85 degrees C). However, it increased to 75 degrees C by the addition of polyamine molecules, such as putrescine, spermine, and spermidine. Analysis of the thermal denaturations of the enzyme and of KOD1-tRNA indicated that neither of them was denatured at temperatures below 70 degrees C. These results suggest polyamine is one of the factors which are required to stabilize the AspRS-tRNA complex in vivo. In order to determine whether the nucleotide triphosphate (NTP) is required for Asp-tRNA synthesis, the aminoacylation was examined in the presence of each of the four NTPs. AspRS most effectively aminoacylated tRNA in the presence of ATP. However, we also found that the enzyme aminoacylated it even in the presence of GTP and UTP as well. Archaeon synthetase may have an interesting system to utilize other NTPs than ATP. The extreme conditions of early life may have given rise to these unique characteristics which then disappeared from developed organisms through evolution.

UV-irradiation-induced DNA immobilization and functional utilization of DNA on nonwoven cellulose fabric.

Immobilization of double-stranded DNA onto nonwoven cellulose fabric by UV irradiation and utilization of DNA-immobilized cloth were examined. The immobilized DNA was found to be stable in water, with the maximum amount of fabric-immobilized DNA being approximately 20 mg/g of nonwoven fabric. The DNA-immobilized cloth could effectively accumulate endocrine disruptors and harmful DNA intercalating pollutants, such as dibenzo-p-dioxin, dibenzofuran, biphenyl, benzo[a]pyrene and ethidium bromide. Additionally, DNA-immobilized cloth was found to bind metal ions, such as Ag+, Cu2+, and Zn2+. The maximum amounts of bound Ag+, Cu2+, and Zn2+ onto DNA-immobilized cloth (1 g) were approximately 5, 2, and 1 mg, respectively. DNA-immobilized cloth containing Ag+ showed antibacterial activity against Escherichia coli and Staphylococcus aureus. DNA-immobilized cloth without metal ion and with Cu2+ or Zn2+ did not show antibacterial activity. These results suggest that immobilized DNA imparts useful functionality to cloth. DNA-immobilized cloth prepared by UV irradiation has potential to serve as a useful biomaterial for medical, engineering, and environmental application.

Characterization of ribonuclease HII from Escherichia coli overproduced in a soluble form.

Escherichia coli RNase HII is composed of 198 amino acid residues. The enzyme has been overproduced in an insoluble form, purified in a urea-denatured form, and refolded with poor yield [M. Itaya (1990) Proc. Natl. Acad. Sci. USA 87, 8587-8591]. To facilitate the preparation of the enzyme in an amount sufficient for physicochemical studies, we constructed an overproducing strain in which E. coli RNase HII is produced in a soluble form. The enzyme was purified from this strain and its biochemical and physicochemical properties were characterized. The good agreement in the molecular weights estimated from SDS-PAGE (23,000) and gel filtration (22,000) suggests that the enzyme acts as a monomer. From the far-UV circular dichroism spectrum, its helical content was calculated to be 23%. The enzyme showed Mn(2+)-dependent RNase H activity. Its specific activity determined using (3)H-labeled M13 RNA/DNA hybrid as a substrate was comparable to but slightly higher than that of the refolded enzyme, indicating that the enzyme overproduced and purified in a soluble form is more suitable for structural and functional analyses than the refolded enzyme.

Identification of the gene encoding esterase, a homolog of hormone-sensitive lipase, from an oil-degrading bacterium, strain HD-1.

The gene encoding an esterase (HDE) was cloned from an oil-degrading bacterium, strain HD-1. HDE is a member of the hormone-sensitive lipase family and composed of 317 amino acid residues with a molecular weight of 33,633. The HDE-encoding gene was expressed in Escherichia coli, and the recombinant protein was purified and characterized. Amino acid sequence analysis indicated that the methionine residue was removed from its NH(2)-terminus. The good agreement of the molecular weights estimated by SDS-PAGE (35,000) and gel filtration (38,000) suggests that it acts in a monomeric form. HDE showed hydrolytic activity towards p-nitrophenyl esters of fatty acids with an acyl chain length of 2 to 14 and tributyrin, whereas it showed little hydrolytic activity towards p-nitrophenyl oleate (C(18)), tricaprylin and triolein. Determination of the kinetic parameters for the hydrolyses of the p-nitrophenyl substrates from C(2) to C(14) indicated that HDE shows a relatively broad substrate specificity. However, comparison of the k(cat)/K(m) values indicated that the C(10)-C(14) substrates are the most preferred ones. Such a preference for substrates with long acyl chains may be a characteristic of HDE.

Stabilities of chimeras of hyperthermophilic and mesophilic glycerol kinases constructed by DNA shuffling.

Glycerol kinases from Thermococcus kodakaraensis KOD1 (Tk-GK) and Escherichia coli (Ec-GK) greatly differ in thermostability. The temperature (T(1/2)) at which the enzymes lose half of their activity upon incubation for 20 min is 50-55 degrees C for Ec-GK and approximately 95 degrees C for Tk-GK. To examine whether the amino acid substitutions that make Tk-GK more stable than Ec-GK are localized in a limited region, the chimeras of two parental genes encoding Tk-GK and Ec-GK were constructed by DNA shuffling. E. coli cells were transformed with a plasmid library harboring these chimeras and screened for those tht produce chimeric enzymes which are more stable than Ec-GK. Four chimeric enzymes were isolated and purified, and their biochemical properties characterized. Replacement of 83 or 93 residues in the C-terminus of Ec-GK with the corresponding ones of Tk-GK increased the T(1/2) value of Ec-GK by 25-30 degrees C. In contrast, replacement of 85 residues in the N-terminus of Ec-GK with the corresponding ones of Tk-GK reduced the T(1/2) value by 5-10 degrees C. In addition, replacement of 10 residues in the C-terminus of Tk-GK with the corresponding ones of Ec-GK reduced the T(1/2) value ot Tk-GK by approximately 15 degrees C. Measurement of the far-UV CD spectra indicates that the three-dimensional structures of the chimeric enzymes, as well as those of the parent enzymes, are similar to one another. These results suggest that the amino acid substitutions responsible for the high stability of Tk-GK are largely localized in the C-terminal region.

Molecular diversities of RNases H.

RNase H is an enzyme that specifically cleaves RNA hybridized to DNA. The enzyme is ubiquitously present in various organisms. Single bacterial and eucaryotic cells often contain two RNases H, whereas single archaeal cells contain only one. To determine whether there is a physiological significance in the ubiquity and multiplicity of the enzyme, and whether all enzymes are evolutionarily diverged from a common ancestor, we carried out phylogenetic analyses of the RNase H sequences. In this report, we demonstrated that RNases H are classified into two major families, Type 1 and Type 2 RNases H, of which only the Type 2 enzymes are present in all living organisms, including bacteria, archaea, and eucaryotes, suggesting that they represent an ancestral form of RNases H. Based on this information, we discuss the evolutionary relationships and possible cellular functions of RNases H.

Isolation and characterization of long-chain-alkane degrading Bacillus thermoleovorans from deep subterranean petroleum reservoirs.

Two extremely thermophilic alkane-degrading bacterial strains, B23 and H41, were respectively isolated from deep subterranean petroleum reservoirs in the Minami-aga (Niigata) and Yabase (Akita) oil fields. Both strains were able to grow at temperatures ranging from 50 to 80 degrees C, with optimal growth at 70 degrees C for B23 and 65 degrees C for H41. From 16S rRNA gene sequence analysis and physiological characterization, both strains were identified as Bacillus thermoleovorans (identities of 99.5% and 99.6% to strain DSM 5366, and 98.3% and 98.7% to the type strain LEH-1(TS), respectively). Strains B23 and H41 effectively (60%) degraded n-alkanes longer than C12 and C15, respectively, at 70 degrees C, while strain LEH-1(TS) degraded undecane (C11) most effectively. When B23 and H41 were cultivated in the presence of heptadecane, heptadecanoate and pentadecanoate were specifically accumulated in the cells. These results strongly suggest that the two strains degraded n-alkanes by a terminal oxidation pathway, followed by a beta-oxidation pathway.

Gene Cloning of an alcohol dehydrogenase from thermophilic alkane-degrading Bacillus thermoleovorans B23.

The gene encoding an alcohol dehydrogenase (Bt-ADH) was cloned from a newly isolated thermophilic alkane-degrading Bacillus thermoleovorans, strain B23. The gene conferred 1-tetradecanol dehydrogenase activity on Escherichia coli cells. Bt-ADH is composed of 249 amino acid residues and the calculated molecular mass is 27,196 Da. A tyrosine residue in the active site and a glycine-rich sequence (GGXXGI/LG) constituting probable nicotinamide adenine dinucleotide (NAD+) or nicotinamide adenine dinucleotide phosphate (NADP+) binding site were completely conserved in the Bt-ADH sequence at positions 155 and 11, respectively. A phylogenetic analysis of Bt-ADH suggested that the enzyme belongs to the zinc-independent ADH Group II. Its highest similarity (48% identical) was to a hypothetical oxidoreductase from a hyperthermophile, Thermotoga maritima.

Gene cloning and characterization of aldehyde dehydrogenase from a petroleum-degrading bacterium, strain HD-1.

The hd-ald gene encoding aldehyde dehydrogenase (hd-ALDH) from an mixotrophic petroleum-degrading bacterium, strain HD-1 was cloned and sequenced. hd-ALDH (506 amino acids) is a member of the NAD+-dependent aldehyde dehydrogenase group. The hd-ald gene was expressed in Escherichia coli, and the recombinant enzyme was purified and characterized biochemically and enzymatically. The molecular weight of the enzyme was estimated to be 55,000 by SDS-PAGE, and 224,000 by gel filtration chromatography, suggesting that it acts as a tetramer. The CD spectrum suggests that the helical content of the enzyme is 10%. hd-ALDH was active on various aliphatic aldehyde substrates. The K(m) values of the enzyme were 6.4 microM for acetaldehyde, 4.2 microM for hexanal, 2.8 microM for octanal, and 0.84 microM for decanal, whereas the kcat values for these substrates were nearly equal (51-64 min(-1)). These results indicate that hd-ALDH acts preferentially on long-chain aliphatic aldehydes.

Characterization of petroleum-degrading bacteria from oil-contaminated sites in Vietnam.

Four petroleum-degrading bacterial strains, 2TN-NB, 6TBX-CL, MVK2-5, and XCK, were isolated from various oil-contaminated sites in Vietnam. Determination of the nucleotide sequence of the gene encoding 16S rRNA allowed 2TN-NB to be identified as Acinetobacter sp. and the other three stains as Pseudomonas sp. Among the four isolates, 2TN-NB was most effective in degrading crude oil: in 1 d, it degraded 95% of the crude oil in the culture medium (5%, v/v). The isolated strains could also degrade a sulfur-containing aromatic hydrocarbon, dibenzothiophene (DBT), with low efficiency. Except for MVK2-5, which degraded crude oil least efficiently, the isolates produced biosurfactants in amounts sufficient for structural analysis. FT-IR measurement suggested that strains 6TBX-CL and XCK produced glycolipid-type biosurfactants while that produced by 2TN-NB was of the polysaccharide type.