Identification of Ustilago cynodontis as a new producer of glycolipid biosurfactants, mannosylerythritol lipids, based on ribosomal DNA sequences

Mannosylerythritol lipids (MELs) are one of the most promising glycolipid biosurfactants known because of their multifunctionality and biocompatibility. The search for novel producers of MELs was undertaken based on the analysis of ribosomal DNA sequences on basidiomycetous yeasts. The bermuda grass smut fungus Ustilago cynodontis NBRC 7530, which taxonomically relates to Pseudozyma shanxiensis known as a MEL-C producer, was found to accumulate glycolipids in the cultured medium. Under a shake flask culture with soybean oil, the amount of the glycolipids was 1.4 g/L for 7 days at 25 degrees C. As a result of the structural characterization, the main glycolipids was identified as 4-O-[(4'-O-acetyl-3'-O-alka(e)noyl-2'-O-butanoyl)-beta-D-mannopyranosyl]-D-erythritol, and the major fatty acids were C(14) and C(16) ones. The glycolipid was highly hydrophilic MEL-C, and very similar to those produced by P. shanxiensis. The fungi of the genus Ustilago are thus likely to be potential producers of MELs as well as the yeasts of the genus Pseudozyma.     

Packing density of glycolipid biosurfactant monolayers give a significant effect on their binding affinity toward immunoglobulin G

Mannosylerythritol lipid-A (MEL-A) is one of the most promising glycolipid biosurfactants, and abundantly produced by Pseudozyma yeasts. MEL-A gives not only excellent self-assembling properties but also a high binding affinity toward human immunoglobulin G (HIgG). In this study, three kinds of MEL-A were prepared from methyl myristate [MEL-A (m)], olive oil [MEL-A (o)], and soybean oil [MEL-A (s)], and the effect of interfacial properties of each MEL-A monolayer on the binding affinity toward HIgG was investigated using surface plasmon resonance (SPR) and the measurement of surface pressure (pi)-area (A) isotherms. Based on GC-MS analysis, the main fatty acids were C(8) and C(10) acids in all MEL-A, and the content of unsaturated fatty acids was 0% for MEL-A (m), 9.1% for MEL-A (o), 46.3% for MEL-A (s), respectively. Interestingly, the acid content significantly influenced on their binding affinity, and the monolayer of MEL-A (o) gave a higher binding affinity than that of MEL-A (m) and MEL-A (s). Moreover, the mixed MEL-A (o)/ MEL-A (s) monolayer prepared from 1/1 molar ratio, which comprised of 27.8% of unsaturated fatty acids, indicated the highest binding affinity. At the air/water interface, MEL-A (o) monolayer exhibited a phase transition at 13 degrees C from a liquid condensed monolayer to a liquid expanded monolayer, and the area per molecule significantly expanded above 13 degrees C, while the amount of HIgG bound to the liquid expanded monolayer was much higher than that bound to liquid condensed monolayer. The binding affinity of MEL-A toward HIgG is thus likely to closely relate to the monolayer packing density, and may be partly controlled by temperature.     

Identification of Pseudozyma graminicola CBS 10092 as a producer of glycolipid biosurfactants, mannosylerythritol lipids

A basidiomycetous yeast, Pseudozyma graminicola CBS 10092, was found to accumulate a large amount of glycolipids in the cultured medium when grown on soybean oil as the sole carbon source. Based on thin layer chromatography, the extracellular glycolipids gave spots corresponding to those of mannosylerythritol lipids (MELs), which are highly functional and promising biosurfactants. From the structural characterization by 1H and 13C NMR, the main product was identified as 4-O-[(4'-mono-O-acetyl-2', 3'-di-O-alka(e)noyl)-beta-D-mannopyranosyl]-D-erythritol, which is a highly hydrophilic derivative of MELs known as MEL-C. According to high-performance liquid chromatography analysis, the main product, MEL-C, comprised approximately 85% of all the MELs, and the total amount reached approximately 10 g/L for 7 days. The fatty acids of the present MEL-C consisted of mainly C6, C8 and C14 acids, considerably different from those of MEL-C produced by other Pseudozyma strains such as P. antarctica and P. shanxiensis. The observed critical micelle concentration (CMC) and the surface-tension at CMC of the MEL-C were 4.0 x 10(-6) M and 24.2 mN/m, respectively, while those of MEL-A, the most intensively studied MEL, were 2.7 x 10(-6) M and 28.4 mN/m, respectively. This implied that the MEL-C has higher hydrophilicity than conventional MELs hitherto reported. In addition, on a water-penetration scan, the MEL-C efficiently formed the lamella phase (Lalpha) at a wide range of concentrations, indicating its excellent self-assembling properties. From these results, the newly identified MELs produced by P. graminicola are likely to have great potential for use in oil-in-water type emulsifiers and/or washing detergents, and would thus facilitate a broad range of applications for the promising yeast biosurfactants.     

Formation of W/O microemulsion based on natural glycolipid biosurfactant, mannosylerythritol lipid-a

Mannosylerythritol lipid-A (MEL-A) is a glycolipid biosurfactant abundantly produced from soybean oil by microorganisms at a yield of up to 100 g/L. In this study, the formation of water-in-oil (W/O) microemulsion based on the single component of MEL-A was confirmed using dynamic light scattering (DLS) and freeze fracture electron microscopy (FF-EM). DLS and FF-EM measurements revealed that the diameter of the microemulsion increases with an increase in water-to-surfactant mole ratio (W(0)) ranging from 20 to 60 nm, and the maximum W(0) value was found to be 20, which is as high as that of soybean lecithin. Glycolipid biosurfactant has a great potential for the formation of W/O microemulsion without using any cosurfactants.     

Changes in tensile properties for the PET films by the treatment in supercritical carbon dioxide fluid

Poly (ethylene terephthalate) films were treated in supercritical CO₂ (s-CO₂) fluid, and the modifications in tensile properties were investigated. The yield load peaks became ambiguous and the breaking strains and loads decreased for the treated films. However, the tensile modulus and the yield strain were almost unchanged after the treatments. The yield strain is related to the interplay between the amorphous entanglement density and the stability of crystal blocks. WAXD patterns for the films were hardly modified through the treatment. Thus the orientation for the crystal blocks seems to be unchanged. Moreover, laser Raman microprobe spectroscopic analyses revealed that the orientation of molecular chains is almost retained after the treatments. Thus it is natural to assume that the interplay between the amorphous entanglement density and the stability of crystal blocks will also be kept constant even though the swelling of amorphous regions with s-CO₂ occurs during the treatments. From differential scanning calorimetry measurements, it was found that the heat of fusion for the crystallites was a little increased without shifting the peak position after the treatment. Therefore, the increase in crystallinity is not due to the thickening or crystal perfecting for primary lamellae, but due to the secondary crystallization. The increase in crystallinity caused by the secondary lamellae crystallized and combined onto the primary lamellae seems to have reduced the amorphous chains to be deformed easily at tensile tests. Then the breaking strains and loads decreased for the treated films.     

Complete genome sequence and characterization of the N-acylhomoserine lactone-degrading gene of the potato leaf-associated Solibacillus silvestris

N-Acylhomoserine lactones (AHLs) are used as quorum-sensing signal molecules by many gram-negative bacteria. We have reported that Solibacillus silvestris, which was isolated from the potato leaf, has AHL-degrading activity. To identify the AHL-degrading gene, whole genome sequencing of S. silvestris StLB046 was performed by using pyrosequencing technology. As the result of the BLAST search, one predicted ORF (ahlS) showed slight similarity to AiiA-like AHL lactonase from Bacillus cereus group. Escherichia coli harboring the ahlS-expressing plasmid showed high AHL-degrading activity. The ahlS-cording region was also amplified by PCR from the other potato leaf-associated and AHL-degrading S. silvestris strains. Purified AhlS as a maltose binding fusion protein showed high AHL-degrading activity and catalyzes AHL ring opening by hydrolyzing lactones. In addition, expression of ahlS in plant pathogen Pectobacterium carotovorum subsp. carotovorum attenuated maceration of the potato slices. Our results suggest that AHL-degrading activity of ahlS might perform useful functions such as useful biocontrol agents.     

Identification of a methanol-inducible promoter from Rhodococcus erythropolis PR4 and its use as an expression vector

The genus Rhodococcus exhibits a broad range of catalytic activity and is tolerant to various kinds of organic solvents. This property makes rhodococci suitable for use as a whole-cell catalyst. Various tools for genetic engineering have been developed to use Rhodococcus erythropolis as a host for bioconversion. In this study, we investigated the protein expression responses of R. erythropolis strains and found that isocitrate lyase production in R. erythropolis PR4 (ICL(Re)) was induced by methanol. By analyzing the regulation mechanisms of icl(Re) expression, the ~200-bp upstream region from the first nucleotide of the translation initiation codon of icl(Re) was shown to be sufficient for the methanol-inducible expression. Also, the ~100-bp upstream region exhibited strong constitutive promoter activity by an unknown mechanism(s). By investigating proteins that bound to the upstream region of icl(Re)in vitro, a RamB homologue of R. erythropolis PR4 (RamB(Re)) was identified. Moreover, 2 putative RamB(Re) binding sites were identified in the upstream region of icl(Re) through pull-down assays. A ramB(Re) knockout experiment suggested that RamB(Re) negatively controlled the expression of icl(Re) and that RamB(Re) regulation was dependent on the availability of a carbon source. On the basis of these findings, we were able to create novel methanol-inducible and strong constitutive expression vectors.     

Sphingomyelinase C from Streptomyces sp. A9107: Unusual primary structure for bacterial sphingomyelinase C

A sphingomyelinase C (SMase) was identified in the culture supernatant of Streptomyces sp. A9107 (S-SMase). Although S-SMase seems to be a typical bacterial SMase, the primary structure of S-SMase was unusual for known bacterial SMase. The gene was functionally overexpressed in the culture medium of recombinant Rhodococcus erythropolis.     

Effect of Risk Information Exposure on Consumers’ Responses to Foods with Insect Contamination

This study explores the impact that scientific information about insect contamination of food has on consumer perceptions. Participants (n = 320, Japanese consumers) were randomly assigned to 1 of 8 information‐type conditions: (1) information about insect type, (2) information about contamination processes, (3) information about the safety of contaminated food, (4, 5, 6) combinations of 2 of (1), (2), and (3) above, (7) all information, and (8) no‐information, and asked to rate their valuation, behavioral intention, and attitude toward food with insect contamination. Results demonstrated that some combinations of scientific information that include the safety of the contaminated food are effective to reduce consumers’ compulsive rejection of insect contamination in food, whereas the single presentation of information about insect type increases consumers’ explicit rejection of both the contaminated product and the manufacturer. These findings have implications for the coordination of risk communication strategies.     

Degradation evaluation of poly-Si TFTs by comparing normal and reverse characteristics and behavior analysis of hot-carrier degradation

We propose degradation evaluation of poly-Si TFTs by comparing normal and reverse characteristics. Since symmetrical normal and reverse characteristics indicate Joule-heating degradation whereas asymmetrical characteristics indicate hot-carrier degradation, they can be clearly and easily classified. Moreover, degradation occurrence is contrasted between standard and fine TFTs. Finally, behavior of the hot-carrier degradation is analyzed.