Identification of the gene PaEMT1 for biosynthesis of mannosylerythritol lipids in the basidiomycetous yeast Pseudozyma antarctica

The yeast Pseudozyma antarctica produces a large amount of glycolipid biosurfactants known as mannosylerythritol lipids (MELs), which show not only excellent surface‐active properties but also versatile biochemical actions. To investigate the biosynthesis of MELs in the yeast, we recently reported expressed sequence tag (EST) analysis and estimated genes expressing under MEL production conditions. Among the genes, a contiguous sequence of 938 bp, PA_004, showed high sequence identity to the gene emt1, encoding an erythritol/mannose transferase of Ustilago maydis, which is essential for MEL biosynthesis. The predicted translation product of the extended PA_004 containing the two introns and a stop codon was aligned with Emt1 of U. maydis. The predicted amino acid sequence shared high identity (72%) with Emt1 of U. maydis, although the amino‐terminal was incomplete. To identify the gene as PaEMT1 encoding an erythritol/mannose transferase of P. antarctica, the gene‐disrupted strain was developed by the method for targeted gene disruption, using hygromycin B resistance as the selection marker. The obtained ΔPaEMT1 strain failed to produce MELs, while its growth was the same as that of the parental strain. The additional mannosylerythritol into culture allowed ΔPaEMT1 strain to form MELs regardless of the carbon source supplied, indicating a defect of the erythritol/mannose transferase activity. Furthermore, we found that MEL formation is associated with the morphology and low‐temperature tolerance of the yeast. Copyright © 2010 John Wiley & Sons, Ltd.     

Study on multi-recycle transmutation of LLFP in light water reactor

The effectiveness of transmutation for long-lived fission products (LLFP) in light water reactors (LWR), i.e. both BWR and PWR, considering the large capture cross-section of FPs in thermal region was evaluated. Calculation results of iodine and technetium transmutation in BWR and PWR suggested an effective use of BWR as compared to PWR. To obtain transmutation fraction [TF] of 30 to 40%, the irradiation period needed for ⁹⁹Tc transmutation was estimated as 10 to 15 years, and the period for ¹²⁹I transmutation was estimated as 30 to 40 years, respectively. The evaluations bring a new concept of multi-recycle LLFP transmutation using LWR_TR (LWR for transmutation).     

Efficient preparation of liposomes encapsulating food materials using lecithins by a mechanochemical method

In order to evaluate to the feasibility of using lecithins for nanocapsules including functional food materials, liposomes were prepared from different commercially available lecithins (SLP-WHITE, SLP-PC70 and PL30S) by the Bangham method, and their physicochemical properties were examined by using a confocal laser scanning microscopy (CLSM) and the measurements of trapping efficiency. There was little difference in the trapping efficiency among the three types of liposomes. In all cases, the trapping efficiency clearly increased with an increase of the lecithin concentration up to 10 wt % , and the maximum efficiency reached at approximately 15%. CLSM observation showed the particle size of liposomes prepared from SLP-WHITE is significantly smaller than that prepared from other lecithins. In addition, liposomal solution prepared from SLP-WHITE remained well dispersed for at least 30 days, while two other liposomal solutions showed a phase separation due to aggregation and/or fusion of liposomes. These results indicated that SLP-WHITE is the most appropriate for the preparation of stable liposomes with well dispersed among the lecithins tested. SLP-WHITE liposomes were then prepared by the mechanochemical method using a homogenizer and microfluidizer, aiming at improving the preparation efficiency and liposome stability. The particle size of the prepared SLP-WHITE liposomes decreased with increasing inlet pressure and the number of processed cycles, and reached between 73 and 123 nm based on the measurement using dynamic light scattering. Moreover, freeze-fracture transmission electron microscopy revealed that the prepared liposomes are small unilamellar vesicles (SUV) with a diameter of approximately 100 nm. The extract of Curcuma longa Linn. (Ukon), which contains curcumins as a functional food material, was then subjected to the mechanochemical method with SLP-WHITE to give liposomes including the functional materials. Interestingly, the trapping efficiency of the liposomes for curcumins was found to reach over 85%. From these results, the present mechanochemical method is very likely to allow us to efficiently prepare stable and functional liposomes from the low-cost lecithin. The method may thus have a potential for manufacturing practical nanocapsules, which serves as a novel carrier of functional food materials.     

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.     

Processing of ethanol fermentation broths by Candida krusei to separate bioethanol by pervaporation using silicone rubber‐coated silicalite membranes

BACKGROUND: Pervaporation employing ethanol‐permselective silicalite membranes as an alternative to distillation is a promising approach for refining low‐concentration bioethanol solutions. However, to make the separation process practicable, it is extremely important to avoid the problems caused by the adsorption of succinate on the membrane during the separation process. In this work, the pervaporation of an ethanol fermentation broth without succinate was investigated, as well as the influence of several fermentation broth nutrient components. RESULTS: Candida krusei IA‐1 produces an extremely low level of succinate. The decrease in permeate ethanol concentration through a silicone rubber‐coated silicalite membrane during the separation of low‐succinate C. krusei IA‐1 fermentation broth was significantly improved when compared with that obtained using Saccharomyces cerevisiae broth. By treating the fermentation broth with activated carbon, bioethanol was concentrated as efficiently as with binary mixtures of ethanol/water. The total flux was improved upto 56% of that obtained from the separation of binary mixtures, compared with 43% before the addition of activated carbon. Nutrients such as peptone, yeast extract and corn steep liquor had a negative effect on pervaporation, but this response was distinct from that caused by succinate. CONCLUSION: For consistent separation of bioethanol from C. krusei IA‐1 fermentation broth by pervaporation, it is useful to treat the low nutrient broth with activated carbon. To further improve pervaporation performance, it will be necessary to suppress the accumulation of glycerol. Copyright © 2009 Society of Chemical Industry     

Cellular and transcriptional responses of yeast to the cleavage of cytosolic tRNAs induced by colicin D

Colicin D is a plasmid‐encoded antibacterial protein that specifically cleaves the anticodon loops of four Escherichia coli tRNA^Arg species. Here, we report that the catalytic domain of colicin D, which is expressed in Saccharomyces cerevisiae, impairs cell growth by cleaving specific tRNAs. DNA microarray analysis revealed that mating‐related genes were upregulated, while genes involved in a range of metabolic processes were downregulated, thereby impairing cell growth. The pheromone‐signalling pathway was activated only in α cells by tRNA cleavage, which was not observed in ‘a’ cells or diploid cells. On the basis of these results and on the recent identification of two killer toxins that cleave specific tRNAs, the relationship between tRNA depletion and the resultant cellular response is discussed. Copyright © 2009 John Wiley & Sons, Ltd.     

Vacuolar H(+)-ATPase and plasma membrane H(+)-ATPase contribute to the tolerance against high-pressure carbon dioxide treatment in Saccharomyces cerevisiae

As a non-thermal sterilization process, high-pressure carbon dioxide treatment (HPCT) is considered to be promising. The main sterilizing effect of HPCT is thought to be acidification in cytoplasm of microorganisms. We investigated the tolerance mechanism of Saccharomyces cerevisiae to HPCT with special reference to vacuolar and plasma membrane H(+)-ATPases. HPCT was imposed at 35 degrees C, 4 to 10 MPa, for 10 min. slp1 mutant defective in vacuole morphogenesis was more sensitive to HPCT than its isogenic parent. Concanamycin A, a specific inhibitor of vacuolar H(+)-ATPase (V-ATPase), at 10 microM rendered the parent more HPCT-sensitive to the level of slp1. To confirm further the contribution of V-ATPase to the tolerance against HPCT in S. cerevisiae, we compared vma1 mutant of V-ATPase with its isogenic parent for their HPCT sensitivity. vma1 mutant was more sensitive to HPCT than its parent. Addition of 10 microM vanadate, an inhibitor of plasma membrane H(+)-ATPase (P-ATPase), to the wild type strains also increased the inactivation ratio. These results clearly show that V- and P-ATPases contribute to the tolerance against HPCT in S. cerevisiae by accumulating excess H(+) from cytoplasm to vacuole and excluding H(+) outside of the cell, respectively.     

Bactericidal effects of konjac fluid on several food-poisoning bacteria

In this study, the bactericidal effects of Japanese alkaline foods on food-poisoning bacteria were evaluated. Konjac is an alkaline food soaked in calcinated calcium (the pH of konjac fluid ranges from 11.42 to 12.53). Konjac fluids completely inactivated Escherichia coli, enterohemorrhagic E. coli O157:H7 and E. coil O26:H9, Salmonella Enteritidis, Vibrio parahemolyticus. and Staphylococcus aureus. The initial level of 6 log CFU/ml dramatically decreased after incubation with konjac fluid, and no viable gram-negative bacterium cells could be detected within 1 to 2 days and no viable S. aureus cells could be detected within 3 to 5 days. On the other hand, treatment with konjac fluid was also effective in reducing levels of spore-forming bacteria (Bacillus subtilis, Bacillus cereus, Clostridium perfringens, and Clostridium botulinum type E and type A). At least a 4-log reduction of spore-forming bacteria was obtained in konjac fluid within 7 to 14 days. Vegetative cells were more susceptible to konjac fluid than spores were. When the initial cell count was 6 log CFU/ml, a few surviving spores remained for 60 to 90 days, but no spores could be detected after 120 days. When the initial count of spore-forming bacteria was 3 to 4 log CFU/ml, the cells considered vegetative were completely inactivated within I to 3 days. Repeated treatment with konjac fluid caused complete inactivation of spores in less than 1 to 3 days. Our studies indicate that konjac fluid, which has a long history of use in food, will control food-poisoning bacterial contamination during the production or preservation of konjac and other foods and has a preventive effect on bacteria that can cause severe disease at uniquely low levels.