Tomotake Morita

National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki, Japan

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Publications (101)194.84 Total impact

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    ABSTRACT: Yeast host-vector systems are useful tools for the production of recombinant proteins. Here, we report the construction of a new high-level expression plasmid pPAX1-neo for the basidiomycetous yeast, Pseudozyma antarctica. pPAX1-neo harbours a xylose-inducible expression cassette under control of the xylanase promoter and terminator of P. antarctica T-34, a selection cassette of neomycin/G418 with an Escherichia coli neomycin resistance gene under control of the homocitrate synthase promoter of strain T-34, and an autonomously replicating sequence fragment of Ustilago maydis (UARS). Biodegradable plastic (BP)-degrading enzymes of P. antarctica JCM10317 (PaE) and Paraphoma-related fungal strain B47-9 (PCLE) were used as reporter proteins and inserted into pPAX1-neo, resulting in pPAX1-neo::PaCLE1 and pPAX1-neo::PCLE, respectively. Homologous and heterologous BP-degrading enzyme production of transformants of P. antarctica T-34 were detected on agar plates containing xylose and emulsified BP. Recombinant PaE were also produced by transformants of other Pseudozyma strains including Pseudozyma aphidis, Pseudozyma rugulosa, and Pseudozyma tsukubaensis. To improve the stability of transformed genes in cells, the UARS fragment was removed from linearized pPAX1-neo::PaCLE1 and integrated into the chromosome of the P. antarctica strain, GB-4(0), which was selected as a PaE producer in xylose media. Two transformants, GB-4(0)-X14 and X49, had an 11-fold higher activity compared with the wild type strain in xylose-containing liquid media. By xylose fed-batch cultivation using a 3-L jar fermentor, GB-4(0)-X14 produced 73.5 U mL(-1) of PaE, which is 13.4-fold higher than that of the wild type strain GB-4(0), which produced 5.5 U mL(-1) of PaE.
    No preview · Article · Dec 2015 · Applied Microbiology and Biotechnology
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    ABSTRACT: Bioethanol production using lignocellulosic biomass generates lignocellulosic bioethanol distillery wastewater (LBDW) that contains a large amount of xylose, making it a potential inexpensive source of xylose for biomaterials production. The main goal of this study was the production of useful enzymes from LBDW during treatment of this wastewater. In this study, we found that xylose strongly induced two yeast strains, Pseudozyma antarctica T-34 and GB-4(0), to produce novel xylanases, PaXynT and PaXynG, respectively. The nucleotide sequence of PaXynT [accession No. DF196774 (GAC73192.1)], obtained from the genome database of strain T-34 using its N-terminal amino acid sequence, was 91% identical to that of PaXynG (accession No. AB901085), and the deduced amino acid sequence is 98% identical. The specific activities of the purified PaXynT and PaXynG were about 52 U/mg. The optimal pH and temperature for both enzymes' activities were 5.2 and 50°C, respectively. They hydrolyzed xylan to xylose and neither had β-xylosidase (EC activity, indicating that they are endo-β-xylanases (EC With these results, we expect that PaXyns can be employed in saccharizing lignocellulosic biomass materials for the production of useful products just like other endoxylanases. After 72 h of LBDW fed-batch cultivation using a jar-fermentor, strain GB-4(0) produced 17.3 U/ml (corresponding to about 0.3 g/l) of PaXynG and removed 63% of dissolved organic carbon and 87% of dissolved total phosphorus from LBDW. These results demonstrate the potential of P. antarctica for xylanase production during LBDW treatment.
    Preview · Article · Dec 2015 · AMB Express
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    ABSTRACT: To develop a structural homolog of mannosylerythritol lipids (MELs), Pseudozyma tsukubaensis JCM16987 (known to be a specific producer of the diastereomer type of mono-acetylated MEL (MEL-B)) was cultivated in medium containing 4 % (w/v) olive oil as the primary carbon source and 4 % L-arabitol as the supplemental sugar alcohol. Based on thin-layer chromatography (TLC), the glycolipid extract showed two major spots corresponding to MEL-B and an unknown glycolipid (GL1). Based on high-performance liquid chromatography after acid hydrolysis, GL1 from the L-arabitol culture showed two primary peaks identical to mannose and arabitol using the sugar analysis column, and one peak identical to L-arabitol was detected using the chiral resolution column. Based on NMR analysis, GL1 was identified as mono-acetylated mannosyl-L-arabitol lipid (MLAL-B) consisting of mannose, with L-arabitol as the sugar moiety. The observed critical micelle concentration (CMC) and surface tension at the CMC (γCMC) of MLAL-B were 1.2 × 10(-5) M and 32.8 mN/m, which were significantly higher than MEL-B (CMC = 3.1 × 10(-6) M and γcmc = 26.1 mN/m). Furthermore, based on a water-penetration scan, MLAL-B efficiently formed lamellar phase (Lα) and myelins at a broad concentration range. Thus, the present glycolipid showed higher hydrophilicity and/or water solubility and increased our understanding of environmentally advanced biosurfactants.
    No preview · Article · May 2015 · Applied Microbiology and Biotechnology
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    ABSTRACT: Nineteen levulinic acid (LA)-utilizing bacteria were isolated from environmental samples. Following examination of the use of 80 g/L LA by some isolated strains, Brevibacterium epidermidis LA39-2 consumed 62.6 g/L LA following 8 days incubation. The strain also utilized both 90 and 100 g/L LA, with consumption ratio of 84.3 and 53.3%, respectively, after 10 days incubation.
    No preview · Article · Apr 2015 · Bioscience Biotechnology and Biochemistry
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    ABSTRACT: The yeast Starmerella bombicola NBRC10243 is an excellent producer of sophorolipids (SLs) from various feedstocks. Here, we report the draft genome sequence of S. bombicola NBRC10243. Analysis of the sequence may provide insight into the properties of this yeast that make it superior for use in the production of functional glycolipids and biomolecules, leading to the further development of S. bombicola NBRC10243 for industrial applications.
    Preview · Article · Mar 2015 · Genome Announcements
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    Tomotake Morita · Tokuma Fukuoka · Tomohiro Imura · Dai Kitamoto
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    ABSTRACT: Mannosylerythritol lipids (MELs) are a glycolipid class of biosurfactants produced by a variety yeast and fungal strains that exhibit excellent interfacial and biochemical properties. MEL-producing fungi were identified using an efficient screening method for the glycolipid production and taxonomical classification on the basis of ribosomal RNA sequences. MEL production is limited primarily to the genus Pseudozyma, with significant variability among the MEL structures produced by each species. Outside of Pseudozyma, one recently isolated strain, Ustilago scitaminea, has been shown to exhibit abundant MEL-B production from sugarcane juice. Structural analyses of these compounds suggest a role for MELs in numerous cosmetic applications. MELs act as effective topical moisturizers and can repair damaged hair. Furthermore, these compounds have been shown to exhibit both protective and healing activities, to activate fibroblasts and papilla cells, and to act as natural antioxidants. In this review, we provide a brief summary of MEL research over the past few decades, focusing on the identification of MEL-producing fungi, the structural characterization of MELs, the use of alternative compounds as a primary carbon source, and the use of these compounds in cosmetic applications.
    Preview · Article · Feb 2015 · Journal of oleo science
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    ABSTRACT: Levulinic acid (LA) is a platform chemical derived from cellulosic biomass, and the expansion of LA utilization as a feedstock is important for production of a wide variety of chemicals. To investigate the potential of LA as a substrate for microbial conversion to chemicals, we isolated and identified LA-utilizing bacteria. Among the six isolated strains, Pseudomonas sp. LA18T and Rhodococcus hoagie LA6W degraded up to 70g/L LA in a high-cell-density system. The maximal accumulation of acetic acid by strain LA18T and propionic acid by strain LA6W was 13.6g/L and 9.1g/L, respectively, after a 4-day incubation. Another isolate, Burkholderia stabilis LA20W, produced trehalose extracellularly in the presence of 40g/L LA to approximately 2g/L. These abilities to produce useful compounds supported the potential of microbial LA conversion for future development and cellulosic biomass utilization. Copyright © 2014 Elsevier Ltd. All rights reserved.
    No preview · Article · Nov 2014 · Bioresource Technology
  • Shun Sato · Tomotake Morita · Tokuma Fukuoka · Dai Kitamoto · Hiroshi Habe
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    ABSTRACT: To produce l-glyceric acid (l-GA) from dl-GA, microbial resolution was investigated using newly isolated bacterial strains capable of enantiospecific degradation of d-GA. Strains GA3R and GA72P, identified as Serratia and Pseudomonas species, respectively, exhausted d-GA within 72 h, resulting in production of l-GA with enantiomeric purity ≥89%.
    No preview · Article · Nov 2014 · Journal of Bioscience and Bioengineering
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    Hideaki Koike · Shun Sato · Tomotake Morita · Tokuma Fukuoka · Hiroshi Habe
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    ABSTRACT: Here we report the 3.7-Mb draft genome sequence of Acetobacter tropicalis NBRC16470T, which can produce optically pure d-glyceric acid (d-GA; 99% enantiomeric excess) from raw glycerol feedstock derived from biodiesel fuel production processes. FOOTNOTES Address correspondence to Hiroshi Habe, hiroshi.habe{at} Citation Koike H, Sato S, Morita T, Fukuoka T, Habe H. 2014. Draft genome sequence of Acetobacter tropicalis type strain NBRC16470, a producer of optically pure d-glyceric acid. Genome Announc. 2(6):e01329-14. doi:10.1128/genomeA.01329-14. Received 12 November 2014. Accepted 13 November 2014. Published 18 December 2014. Copyright © 2014 Koike et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 3.0 Unported license.
    Preview · Article · Nov 2014 · Genome Announcements
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    ABSTRACT: Cellobiose lipids (CLs) are asymmetric bolaform biosurfactants, which are produced by Cryptococcus humicola JCM 10251 and have fungicidal activity. In this study, the sodium salts of CLs (CLNa) were prepared to improve aqueous solubility of the CLs, and their surface and gelation properties in aqueous solutions were examined by surface tension, rheology, and freeze-fracture transmission electron microscopy (FF-TEM) measurements. The surface tension measurements revealed that the CLNa have high surface activity: CMC1 and γCMC1 are 0.1 mg/ml and 34.7 mN/m, respectively. It was also found that the CLNa form giant micelles above their CMC, whose average size is 116.6 ± 31.9 nm. Unlike conventional surfactants, the surface tension reduced further with an increase in concentration and the aqueous solution became viscous at the minimum gelation concentration (MGC: 5.0 mg/ml). In rheological studies, the obtained gels proved to be rather soft and their sol-gel temperature was found to be approximately 50°C. FF-TEM observation of the gels showed 3D supramolecular structures with an entangled fibrous network. Since the present CLNa aqueous gels have a degree of fungicidal activity, they could be useful for novel multifunctional soft materials applicable to the food and cosmetic industries.
    No preview · Article · Sep 2014 · Journal of oleo science
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    ABSTRACT: The basidiomycetous yeast Pseudozyma antarctica is known as a producer of industrial enzymes and the extracellular glycolipids, mannosylerythritol lipids. Here, we report the draft genome sequence of the type strain JCM10317. The draft genome assembly has a size of 18.1 Mb and a G+C content of 60.9%, and it consists of 197 scaffolds.
    Preview · Article · Sep 2014 · Genome Announcements
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    ABSTRACT: The biological function of mannosylerythritol lipids (MELs) towards their producer, Pseudozyma antarctica, on plant surfaces was investigated. MEL-producing wild-type strain and its MEL production-defective mutant strain (ΔPaEMT1) were compared in terms of their phenotypic traits on the surface of plastic plates, onion peels, and fresh leaves of rice and wheat. While wild-type cells adhering on plastic surfaces and onion peels changed morphologically from single cells to elongated ones for a short period of about 4 h and 1 day, respectively, ΔPaEMT1 cells did not. Microscopic observation of both strains grown on plant leaf surfaces verified that the wild type colonized a significantly bigger area than that of ΔPaEMT1. However, when MELs were exogenously added to the mutant cells on plant surfaces, their colonized area became enlarged. High-performance liquid chromatography analysis revealed a secretion of higher amount of MELs in the cell suspension incubated with wheat leaf cuttings compared to that in the suspension without cuttings. Transcriptional analysis by real-time reverse transcriptase PCR verified that the expression of erythritol/mannose transferase gene and MELs transporter gene of P. antarctica increased in the cells inoculated onto wheat leaves at 4, 6, and 8 days of incubation, indicating a potential of P. antarctica to produce MELs on the leaves. These findings demonstrate that MELs produced by P. antarctica on plant surfaces could be expected to play a significant role in fungal morphological development and propagation on plant surfaces.
    No preview · Article · Apr 2014 · Applied Microbiology and Biotechnology
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    ABSTRACT: The isolation of biosurfactant-producing yeasts from food materials was accomplished. By a combination of a new drop collapse method and thin-layer chromatography, 48 strains were selected as glycolipid biosurfactant producers from 347 strains, which were randomly isolated from various vegetables and fruits. Of the producers, 69% were obtained from vegetables of the Brassica family. Of the 48 producers, 15 strains gave relatively high yields of mannosylerythritol lipids (MELs), and were identified as Pseudozyma yeasts. These strains produced MELs from olive oil at yields ranging from 8.5 to 24.3 g/L. The best yield coefficient reached 0.49 g/g as to the carbon sources added. Accordingly, MEL producers were isolated at high efficiency from various vegetables and fruits, indicating that biosurfactant producers are widely present in foods. The present results should facilitate their application in the food and related industries.
    No preview · Article · Mar 2014 · Bioscience Biotechnology and Biochemistry
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    ABSTRACT: Pseudozyma antarctica is a non-pathogenic phyllosphere yeast known as an excellent producer of mannosylerythritol lipids (MELs), multi-functional extracellular glycolipids, from vegetable oils. To clarify the genetic characteristics of P. antarctica, we analyzed the 18 Mb genome of P. antarctica T-34. On the basis of KOG analysis, the number of genes (219 genes) categorized into lipid transport and metabolism classification in P. antarctica was one and a half times larger than that of yeast Saccharomyces cerevisiae (140 genes). The gene encoding an ATP/citrate lyase (ACL) related to acetyl-CoA synthesis conserved in oleaginous strains was found in P. antarctica genome: the single ACL gene possesses the four domains identical to that of the human gene, whereas the other oleaginous ascomycetous species have the two genes covering the four domains. P. antarctica genome exhibited a remarkable degree of synteny to U. maydis genome, however, the comparison of the gene expression profiles under the culture on the two carbon sources, glucose and soybean oil, by the DNA microarray method revealed that transcriptomes between the two species were significantly different. In P. antarctica, expression of the gene sets relating fatty acid metabolism were markedly up-regulated under the oily conditions compared with glucose. Additionally, MEL biosynthesis cluster of P. antarctica was highly expressed regardless of the carbon source as compared to U. maydis. These results strongly indicate that P. antarctica has an oleaginous nature which is relevant to its non-pathogenic and MEL-overproducing characteristics. The analysis and dataset contribute to stimulate the development of improved strains with customized properties for high yield production of functional bio-based materials.
    Preview · Article · Feb 2014 · PLoS ONE
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    ABSTRACT: In this study, spontaneous vesicle formation from the sodium salt of acidic sophorolipid (SLNa) was observed, and its potential application as a skin penetration enhancer for triterpene glycosides extracted from the fruits of Siraitia grosvenorii Swingle was then investigated. Dynamic light scattering (DLS) measurements of the SLNa assemblies prepared by the gentle mixing of SLNa with water (1%) showed their hydrodynamic radius (Rh) to be 96.2 nm, and their structure was assigned to be vesicles by freeze-fracture electron microscopy (FF-TEM). DLS and FF-TEM also revealed that the size of the vesicles increased with an increase in the concentration of the triterpene glycosides, indicating that the triterpene glycosides were incorporated into the SLNa vesicles. The results of an in vitro skin permeation assay, after loading the SLNa vesicles on a 3D cultured skin model, showed that the amount of SLNa that penetrated though the skin model increased with time. It was also found that the amount of permeated mogroside, which is the main active component of triterpene glycosides, was significantly enhanced by the SLNa vesicle formulation. These results clearly demonstrated that spontaneously formed vesicles composed of the bolaamphiphile SLNa are useful for application as penetration enhancers for active ingredients such as mogroside V.
    No preview · Article · Jan 2014 · Journal of oleo science
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    ABSTRACT: To promote the effective use of raw glycerol, 13 yeast strains with the ability to produce mannitol from glycerol were isolated from environmental samples. Of the 13 strains, strain 7-12G was selected as an efficient mannitol producer from 25% (w/v) glycerol and was identified as Candida azyma by morphological, physicochemical, and phylogenetic analyses. When the ability to produce mannitol from raw glycerol in flask culture was compared among strains 7-12G, NBRC10406 (the type strain of C. azyma), and related strains, strain NBRC10406 exhibited the highest production level (31.8 g/l). Culture in jar fermentors was next investigated, and mannitol production reached 50.8 g/l over 7 days, corresponding to 0.30 g/g-glycerol. To the best of our knowledge, this is the highest reported level of mannitol produced by a microbe from glycerol under batch-type culture conditions.
    No preview · Article · Dec 2013 · Journal of Bioscience and Bioengineering
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    ABSTRACT: The synergic effect and miscibility of the lactonic and acidic forms of sophorolipids (SLs) produced by Starmerella bombicola NBRC 10243 were first evaluated through atomic force microscopy (AFM), together with the Langmuir monolayer technique. The π-A isotherm of a pure lactonic sophorolipid (LS) monolayer mostly exhibited a liquid expanded monolayer, while that of an acidic sophorolipid (AS) monolayer showed a liquid condensed monolayer, suggesting that the lactonization of SLs makes the molecules more bulky and prevents them from adopting a close-packed arrangement. Plots of the mean area per molecule of mixed LS/AS monolayers gave positive deviations from the ideal curves, implying that the LS and AS molecules are miscible. Interestingly, the positive deviation of excess area (Aex) from ideality was most significant at a mole fraction (XAS) of 0.3, which closely resembles the composition of the LS/AS mixture secreted by S. bombicola in culture. The AFM images of mixed LS/AS monolayers transferred at 20 mN/m revealed no phase-separated microdomain structures, but rather showed small protruding objects for all compositions, indicating that LS and AS are partially miscible, as predicted by the positive deviations from the ideal curves. Cross-section analysis of the AFM images indicated that the observed protruding objects are AS-rich monolayers formed on the LS/AS monolayer. Our results clearly demonstrate that AFM combined with the Langmuir technique is useful for the exploration of the miscibility and synergic effects of microbial products.
    No preview · Article · Dec 2013 · Journal of oleo science
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    ABSTRACT: To promote the effective use of raw glycerol (a by-product of biodiesel production), 110 yeast strains that produce D-arabitol from glycerol were isolated from environmental samples. Among them, strain 17-2A was an effective D-arabitol producer in the presence of 250 g/l glycerol and was identified as Candida quercitrusa based on morphological, physicochemical, and phylogenetic analyses. C. quercitrusa type strain NBRC1022 produced the greatest quantity of D-arabitol (41.7 g/l) when the ability to produce D-arabitol from raw glycerol was compared among C. quercitrusa 17-2A and its phylogenetically related strains in flask culture. Under optimized culture conditions, strain NBRC1022 produced D-arabitol at a concentration of 58.2 g/l after a 7-day cultivation in 250 g/l glycerol, 6 g/l yeast extract, and 2 g/l CaCl2. The culture conditions were further investigated with raw glycerol using a jar fermenter; the concentration of D-arabitol reached 67.1 g/l after 7 days and 85.1 g/l after 10 days, respectively, which corresponded to 0.40 g/g of glycerol. To our knowledge, the present D-arabitol yield from glycerol is higher than reported previously using microbial production.
    No preview · Article · Dec 2013 · Applied Microbiology and Biotechnology
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    ABSTRACT: Mannosylerythritol (ME) is the hydrophilic backbone of mannosylerythritol lipids as the most promising biosurfactants produced by different Pseudozyma yeasts, and has been receiving attention as a new sugar alcohol. Different Pseudozyma yeasts were examined for the sugar alcohol production using glucose as the sole carbon source. P. hubeiensis KM-59 highly produced a conventional type of ME, i.e., 4-O-β-D-mannopyranosyl-D-erythritol (4-ME). Interestingly, P. tsukubaensis KM-160 produced a diastereomer of 4-ME, i.e., 1-O-β-D-mannopyranosyl-D-erythritol (1-ME). In shake flask culture with 200 g/l of glucose, strain KM-59 produced 4-ME at a yield of 33.2 g/l (2.2 g/l/day of the productivity), while strain KM-160 produced 1-ME at 30.0 g/l (2.0 g/l/day). Moreover, the two strains were found to produce ME from glycerol; the maximum yields of 4-ME and 1-ME from 200 g/l of glycerol were 16.1 g/l (1.1 g/l/day) and 15.8 g/l (1.1 g/l/day), respectively. The production of 1-ME as the new diastereomer was further investigated in fed batch culture using a 5-l jar-fermenter. Compared to the flask culture, strain KM-160 gave three times higher productivity of 1-ME at 38.0 g/l (6.3 g/l/day) from glucose and at 31.1 g/l (3.5 g/l/day) from glycerol, respectively. This is the first report on the selective production of two diastereomers of ME, and should thus facilitate the functional development and application of the disaccharide sugar alcohol in the food and relative industries.
    No preview · Article · Nov 2013 · Applied Microbiology and Biotechnology
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    ABSTRACT: To facilitate the development of bio-based chemicals from renewable and inexpensive natural resources, we sought to produce biosurfactants using non-edible jatropha oil. Twenty yeasts known to produce biosurfactants were tested in this study, and Stamerella bombicola NBRC 10243 was found to use jatropha oil efficiently to produce sophorolipids (SLs) as a mixture of lactone-form SL (L-SL) and acid-form SL (A-SL). Under culture conditions using rice bran as the source of organic nutrients, the yield of SLs reached 122.6 g/L in 5-L jar fermentors after 9 d in culture. HPLC analysis of the culture medium showed that the levels of phorbol esters (PEs), major toxic components of the oil, decreased markedly with an increase in culture time, suggesting that the yeast degrades PEs. Although the SLs obtained by solvent extraction of the culture medium contained a small amount of PEs, the sodium salt of A-SL (A-SL-Na) obtained by alkaline treatment (5N NaOH, 80°C) showed no PE peaks upon HPLC analysis. A-SL-Na had excellent surface activity with low CMC (9.0×10(-4) M) and γCMC (29.6 mN/m), which are lower than that of sodium dodecyl sulfate (SDS). The solubilizing ability of A-SL-Na toward for octanoic acid ([octanoic acid]/[A-SL-Na]) was found to be 2.0, which is half that of SDS. Our findings should help improve SL production from non-edible feedstock and broaden the use of promising bio-based surfactants.
    No preview · Article · Oct 2013 · Journal of oleo science

Publication Stats

2k Citations
194.84 Total Impact Points


  • 2006-2015
    • National Institute of Advanced Industrial Science and Technology
      • Research Institute for Innovation in Sustainable Chemistry
      Tsukuba, Ibaraki, Japan
  • 2003-2010
    • Kagawa University
      • • Department of Life Sciences
      • • Faculty of Agriculture
      Takamatu, Kagawa, Japan
    • Kochi University
      • Department of Bioresources Science
      Kôti, Kōchi, Japan