Dai Kitamoto

Functions and potential applications of glycolipid biosurfactants--from energy-saving materials to gene delivery carriers.

Biosurfactants (BS) produced by various microorganisms show unique properties (e.g., mild production conditions, lower toxicity, higher biodegradability and environmental compatibility) compared to their chemical counterparts. The numerous advantages of BS have prompted applications not only in the food, cosmetic, and pharmaceutical industries but in environmental protection and energy-saving technology as well. Glycolipid BS are the most promising, due to high productivity from renewable resources and versatile biochemical properties. Mannosylerythritol lipids (MEL), which are glycolipid BS produced by a yeast Candida antarctrica, exhibit not only excellent interfacial properties but also remarkable differentiation-inducing activities against human leukemia cells. MEL also show a potential anti-agglomeration effect on ice particles in ice slurry used for cold thermal storage. Recently, the cationic liposome bearing MEL has been demonstrated to increase dramatically the efficiency of gene transfection into mammalian cells. These features of BS should broaden its applications in new advanced technologies. The current status of research and development on glycolipid BS, especially their function and potential applications, is discussed.

Surface active properties and antimicrobial activities of mannosylerythritol lipids as biosurfactants produced by Candida antarctica

Abstract The interfacial and antimicrobial properties of two kinds of mannosylerythritol lipids (MEL-A and B), which were produced as biosurfactants from soybean oil by a yeast strain of Candida antarctica T-34, were investigated. MEL showed excellent surface and interfacial tension lowering actions and critical micelle concentrations (CMC). At CMCs, they reduced the surface tension and the interfacial tension against n-tetradecane to about 28 and 2 mN m−1, respectively. These interfacial properties are equivalent to those of other biosurfactants reported and ordinary glycolipid-type synthetic surfactants. MEL also exhibited antimicrobial activity particularly against Gram-positive bacteria, and their minimum inhibitory concentrations were significantly smaller than those of sucrose and sorbitan monoesters of fatty acids.

Microbial conversion of n-alkanes into glycolipid biosurfactants, mannosylerythritol lipids, by Pseudozyma (Candida antarctica)

Abstractn-Alkanes ranging from C12 to C18 were converted into glycolipid biosurfactants, mannosylerythritol lipids (MEL), by resting cells of Pseudozyma (Candida) antarctica T-34. The highest yield (0.87 g g−1 substrate) was obtained from 6% (v/v) of n-octadecane after 7 days reaction. The amount of MEL reached 140 g l−1 by intermittent feeding of the substrate.

Differentiation of human promyelocytic leukemia cell line HL60 by microbial extracellular glycolipids

Microbial extracellular glycolipids, succinoyl trehalose lipid (STL), and mannosylerythritol lipid (MEL) inhibited the growth of a human promyelocytic leukemia cell line, HL60, and induced their morphological changes. The results of specific and nonspecific leukocyte esterase activities showed that STL induced monocytotic differentiation while MEL induced granulocytic differentiation. STL and MEL markedly increased common differentiation-associated characteristics in monocytes and granulocytes, such as nitroblue tetrazolium (NBT) reducing ability, expression of Fc receptors, and phagocytic activities in HL60 cells, respectively. Neither sugar moieties nor fatty acids in the free form, the individual components of STL and MEL, were effective at inducing the differentiation of HL60 cells. The induction of differentiation was not due to surface activities of STL and MEL on the basis of the complete ineffectiveness of the analogues tested. The composition of cell surface glycosphingolipids (GSL) changed such that the GM3/LacCer ratio increased in STL-treated cells, whereas it decreased in MEL-treated cells. HL60 cells treated with STL and MEL exhibited a significant decrease in the activity of the intracellular phospholipid- and Ca2+-dependent protein kinase (protein kinase C). Furthermore, the serine/threonine phosphorylations in intact HL60 cells were clearly inhibited by the presence of GM3 and MEL, but not by LacCer and STL. These results suggest that the differentiation-inducing activity of STL and MEL is not due to a simple detergent-like effect but due to a specific action on the plasma membrane. The inhibitory effect of STL on protein kinase activity was through increasing GM3, but MEL had a direct inhibitory effect.

Improvement of ethanol selectivity of silicalite membrane in pervaporation by silicone rubber coating

In order to improve the pervaporation performance of silicalite membrane, two types of silicone rubber, KE45 and KE108, were coated on the membrane surface. The initial molecular weight of KE108 is high and vulcanizing starts when it comes into contact with moisture in air, whereas the initial molecular weight of KE45 is low and vulcanizing starts when it is mixed with a catalyst. KE108 was found to be more effective than KE45 in enhancing the ethanol selectivity of silicalite membranes. A membrane coated using a 3 wt.% KE108 hexane solution showed separation factor of α=125 with a total flux of 0.14 kg/m2 h.

Improved crown ether-based chiral stationary phase

Abstract An improved crown ether-based chiral stationary phase (CSP) was prepared by dynamic coating of a reversed-phase silica gel with a new chiral crown ether which was designed to have more lipophilicity than the previously used one, while preserving the basic structure responsible for chiral recognition. The CSP showed not only excellent enantioselectivity for amino acids, but also higher stability against organic solvents in the mobile phase. No desorption of the crown ether from the support was observed in a mobile phase containing up to 40% of methanol. An increase in methanol concentration in the mobile phase gave rise to a decrease in the retention of amino acids and an increase in the “apparent” enantioselectivity, i.e., the separation coefficient and the resolution factor. A possible retention mechanism is proposed to explain these behaviours.

Microbial Production of Glyceric Acid, an Organic Acid That Can Be Mass Produced from Glycerol

ABSTRACT Glyceric acid (GA), an unfamiliar biotechnological product, is currently produced as a small by-product of dihydroxyacetone production from glycerol by Gluconobacter oxydans. We developed a method for the efficient biotechnological production of GA as a target compound for new surplus glycerol applications in the biodiesel and oleochemical industries. We investigated the ability of 162 acetic acid bacterial strains to produce GA from glycerol and found that the patterns of productivity and enantiomeric GA compositions obtained from several strains differed significantly. The growth parameters of two different strain types, Gluconobacter frateurii NBRC103465 and Acetobacter tropicalis NBRC16470, were optimized using a jar fermentor. G. frateurii accumulated 136.5 g/liter of GA with a 72% d-GA enantiomeric excess (ee) in the culture broth, whereas A. tropicalis produced 101.8 g/liter of d-GA with a 99% ee. The 136.5 g/liter of glycerate in the culture broth was concentrated to 236.5 g/liter by desalting electrodialysis during the 140-min operating time, and then, from 50 ml of the concentrated solution, 9.35 g of GA calcium salt was obtained by crystallization. Gene disruption analysis using G. oxydans IFO12528 revealed that the membrane-bound alcohol dehydrogenase (mADH)-encoding gene (adhA) is required for GA production, and purified mADH from G. oxydans IFO12528 catalyzed the oxidation of glycerol. These results strongly suggest that mADH is involved in GA production by acetic acid bacteria. We propose that GA is potentially mass producible from glycerol feedstock by a biotechnological process.

Supported liquid membranes for enantioselective transport of amino acid mediated by chiral crown ether - effect of membrane solvent on transport rate and membrane stability

Abstract The effect of membrane solvent on transport efficiency and membrane stability was investigated in a crown ether-mediated enantioselective amino acid transport system. Relatively non-volatile organic liquids were evaluated as membrane solvents in a supported liquid membrane (SLM) using a microporous polymer film as solid support. For the organic liquids giving high fluxes and a high enantioselectivity, the membrane stability was assessed by operating the membranes for periods of up to 90 days. During these experiments, test-runs were repeated daily and thus the experiments were termed ‘repeated-run’ experiments. The best organic liquids were found to be o -nitrophenyl octyl ether (ONPOE) and p -nitrophenyl heptyl ether (PNPHE). SLMs with these organics were stable for more than 50 repeated-runs. The factors controlling the membrane efficiency are discussed on the basis of the physico-chemical properties of the organic liquids, and it is found that the membrane solvent must have both a high dielectric constant and low solubility in water for the SLMs to be highly stable and permeable.

Production of highly concentrated ethanol in a coupled fermentation/pervaporation process using silicalite membranes

The fermentation performance of a coupled fermentation/pervaporation process using silicalite membranes, which are ethanol permselective for an ethanol/water solution, was studied. The process exhibited about a 20% increase in an average glucose consumption rate as compared with that without the pervaporation unit. A strong correlation was observed between the membrane flux and the consumption rate. Ethanol concentrations in the permeates reached a maximum of 85% (v/v).

Preparation of asymmetric polyimide membrane for water/ethanol separation in pervaporation by the phase inversion process

The preparation of polyimide pervaporation membranes for the separation of aqueous ethanol solutions was investigated. Asymmetric membranes were prepared by the phase inversion process from casting solutions composed of aromatic polyimide (PI-2080), N,N′-dimethylformamide (DMF) and 1,4-dioxane. The membranes were annealed at 300°C in a vacuum oven and were measured at 25°C for an aqueous 95 vol% ethanol solution in pervaporation. Membranes made from a casting solution composed of 25 wt% polyimide, 37.5 wt% DMF and 37.5 wt% dioxane, showed a molecular weight cut-off of 600. The membranes which were annealed at 300°C for 3 h exhibited a separation factor α(H2O/EtOH)=900 with a flux of 1.0 kg/m2 h for an aqueous 95 vol% ethanol solution at 60°C.