Kazunori Nakashima

Water-in-ionic liquid microemulsions as a new medium for enzymatic reactions

The insolubility of enzymes in most ionic liquids has been overcome by the formation of aqueous microemulsion droplets in a hydrophobic ionic liquid stabilized by a layer of anionic surfactant sodium bis(2-ethyl-1-hexyl) sulfosuccinate (AOT) in the presence of 1-hexanol as a cosurfactant and the catalytic activity of one of the enzymes studied (lipase PS) became higher than in microemulsions of AOT in isooctane.

Versatile Supramolecular Gelators That Can Harden Water, Organic Solvents and Ionic Liquids

We developed novel supramolecular gelators with simple molecular structures that could harden a broad range of solvents: aqueous solutions of a wide pH range, organic solvents, edible oil, biodiesel, and ionic liquids at gelation concentrations of 0.1-2 wt %. The supramolecular gelators were composed of a long hydrophobic tail, amino acids and gluconic acid, which were prepared by liquid-phase synthesis. Among seven types of the gelators synthesized, the gelators containing L-Val, L-Leu, and L-Ile exhibited high gelation ability to various solvents. These gelators were soluble in aqueous and organic solvents, and also in ionic liquids at high temperature. The gelation of these solvents was thermally reversible. The microscopic observations (TEM, SEM, and CLSM) and small-angle X-ray scattering (SAXS) measurements suggested that the gelator molecules self-assembled to form entangled nanofibers in a large variety of solvents, resulting in the gelation of these solvents. Molecular mechanics and density functional theory (DFT) calculations indicated the possible molecular packing of the gelator in the nanofibers. Interestingly, the gelation of an ionic liquid by our gelator did not affect the ionic conductivity of the ionic liquid, which would provide an advantage to electrochemical applications.

Short time ionic liquids pretreatment on lignocellulosic biomass to enhance enzymatic saccharification.

The potential of 1-buthyl-3-methylpyridinium chloride, [Bmpy][Cl], as a pretreatment solvent for lignocellulosic biomasses, Bagasse and Eucalyptus, was investigated. The yields of regenerated biomasses ranged between 35% and 96%, and varied according to the pretreatment time, type of ionic liquid (IL) and biomass. The pretreatment of the biomass with [Bmpy][Cl] resulted in up to 8-fold increase in the cellulose conversion when compared with the untreated biomass. For a short pretreatment period (i.e., 10 min), [Bmpy][Cl] showed better performance than 1-ethyl-3-methylimidazolium acetate ([Emim][OAc]) with respect to the initial enzymatic saccharification rates. The increase in the reaction rates with [Emim][OAc] treatment was because of a reduction in the cellulose crystallinity. In contrast, a decrease in the crystallinity index was not clearly observed for the biomass pretreated with [Bmpy][Cl], and the enhancement of the enzymatic saccharification rates using this IL is presumably due to a reduction in the degree of polymerization of cellulose in the biomass.

Development of an Aspergillus oryzae whole-cell biocatalyst coexpressing triglyceride and partial glyceride lipases for biodiesel production

An Aspergillus oryzae whole-cell biocatalyst which coexpresses Fusarium heterosporum lipase (FHL) and mono- and di-acylglycerol lipase B (mdlB) in the same cell has been developed to improve biodiesel production. By screening a number of transformants, the best strain was obtained when FHL gene was integrated into A. oryzae chromosome using sC selection marker while mdlB was integrated using niaD selection marker. The reaction system using the lipase-coexpressing whole-cells was found to be superior in biodiesel production to others such as lipase-mixing and two-step reactions, affording the highest reaction rate and the highest ME content (98%). Moreover, an ME content of more than 90% was maintained during 10 repeated batch cycles. The whole-cell biocatalyst developed in this work would be promising biocatalysts for efficient biodiesel production.

Homogeneous enzymatic reactions in ionic liquids with poly(ethylene glycol)-modified subtilisin

Subtilisin Carlsberg was covalently modified with comb-shaped poly(ethylene glycol) (PM13). PM13-modified subtilisin (PM13-Sub) was readily solubilized in three different ionic liquids (ILs), i.e., [Emim][Tf2N], [C2OC1mim][Tf2N] and [C2OHmim][Tf2N]. Analysis of homogeneous enzymatic reactions in the ILs revealed that PM13-Sub exhibited excellent catalytic performance while the native enzyme suspended in ILs showed no activity. Hydrophobicity of ILs slightly affected enzyme activity, and the relatively hydrophobic IL [Emim][Tf2N] was the preferred medium for enzymatic reactions, similar to enzymatic reactions in conventional organic solvents. Enzyme activity was much higher in [Emim][Tf2N] than in conventional organic solvents, and excellent activity was associated with unique properties of ILs such as hydrophobicity and high polarity. Furthermore, PM13-Sub showed good stability in [Emim][Tf2N], and maintained 80% of its initial activity after 60 h.

Production of biodiesel from plant oil hydrolysates using an Aspergillus oryzae whole-cell biocatalyst highly expressing Candida antarctica lipase B

For enzymatic biodiesel production from plant oil hydrolysates, an Aspergillus oryzae whole-cell biocatalyst that expresses Candida antarctica lipase B (r-CALB) with high esterification activity was developed. Each of soybean and palm oils was hydrolyzed using Candida rugosa lipase, and the resultant hydrolysates were subjected to esterification where immobilized r-CALB was used as a catalyst. In esterification, r-CALB afforded a methyl ester content of more than 90% after 6 h with the addition of 1.5 M equivalents of methanol. Favorably, stepwise additions of methanol and a little water were unnecessary for maintaining the lipase stability of r-CALB during esterification. During long-term esterification in a rotator, r-CALB can be recycled for 20 cycles without a significant loss of lipase activity, resulting in a methyl ester content of more than 90% even after the 20th batch. Therefore, the presented reaction system using r-CALB shows promise for biodiesel production from plant oil hydrolysates.

Activation of lipase in ionic liquids by modification with comb-shaped poly(ethylene glycol)

Abstract Outstanding activation of an enzyme in ionic liquids (ILs) has been demonstrated by covalent modification with comb-shaped poly(ethylene glycol) (PEG) (PM13). Candida rugosa lipase modified with PM13 (PM13–CRL) was readily solubilized in all the ILs tested ([Emim][Tf2N], [C2OC1mim][Tf2N] and [C2OHmim][Tf2N]) containing 0.5% (v/v) of water, whereas native lipase did not dissolve in any of the ILs. The results for transesterification of 2-phenyl-1-propanol with vinyl acetate using lipase in ILs revealed that the PM13–CRL conjugate exhibits a high catalytic activity while suspended native lipase shows little activity. The hydrophobicity of ILs somewhat affected the enzyme activity and a more hydrophobic IL such as [Emim][Tf2N] was preferable for the lipase reaction, as was also observed in enzymatic reaction in conventional organic solvents. The enzyme activities in ILs were much higher than those in organic solvents, the excellent activity being associated with unique properties such as the hydrophobicity and the high polarity of ILs. Furthermore, the PM13-–CRL conjugate exhibited a high storage stability in [Emim][Tf2N].

Comb-shaped poly(ethylene glycol)-modified subtilisin Carlsberg is soluble and highly active in ionic liquids.

Subtilisin Carlsberg conjugated with comb-shaped poly(ethylene glycol) was solubilized in common ionic liquids without adding water, and exhibited higher transesterification activity in an ionic liquid, [Emim][Tf2N], than in organic solvents commonly used for enzymatic biotransformation.

Simultaneous production of high quality biodiesel and glycerin from Jatropha oil using ion-exchange resins as catalysts and adsorbent.

The simultaneous production of high quality biodiesel and glycerin was realized by a bench-scale process using expanded-bed reactors packed with cation- and anion-exchange resins. The mixed-solution of crude Jatropha oil and methanol at a stoichiometric molar ratio was supplied to the process. The free fatty acid as well as triglyceride was completely converted to biodiesel. All by-products were adsorbed on the resin and the effluent from the process was free from them. The effluent fully met the international biodiesel standard specifications without any downstream purification processes except for removing methanol. The glycerin adsorbed on the resin was completely recovered as a transparent methanol solution during regeneration of the resin.

Pretreatment combining ultrasound and sodium percarbonate under mild conditions for efficient degradation of corn stover.

Ultrasound (US) can be used to disrupt microcrystalline cellulose to give nanofibers via ultrasonic cavitation. Sodium percarbonate (SP), consisting of sodium carbonate and hydrogen peroxide, generates highly reactive radicals, which cause oxidative delignification. Here, we describe a novel pretreatment technique using a combination of US and SP (US-SP) for the efficient saccharification of cellulose and hemicellulose in lignocellulosic corn stover. Although US-SP pretreatment was conducted under mild condition (i.e., at room temperature and atmospheric pressure), the pretreatment greatly increased lignin removal and cellulose digestibility. We also determined the optimum US-SP treatment conditions, such as ultrasonic power output, pretreatment time, pretreatment temperature, and SP concentration for an efficient cellulose saccharification. Moreover, xylose could be effectively recovered from US-SP pretreated biomass without the formation of microbial inhibitor furfural.