For the first time, CO2-expanded bio-based liquids were reported as novel and sustainable solvents for biocatalysis. Herein, it was found that by expansion with CO2, 2-methyltetrahydrofuran (MeTHF), and other bio-based liquids, which were not favorable solvents for immobilized Candida antarctica lipase B (Novozym 435) catalyzed transesterification, were tuned into excellent reaction media. Especially, for the kinetic resolution of challenging bulky secondary substrates such as rac-1-adamantylethanol, the lipase displayed very high activity with excellent enantioselectivity (E value > 200) in CO2-expanded MeTHF (MeTHF concentration 10% v/v, 6 MPa), whereas there was almost no activity observed in conventional organic solvents.Download high-res image (307KB)Download full-size image

Secondary alcohols having bulky substituents on both sides of the chiral center are often poor substrates for most lipases. Here we reported that substrate scopes of two of the most used lipases, Candida antarctica lipase B and Burkholderia cepacia lipase, were found to be expanded toward more bulky secondary alcohols such as 1-phenyl-1-dodecanol and 2-methyl-1-phenyl-1-propanol by simply using them in liquid carbon dioxide as a solvent. The effects of solvents, reaction pressure, and pre-treatment of the enzyme with liquid CO2 on this acceleration phenomenon were also studied.

The transesterification of alcohols catalyzed by immobilized Candida antarctica lipase B (Novozym 435®) was found to be effectively enhanced using a liquid CO2 medium when it was compared with that using organic solvents. The large-scale kinetic-resolution of secondary alcohol by the immobilized lipase was also successfully performed with a continuous packed-column reactor that stably afforded corresponding enantiopure products. Herein, liquid CO2 was proved for the first time to be superior to conventional organic solvents for biotransformation.

The gene encoding acetophenone reductase (APRD), a useful biocatalyst for producing optically pure alcohols, was cloned from the cDNA of Geotrichum candidum NBRC 4597. The gene contained an open reading frame that consisted of 1,029 nucleotides corresponding to 342 amino acid residues. The subunit molecular weight was calculated to be 36.7 kDa. The predicted amino acid sequence did not have significant similarity to those of the acetophenone reductase reported previously. The gene was inserted into the pET-21b(+) expression vector and expressed in Escherichia coli Rosetta™(DE3)pLysS by induction with 1 mM of isopropyl-β-d-thiogalactopyranoside. E. coli cell-free extract gave 21.9 U/mg APRD activity, which was 81 times that of the G. candidum cell-free extract. The enzyme was purified with a HisTrap FF crude column. The enzyme exhibited the highest activity at 60 °C, and optimum reducing and oxidizing activity were observed in a pH range around 7.0–8.0 and 8.5, respectively. The enzyme was most stable at 60 °C and pH 6.5–7.5. The Vmax and the apparent Km value of the reductase were 67.6 μmol/min per milligram of protein and 0.146 mM for acetophenone, respectively. From 4 % (v/v) 4-phenyl-2-butanone, (S)-4-phenyl-2-butanol was obtained with a yield >80 % and an enantiomeric excess >99 % in a 20 h reaction recycling NADH with 15 % (v/v) 2-propanol.

Latest advances for asymmetric synthesis through reduction and oxidation including deracemization by biocatalysts are reviewed. Newly developed methodologies as well as practical applications are covered.Recent developments in biocatalytic approach to reduction, oxidation, dynamic kinetic resolution, and deracemization are reviewed.

The enantioselectivity for the reduction of ketones by Geotrichum candidum NBRC 5767 was improved upon immobilization of the whole cell onto an ion exchange resin with polyallylamine. Furthermore, immobilization of the cell enhanced the stability of the enzyme and enabled a continuous-flow reaction under normal aqueous conditions. The biocatalyst was also applied to the reaction in supercritical carbon dioxide.

Kinetic resolution of racemic P-chiral hydroxymethanephosphinates via their lipase-promoted acetylation in supercritical carbon dioxide as the reaction medium was for the first time investigated under various conditions. The reactivity and selectivity could be controlled by changing the pressure.Methyl hydroxymethanephenylphosphinateC8H11O3PEe = 4%[α]D = −1.1 (c 1.1, CHCl3)Source of chirality: enzymatic kinetic resolution in scCO2Absolute configuration: RMethyl acetoxymethanephenylphosphinateC10H3O4PEe = 7%[α]D = +3.4 (c 2.2, CHCl3)Source of chirality: enzymatic kinetic resolution in scCO2Absolute configuration: SEthyl hydroxymethanephenylphosphinateC9H13O3PEe = 88%[α]D = −13.4 (c 1.9, CHCl3)Source of chirality: enzymatic kinetic resolution in scCO2Absolute configuration: REthyl acetoxymethanephenylphosphinateC11H15O4PEe = 6%[α]D = +2.9 (c 2.4, CHCl3)Source of chirality: enzymatic kinetic resolution in scCO2Absolute configuration: Si-Propyl hydroxymethanephenylphosphinateC10H15O3PEe = 28%[α]D = −6.2 (c 1.2, CHCl3)Source of chirality: enzymatic kinetic resolution in scCO2Absolute configuration: Ri-Propyl acetoxymethanephenylphosphinateC12H17O4PEe = 27%[α]D = +10.4 (c 2.1, CHCl3)Source of chirality: enzymatic kinetic resolution in scCO2Absolute configuration: S

The use of hydrolytic enzymes in supercritical carbon dioxide (scCO2), an environmentally friendly solvent with many uses, is an attractive approach to asymmetric synthesis: several examples are reviewed here.The use of hydrolytic enzymes in scCO2 is an attractive approach to asymmetric synthesis, and several examples are reviewed here.

The latest advances in biocatalysis using supercritical carbon dioxide (scCO2) are reviewed. Stability and stabilization methodologies of enzymes in scCO2 as well as reactions for organic synthesis are described. Especially, varieties of examples for lipase-catalyzed synthesis of chiral compounds using scCO2 are given. Furthermore, asymmetric reduction by alcohol dehydrogenase in scCO2 and carboxylation by decarboxylase in scCO2 are also introduced.