Modifying Surface Charges of a Thermophilic Laccase Toward Improving Activity and Stability in Ionic Liquid.

The multicopper oxidase enzyme laccase holds great potential to be used for biological lignin valorization alongside a biocompatible ionic liquid (IL). However, the IL concentrations required for biomass pretreatment severely inhibit laccase activity. Due to their ability to function in extreme conditions, many thermophilic enzymes have found use in industrial applications. The thermophilic fungal laccase from Myceliophthora thermophila was found to retain high levels of activity in the IL [C2C1Im][EtSO4], making it a desirable biocatalyst to be used for lignin valorization. In contrast to [C2C1Im][EtSO4], the biocompatibility of [C2C1Im][OAC] with the laccase was markedly lower. Severe inhibition of laccase activity was observed in 15% [C2C1Im][OAc]. In this study, the enzyme surface charges were modified via acetylation, succinylation, cationization, or neutralization. However, these modifications did not show significant improvement in laccase activity or stability in [C2C1Im][OAc]. Docking simulations show that the IL docks close to the T1 catalytic copper, likely interfering with substrate binding. Although additional docking locations for [OAc]- are observed after making enzyme modifications, it does not appear that these locations play a role in the inhibition of enzyme activity. The results of this study could guide future enzyme engineering efforts by showing that the inhibition mechanism of [C2C1Im][OAc] toward M. thermophila laccase is likely not dependent upon the IL interacting with the enzyme surface.

Biocatalysis in ionic liquids for lignin valorization: Opportunities and recent developments.

Lignin holds tremendous potential as a renewable feedstock for upgrading to a number of high-value chemicals and products that are derived from the petroleum industry at present. Since lignin makes up a significant fraction of lignocellulosic biomass, co-utilization of lignin in addition to cellulose and hemicelluloses is vital to the economic viability of cellulosic biorefineries. The recalcitrant nature of lignin, originated from the molecule's compositional and structural heterogeneity, however, poses great challenges toward effective and selective lignin depolymerization and valorization. Ionic liquid (IL) is a powerful solvent that has demonstrated high efficiency in fractionating lignocellulosic biomass into sugar streams and a lignin stream of reduced molecular weight. Compared to thermochemical methods, biological lignin deconstruction takes place at mild temperature and pressure while product selectivity can be potentially improved via the specificity of biocatalysts (lignin degrading enzymes, LDEs). This review focuses on a lignin valorization strategy by harnessing the biomass fractionating capabilities of ILs and the substrate and product selectivity of LDEs. Recent advances in elucidating enzyme-IL interactions as well as strategies for improving enzyme activity in IL are discussed, with specific emphases on biocompatible ILs, thermostable and IL-tolerant enzymes, enzyme immobilization, and surface charge engineering. Also reviewed is the protein engineering toolsets (directed evolution and rational design) to improve the biocatalysts' activity, stability and product selectivity in IL systems. The alliance between IL and LDEs offers a great opportunity for developing a biocatalytic route for lignin valorization.

Stereoselective Synthesis of Diverse Lactones through a Cascade Reaction of Rhodium Carbenoids with Ketoacids.

A convergent cascade approach for the stereoselective synthesis of diverse lactones is described. The Rh2(TFA)4-catalyzed cascade reaction proceeds via a carboxylic acid O-H insertion/aldol cyclization with high chemo-, regio-, and diastereoselectivity. The cascade reaction provides quick access to highly functionalized γ-butyro- and δ-valerolactones from readily accessible ketoacid and diazo synthons. To demonstrate the utility of this approach, a thermally induced oxy-Cope ring-expansion strategy has been incorporated in the cascade sequence to access medium-sized lactones, which can undergo a serendipitous rearrangement to form spiro-lactones through an intramolecular aldol/trans-lactonization sequence. The reaction has proven to be general, with a range of ketoacids and diazo carbonyls to provide functionalized lactones of varying ring sizes.

A Convergent Approach to Diverse Spiroethers through Stereoselective Trapping of Rhodium Carbenoids with Gold-Activated Alkynols.

A convergent approach for the stereoselective synthesis of diverse spiroethers is described. The reaction involves stereoselective trapping of diazo-derived rhodium carbenoids with gold-activated alkynols for the installation of spiro cores. The reaction has proven general with a range of readily accessible homopropargylic alcohols and diazo carbonyls to provide functionalized spiroether cores of bioactive scaffolds such as spirobarbiturates, spirooxindoles, and pseurotin natural products.

Decline of tactile acuity in aging: a study of body site, blood flow, and lifetime habits of smoking and physical activity.

Tactile acuity of 60 older subjects (> or = 65 years) and 19 younger subjects (18-28 years) was assessed by two-point gap thresholds at the upper and lower surfaces of the forefinger, at the upper and lower surfaces of the feet, and at the volar surface of the forearm. The older subjects were assigned to one of four groups of 15 subjects each, depending on reported lifetime habits of physical activity and smoking: (1) active smokers, (2) active nonsmokers, (3) inactive smokers, and (4) inactive nonsmokers. Peripheral blood flow was assessed at the forefinger, foot, and forearm by means of laser-Doppler imaging and skin temperature recordings, under resting conditions and during and after a 5-min exposure to mild cooling (28 degrees C). Consistent with previous studies, tactile acuity thresholds in the foot and finger averaged about 80% higher in the older subjects than in the younger subjects, but only about 22% higher in the forearm. Although the upper surface of the fingertip was more sensitive than the lower surface in both younger and older subjects, the age-related decline in tactile acuity was nearly identical on both sides of the finger and foot. The latter finding refutes the hypothesis that the larger effect of aging in the extremities results from greater physical wear and tear on the contact surfaces of the hands and feet. Self-reported lifetime histories of physical activity and smoking were not significantly associated with measures of cutaneous blood flow or tactile thresholds. Possible reasons for this lack of association are discussed, including the inherent limitations of testing only healthy older subjects, and the concept of "successful aging".