Succinic acid (SA), a dicarboxylic acid of industrial importance, can be efficiently produced by metabolically engineered Mannheimia succiniciproducens. Malate dehydrogenase (MDH) is one of the key enzymes for SA production, but has not been well characterized. Here we report biochemical and structural analyses of various MDHs and development of hyper-SA producing M. succiniciproducens by introducing the best MDH. Corynebacterium glutamicum MDH (CgMDH) shows the highest specific activity and least substrate inhibition, whereas M. succiniciproducens MDH (MsMDH) shows low specific activity at physiological pH and strong uncompetitive inhibition toward oxaloacetate (ki of 67.4 and 588.9 μM for MsMDH and CgMDH, respectively). Structural comparison of the two MDHs reveals a key residue influencing the specific activity and susceptibility to substrate inhibition. A high-inoculum fed-batch fermentation of the final strain expressing cgmdh produces 134.25 g L −1 of SA with the maximum productivity of 21.3 g L −1 h −1 , demonstrating the importance of enzyme optimization in strain development.

Two types of photoinitiators were synthesized: (1) a ␣,-telechelic oligomeric photoinitiator, by the reaction of poly(propylene glycol) diglycidylether (PPGDGE) and 2-hydroxy-2-methyl-1-phenyl-propan-1-one (Darocur 1173), and (2) a polymeric photoinitiator, by copolymerization of a monomer that had a liquid crystalline property, 4-[-(2-methylpropenoyloxy)decanoxy]-4Ј-cyanobiphenyl, with a vinyl monomer that had a photosensitive group. For comparison, low-molecular-weight (low-MW) photoinitiator (Darocur 1173) also was used. Attention was directed to the structural effect of the photoinitiators on the electro-optical properties of polymer-dispersed liquid crystal (PDLC) film in which the LC phase occupied a major volume (80 wt % of the composite film). For the preparation of PDLC films by the polymerization-induced phase separation method, the optimum UV-curing temperature was observed at 50°C, a temperature slightly higher than the cloud temperature (T cloud ) of the low-MW LC/matrix-forming material mixture. It was found that the electro-optical performance of the PDLC cell fabricated with the oligomeric or polymeric photoinitiator was better than that of the PDLC cell made with a low-MW photoinitiator (Darocur 1173), exhibiting lower driving voltage (V 90 ) and higher contrast ratio under identical formulation conditions. Oligomeric photoinitiators allowed premature phase separation between the LC and matrix phases, resulting in relatively pure LC-rich phases. For the polymeric photoinitiator, incorporation of mesogenic moieties into the photoinitiator resulted in not only a well-defined LC/matrix morphology but also in low driving voltage (V 90 ) because of reduced friction at the LC/matrix interfaces.

The internal structures of thin films, particularly interfaces between different materials, are critical to system properties and performance across many disciplines, but characterization of buried interface topography is often unfeasible. In this work, we demonstrate that Grazing Resonant Soft X-ray Scattering (GRSoXS), a technique using diffusely scattered soft X-rays in grazing incidence geometry, can reveal the statistical topography of buried thin film interfaces. By controlling and predicting the X-ray electric field intensity throughout the depth of the film and simultaneously the scattering contrast between materials, we are able to unambiguously identify the microstructure at different interfaces of a model polymer bilayer system. We additionally demonstrate the use of GRSoXS to selectively measure the topography of the surface and buried polymer-polymer interface in an organic thin film transistor, revealing different microstructure and markedly differing evolution upon annealing. In such systems, where only indirect control of interface topography is possible, accurate measurement of the structure of interfaces for feedback is critically important. While we demonstrate the method here using organic materials, we also show that the technique is readily extendable to any thin film system with elemental or chemical contrasts exploitable at absorption edges.

d-Threonine aldolase (DTA) is a useful biocatalyst that reversibly converts glycine and aldehyde to β-hydroxy-α-d-amino acid. However, low activity and poor diastereoselectivity limit its applications. Here we report DTA from Filomicrobium marinum (FmDTA) that shows much higher activity and Cβ-stereoselectivity in d-threonine production compared with those of other known DTAs. We determine the FmDTA structure at a 2.2 Å resolution and propose a DTA catalytic mechanism with a kernel of the Lys49 inner proton sink and metal ion in the aldol reaction cycle. The enzyme is rationally engineered to have high Cβ-stereoselectivity based on spatial constraint at the anti-specific aldehyde position in the mechanism, and the rational strategy is further applied to other DTAs for syn-production. The final FmDTAG179A/S312A variant exhibits a near-perfect 99.5% de value for d-threonine and maintains the de value above 93% even under kinetically unfavorable conditions. This study demonstrates how a detailed understanding of the reaction mechanism can be used for rational protein engineering.

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