AsiteDesign: a Semirational Algorithm for an Automated Enzyme Design.

With advances in protein structure predictions, the number of available high-quality structures has increased dramatically. In light of these advances, structure-based enzyme engineering is expected to become increasingly important for optimizing biocatalysts for industrial processes. Here, we present AsiteDesign, a Monte Carlo-based protocol for structure-based engineering of active sites. AsiteDesign provides a framework for introducing new catalytic residues in a given binding pocket to either create a new catalytic activity or alter the existing one. AsiteDesign is implemented using pyRosetta and incorporates enhanced sampling techniques to efficiently explore the search space. The protocol was tested by designing an alternative catalytic triad in the active site of Pseudomonas fluorescens esterase (PFE). The designed variant was experimentally verified to be active, demonstrating that AsiteDesign can find alternative catalytic triads. Additionally, the AsiteDesign protocol was employed to enhance the hydrolysis of a bulky chiral substrate (1-phenyl-2-pentyl acetate) by PFE. The experimental verification of the designed variants demonstrated that F158L/F198A and F125A/F158L mutations increased the hydrolysis of 1-phenyl-2-pentyl acetate from 8.9 to 66.7 and 23.4%, respectively, and reversed the enantioselectivity of the enzyme from (R) to (S)-enantiopreference, with 32 and 55% enantiomeric excess (ee), respectively.

Chemoenzymatic Cascade Reaction for the Valorization of the Lignin Depolymerization Product G-C2-Dioxolane Phenol.

Combining solid acid catalysts with enzyme reactions in aqueous environments is challenging because either very acidic conditions inactivate the enzymes, or the solid acid catalyst is neutralized. In this study, Amberlyst-15 encapsulated in polydimethylsiloxane (Amb-15@PDMS) is used to deprotect the lignin depolymerization product G-C2 dioxolane phenol in a buffered system at pH 6.0. This reaction is directly coupled with the biocatalytic reduction of the released homovanillin to homovanillyl alcohol by recombinant horse liver alcohol dehydrogenase, which is subsequently acylated by the promiscuous acyltransferase/hydrolase PestE_I208A_L209F_N288A in a one-pot system. The deprotection catalyzed with Amb-15@PDMS attains up to 97 % conversion. Overall, this cascade enables conversions of up to 57 %.

Human perception of color differences using computer vision system measurements of raw pork loin.

In the food industry, product color plays an important role in influencing consumer choices. Yet, there remains little research on the human ability to perceive differences in product color; therefore, preference testing is subjective rather than based on quantitative colors. Using a de-centralized computer-aided systematic discrimination testing method, we ascertain consumers' ability to discern between systematically varied colors. As a case study, the colors represent the color variability of fresh pork as measured by a computer vision system. Our results indicate that a total color difference (ΔE) of approximately 1 is discriminable by consumers. Furthermore, we ascertain that a change in color along the b*-axis (yellowness) in CIELAB color space is most discernable, followed by the a*-axis (redness) and then the L*-axis (lightness). As developed, our web-based discrimination testing approach allows for large scale evaluation of human color perception, while these quantitative findings on meat color discrimination are of value for future research on consumer preferences of meat color and beyond.