Photocatalytic CO2 Reduction Using CO2-Binding Enzymes.

Novel concepts to utilize carbon dioxide are required to reach a circular carbon economy and minimize environmental issues. To achieve these goals, photo-, electro-, thermal-, and biocatalysis are key tools to realize this, preferentially in aqueous solutions. Nevertheless, catalytic systems that operate efficiently in water are scarce. Here, we present a general strategy for the identification of enzymes suitable for CO2 reduction based on structural analysis for potential carbon dioxide binding sites and subsequent mutations. We discovered that the phenolic acid decarboxylase from Bacillus subtilis (BsPAD) promotes the aqueous photocatalytic CO2 reduction selectively to carbon monoxide in the presence of a ruthenium photosensitizer and sodium ascorbate. With engineered variants of BsPAD, TONs of up to 978 and selectivities of up to 93 % (favoring the desired CO over H2 generation) were achieved. Mutating the active site region of BsPAD further improved turnover numbers for CO generation. This also revealed that electron transfer is rate-limiting and occurs via multistep tunneling. The generality of this approach was proven by using eight other enzymes, all showing the desired activity underlining that a range of proteins is capable of photocatalytic CO2 reduction.

Cell-free chemoenzymatic cascades with bio-based molecules.

For the valorization of various bio-based feedstocks, the combination of different catalytic systems with biocatalysis in chemoenzymatic cascades has been shown to have high potential. However, the development of such integrated catalytic systems is often limited by catalyst incompatibility. Therefore, incorporating novel catalytic concepts into the chemoenzymatic valorization of bio-based feedstocks is currently of great interest. This article provides an overview of the methods/approaches used to advance the development of chemoenzymatic cascades for the catalytic upgrading of bio-based feedstocks. It specifically focuses on recent developments in the combination of enzymes with organo- and chemocatalysis. Furthermore, current applications and future perspectives of integrating novel catalytic systems such as photo- and electrocatalysis toward new synthetic routes for the utilization of the often highly functionalized bio-based compounds are reviewed.

Spectrophotometric and Fluorimetric High-Throughput Assays for Phenolic Acid Decarboxylase.

Biocatalytic decarboxylation of hydroxycinnamic acids yields phenolic styrenes, which are important precursors for antioxidants, epoxy coatings, adhesives and other polymeric materials. Bacillus subtilis decarboxylase (BsPAD) is a cofactor-independent enzyme that catalyzes the cleavage of carbon dioxide from p-coumaric-, caffeic-, and ferulic acid with high catalytic efficiency. Real-time spectroscopic assays for decarboxylase reactions remove the necessity of extensive sample workup, which is required for HPLC, mass spectrometry, gas chromatography, or NMR methods. This work presents two robust and sensitive assays based on photometry and fluorimetry that allow decarboxylation reactions to be followed with high sensitivity while avoiding product extraction and long analysis times. Optimized assay procedures were used to measure BsPAD activity in cell lysates and to determine the kinetic constants (KM and Vmax ) of the purified enzyme for p-coumaric-, caffeic- and ferulic acid. Substrate inhibition was shown for caffeic acid.

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 %.

Recovery of Hydroxytyrosol from Olive Mill Wastewater Using the Promiscuous Hydrolase/Acyltransferase PestE.

Olive mill wastewater (OMWW) is produced annually during olive oil extraction and contains most of the health-promoting 3-hydroxytyrosol of the olive fruit. To facilitate its recovery, enzymatic transesterification of hydroxytyrosol (HT) was directly performed in an aqueous system in the presence of ethyl acetate, yielding a 3-hydroxytyrosol acetate rich extract. For this, the promiscuous acyltransferase from Pyrobaculum calidifontis VA1 (PestE) was engineered by rational design. The best mutant for the acetylation of hydroxytyrosol (PestE_I208A_L209F_N288A) was immobilized on EziG2 beads, resulting in hydroxytyrosol conversions between 82 and 89 % in one hour, for at least ten reaction cycles in a buffered hydroxytyrosol solution. Due to inhibition by other phenols in OMWW the conversions of hydroxytyrosol from this source were between 51 and 62 %. In a preparative scale reaction, 13.8 mg (57 %) of 3-hydroxytyrosol acetate was extracted from 60 mL OMWW.

α-Dioxygenases (α-DOXs): Promising Biocatalysts for the Environmentally Friendly Production of Aroma Compounds.

Fatty aldehydes (FALs) can be derived from fatty acids (FAs) and related compounds and are frequently used as flavors and fragrances. Although chemical methods have been conventionally used, their selective biotechnological production aiming at more efficient and eco-friendly synthetic routes is in demand. α-Dioxygenases (α-DOXs) are heme-dependent oxidative enzymes biologically involved in the initial step of plant FA α-oxidation during which molecular oxygen is incorporated into the Cα -position of a FA (Cn ) to generate the intermediate FA hydroperoxide, which is subsequently converted into the shortened corresponding FAL (Cn-1 ). α-DOXs are promising biocatalysts for the flavor and fragrance industries, they do not require NAD(P)H as cofactors or redox partner proteins, and they have a broad substrate scope. Here, we highlight recent advances in the biocatalytic utilization of α-DOXs with emphasis on newly discovered cyanobacterial α-DOXs as well as analytical methods to measure α-DOX activity in vitro and in vivo.

Rational Design for Enhanced Acyltransferase Activity in Water Catalyzed by the Pyrobaculum calidifontis VA1 Esterase.

Biocatalytic transesterification is commonly carried out employing lipases in anhydrous organic solvents since hydrolases usually prefer hydrolysis over acyl transfer in bulk water. However, some promiscuous acyltransferases can catalyze acylation in an aqueous solution. In this study, a rational design was performed to enhance the acyltransferase selectivity and substrate scope of the Pyrobaculum calidifontis VA1 esterase (PestE). PestE wild type and variants were applied for the acylation of monoterpene alcohols. The mutant PestE_I208A is selective for (-)-menthyl acetate (E-Value = 55). Highly active acyltransferases were designed, allowing for complete conversion of (-)-citronellol to citronellyl acetate. Additionally, carvacrol was acetylated but with lower conversions. To the best of our knowledge, this is the first example of the biocatalytic acylation of a phenolic alcohol in bulk water. In addition, a high citronellol conversion of 92% was achieved with the more environmentally friendly and inexpensive acyl donor ethyl acetate using PestE_N288F as a catalyst. PestE_N288F exhibits good acyl transfer activity in an aqueous medium and low hydrolysis activity at the same time. Thus, our study demonstrates an alternative synthetic strategy for acylation of compounds without organic solvents.