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Low yields commonly obtained during enzymatic conversion of CO2 to methanol are attributed to low CO2 solubility in water. In this study, four selected ionic liquids with high CO2 solubility were separately added to the multi-enzyme reaction mixture and the yields were compared to the pure aqueous system (control). In an aqueous 20% [CH][Glu] system, yield increased ca. 3.5-fold compared to the control (ca. 5-fold if NADH regeneration was incorporated). Molecular dynamics simulation revealed that CO2 remains for longer in a productive conformation in the enzyme in the presence of [CH][Glu], which explains the marked increase of yield that was also confirmed by isothermal titration calorimetry – lower energy (ΔG) binding of CO2 to FDH. The results suggest that the accessibility of CO2 to the enzyme active site depends on the absence/presence and nature of the ionic liquid, and that the enzyme conformation determines CO2 retention and hence final conversion. [ABSTRACT FROM AUTHOR]

Human milk oligosaccharides (HMOs) constitute a unique family of bioactive lactose-based molecules present in human breast milk. HMOs are of major importance for infant health and development but also virtually absent from bovine milk used for infant formula. Among the HMOs, the fucosylated species are the most abundant. Transfucosylation catalysed by retaining α- l -fucosidases is a new route for manufacturing biomimetic HMOs. Seven α- l -fucosidases from glycosyl hydrolase family 29 were expressed, characterized in terms of substrate specificity and thermal stability, and shown to be able to catalyse transfucosylation. The α- l -1,3/4-fucosidase CpAfc2 from Clostridium perfringens efficiently catalysed the formation of the more complex human milk oligosaccharide structure lacto- N- fucopentaose II (LNFP II) using 3-fucosyllactose as fucosyl donor and lacto- N -tetraose as acceptor with a 39% yield. α- l -Fucosidases FgFCO1 from Fusarium graminearum and Mfuc5 from a soil metagenome were able to catalyse transfucosylation of lactose using citrus xyloglucan as fucosyl donor. FgFCO1 catalysed formation of 2′-fucosyllactose, whereas Mfuc5 catalysis mainly produced an unidentified, non-HMO fucosyllactose, reaching molar yields based on the donor substrate of 14% and 18%, respectively. [ABSTRACT FROM AUTHOR]

Nature's smartness and efficient assembling cascade type reactions inspired biologists and chemists all around the world. Tremendous effort has been directed towards the understanding and mimicking of such networks. In recent years considerable progress has been made in developing multistep one-pot reactions combining either advantage of chemo-, regio-, and stereoselectivity of biocatalysts or promiscuity and productivity of chemocatalysts. In this context several concepts, inspired by different disciplines (biocatalysis, metabolic engineering, synthetic chemistry, and material science), have been evolved. This review will focus on major contributions in the field of cascade reactions over the last three years. [ABSTRACT FROM AUTHOR]

The chambers of Trivial Pursuit: Locking immobilized enzymes or encapsulated whole cells into a compartment that is connected to the stirrer enables efficient reactions and facilitates the reuse of biocatalysts by using this SpinChem system. R‐ATA=(R)‐amine transaminase. [ABSTRACT FROM AUTHOR]

β-N-acetylhexosaminidases (EC 3.2.1.52) are retaining hydrolases of glycoside hydrolase family 20 (GH20). These enzymes catalyze hydrolysis of terminal, non-reducing N-acetylhexosamine residues, notably N-acetylglucosamine or N-acetylgalactosamine, in N-acetyl-β-D-hexosaminides. In nature, bacterial β-N-acetylhexosaminidases are mainly involved in cell wall peptidoglycan synthesis, analogously, fungal β-N-acetylhexosaminidases act on cell wall chitin. The enzymes work via a distinct substrate-assisted mechanism that utilizes the 2-acetamido group as nucleophile. Curiously, the β-N-acetylhexosaminidases possess an inherent trans-glycosylation ability which is potentially useful for biocatalytic synthesis of functional carbohydrates, including biomimetic synthesis of human milk oligosaccharides and other glycan-functionalized compounds. In this review, we summarize the reaction engineering approaches (donor substrate activation, additives, and reaction conditions) that have proven useful for enhancing trans-glycosylation activity of GH20 β-N-acetylhexosaminidases. We provide comprehensive overviews of reported synthesis reactions with GH20 enzymes, including tables that list the specific enzyme used, donor and acceptor substrates, reaction conditions, and details of the products and yields obtained. We also describe the active site traits and mutations that appear to favor trans-glycosylation activity of GH20 β-N-acetylhexosaminidases. Finally, we discuss novel protein engineering strategies and suggest potential "hotspots" for mutations to promote trans-glycosylation activity in GH20 for efficient synthesis of specific functional carbohydrates and other glyco-engineered products. [ABSTRACT FROM AUTHOR]

The colonization of land by plants generated opportunities for the rise of new heterotrophic life forms, including humankind. A unique event underpinned this massive change to earth ecosystems—the advent of eukaryotic green algae. Today, an abundant marine green algal group, the prasinophytes, alongside prasinodermophytes and nonmarine chlorophyte algae, is facilitating insights into plant developments. Genome-level data allow identification of conserved proteins and protein families with extensive modifications, losses, or gains and expansion patterns that connect to niche specialization and diversification. Here, we contextualize attributes according to Viridiplantae evolutionary relationships, starting with orthologous protein families, and then focusing on key elements with marked differentiation, resulting in patchy distributions across green algae and plants. We place attention on peptidoglycan biosynthesis, important for plastid division and walls; phytochrome photosensors that are master regulators in plants; and carbohydrate-active enzymes, essential to all manner of carbohydratebiotransformations. Together with advances in algal model systems, these areas are ripe for discovering molecular roles and innovations within and across plant and algal lineages. [ABSTRACT FROM AUTHOR]

Fungal laccases (EC 1.10.3.2) are multi-copper oxidases that oxidize a wide variety of substrates. Despite extensive studies, the molecular basis for their diverse activity is unclear. Notably, there is no current way to rationally predict the activity of a laccase toward a given substrate. Such knowledge would greatly facilitate the rational design of new laccases for technological purposes. We report a study of three datasets of experimental Km values and activities for Trametes versicolor and Cerrena unicolor laccase, using a range of protein modeling techniques. We identify diverse binding modes of the various substrates and confirm an important role of Asp-206 and His-458 (T. versicolor laccase numbering) in guiding substrate recognition. Importantly, we demonstrate that experimental Km values correlate with binding affinities computed by MMGBSA. This confirms the common assumption that the protein-substrate affinity is a major contributor to observed Km. From quantitative structure-activity relations (QSAR) we identify physicochemical properties that correlate with observed Km and activities. In particular, the ionization potential, shape, and binding affinity of the substrate largely determine the enzyme’s Km for the particular substrate. Our results suggest that Km is not just a binding constant but also contains features of the enzymatic activity. In addition, we identify QSAR models with only a few descriptors showing that phenolic substrates employ optimal hydrophobic packing to reach the T1 site, but then require additional electronic properties to engage in the subsequent electron transfer. Our results advance our ability to model laccase activity and lend promise to future rational optimization of laccases toward phenolic substrates. [ABSTRACT FROM AUTHOR]

Review: 17 refs. [ABSTRACT FROM AUTHOR]

Type A chitinases (EC 3.2.1.14), GH family 18, attack chitin ((1 → 4)-2-acetamido-2-deoxy-β- d-glucan) and chito-oligosaccharides from the reducing end to catalyze release of chitobiose ( N, N′-diacetylchitobiose) via hydrolytic cleavage of N-acetyl-β- d-glucosaminide (1 → 4)-β-linkages and are thus ' exo-chitobiose hydrolases.' In this study, the chitinase type A from Serratia marcescens (SmaChiA) was used as a template for identifying two novel exo-chitobiose hydrolase type A enzymes, FbalChi18A and MvarChi18A, originating from the marine organisms Ferrimonas balearica and Microbulbifer variabilis, respectively. Both FbalChi18A and MvarChi18A were recombinantly expressed in Escherichia coli and were confirmed to exert exo-chitobiose hydrolase activity on chito-oligosaccharides, but differed in temperature and pH activity response profiles. Amino acid sequence comparison of the catalytic β/α barrel domain of each of the new enzymes showed individual differences, but ~69% identity of each to that of SmaChiA and highly conserved active site residues. Superposition of a model substrate on 3D structural models of the catalytic domain of the enzymes corroborated exo-chitobiose hydrolase type A activity for FbalChi18A and MvarChi18A, i.e., substrate attack from the reducing end. A main feature of both of the new enzymes was the presence of C-terminal 5/12 type carbohydrate-binding modules (SmaChiA has no C-terminal carbohydrate binding module). These new enzymes may be useful tools for utilization of chitin as an N-acetylglucosamine donor substrate via chitobiose. [ABSTRACT FROM AUTHOR]

Poly-ε-caprolactone (PCL) is chemically produced on an industrial scale in spite of the need for hazardous peracetic acid as an oxidation reagent. Although Baeyer-Villiger monooxygenases (BVMO) in principle enable the enzymatic synthesis of ε-caprolactone (ε-CL) directly from cyclohexanone with molecular oxygen, current systems suffer from low productivity and are subject to substrate and product inhibition. The major limitations for such a biocatalytic route to produce this bulk chemical were overcome by combining an alcohol dehydrogenase with a BVMO to enable the efficient oxidation of cyclohexanol to ε-CL. Key to success was a subsequent direct ring-opening oligomerization of in situ formed ε-CL in the aqueous phase by using lipase A from Candida antarctica, thus efficiently solving the product inhibition problem and leading to the formation of oligo-ε-CL at more than 20 g L−1 when starting from 200 m M cyclohexanol. This oligomer is easily chemically polymerized to PCL. [ABSTRACT FROM AUTHOR]

In industriellem Maßstab wird Poly-ε-caprolacton (PCL) gegenwärtig nur chemisch produziert, wobei mit Peressigsäure ein gefährliches Reagens als Oxidationsmittel genutzt wird. Baeyer-Villiger-Monooxygenasen (BVMOs) ermöglichen im Prinzip die enzymatische Synthese von ε-Caprolacton (ε-CL) direkt ausgehend von Cyclohexanon mit molekularem Sauerstoff, doch gegenwärtige Systeme leiden unter niedriger Produktivität sowie Substrat- und Produktinhibierung. Wir überwanden wesentliche Limitationen eines solchen biokatalytischen Wegs durch die Kombination einer Alkoholdehydrogenase mit einer BVMO für die effiziente Oxidation von Cyclohexanol zu ε-CL. Entscheidend war die direkte Ringöffnungs-Oligomerisierung des in situ gebildeten ε-CL in wässriger Phase unter Nutzung der Lipase A aus Candida antarctica. So wurde das Problem der Produktinhibierung gelöst, und Oligo-ε-CL wurde mit >20 g L−1 ausgehend von 200 m M Cyclohexanol erhalten. Dieses Oligomer konnte chemisch leicht zu PCL polymerisiert werden. [ABSTRACT FROM AUTHOR]

Joined forces: Alcohol dehydrogenase, enoate reductase, and Baeyer–Villiger monooxygenase are combined in a cascade reaction by coexpression in E. coli to have a recombinant whole‐cell biocatalyst. Such an artificial metabolic “mini”‐pathway provides access to functionalized chiral compounds in high yields and optical purities as exemplified for kinetic resolutions, desymmetrizations, and regiodivergent biotransformations. [ABSTRACT FROM AUTHOR]

Fusarium graminearum produces an α-L-fucosidase, FgFCO1, which so far appears to be the only known fungal GH29 α-L-fucosidase that catalyzes the release of fucose from fucosylated xyloglucan. In our quest to synthesize bioactive glycans by enzymatic catalysis, we observed that FgFCO1 is able to catalyze a transglycosylation reaction involving transfer of fucose from citrus peel xyloglucan to lactose to produce 2'-fucosyllactose, an important human milk oligosaccharide. In addition to achieving maximal yields, control of the regioselectivity is an important issue in exploiting such a transglycosylation ability successfully for glycan synthesis. In the present study, we aimed to improve the transglycosylation efficiency of FgFCO1 through protein engineering by transferring successful mutations from other GH29 α-L-fucosidases. We investigated several such mutation transfers by structural alignment, and report that transfer of the mutation F34I from BiAfcB originating from Bifidobacterium longum subsp. infantis to Y32I in FgFCO1 and mutation of D286, near the catalytic acid/base residue in FgFCO1, especially a D286M mutation, have a positive effect on FgFCO1 transfucosylation regioselectivity. We also found that enzymatic depolymerization of the xyloglucan substrate increases substrate accessibility and in turn transglycosylation (i.e., transfucosylation) efficiency. The data include analysis of the active site amino acids and the active site topology of FgFCO1 and show that transfer of point mutations across GH29 subfamilies is a rational strategy for targeted protein engineering of a xyloglucan-active fungal α-L--fucosidase. [ABSTRACT FROM AUTHOR]

Covering: Up to 2019 Phenolic cross-links and phenolic inter-unit linkages result from the oxidative coupling of two hydroxycinnamates or two molecules of tyrosine. Free dimers of hydroxycinnamates, lignans, play important roles in plant defence. Cross-linking of bound phenolics in the plant cell wall affects cell expansion, wall strength, digestibility, degradability, and pathogen resistance. Cross-links mediated by phenolic substituents are particularly important as they confer strength to the wall via the formation of new covalent bonds, and by excluding water from it. Four biopolymer classes are known to be involved in the formation of phenolic cross-links: lignins, extensins, glucuronoarabinoxylans, and side-chains of rhamnogalacturonan-I. Lignins and extensins are ubiquitous in streptophytes whereas aromatic substituents on xylan and pectic side-chains are commonly assumed to be particular features of Poales sensu lato and core Caryophyllales, respectively. Cross-linking of phenolic moieties proceeds via radical formation, is catalyzed by peroxidases and laccases, and involves monolignols, tyrosine in extensins, and ferulate esters on xylan and pectin. Ferulate substituents, on xylan in particular, are thought to be nucleation points for lignin polymerization and are, therefore, of paramount importance to wall architecture in grasses and for the development of technology for wall disassembly, e.g. for the use of grass biomass for production of 2nd generation biofuels. This review summarizes current knowledge on the intra- and extracellular acylation of polysaccharides, and inter- and intra-molecular cross-linking of different constituents. Enzyme mediated lignan in vitro synthesis for pharmaceutical uses are covered as are industrial exploitation of mutant and transgenic approaches to control cell wall cross-linking. [ABSTRACT FROM AUTHOR]

Human milk oligosaccharides (HMOs) signify a unique group of oligosaccharides in breast milk, which is of major importance for infant health and development. The functional benefits of HMOs create an enormous impetus for biosynthetic production of HMOs for use as additives in infant formula and other products. HMO molecules can be synthesized chemically, via fermentation, and by enzymatic synthesis. This treatise discusses these different techniques, with particular focus on harnessing enzymes for controlled enzymatic synthesis of HMO molecules. In order to foster precise and high-yield enzymatic synthesis, several novel protein engineering approaches have been reported, mainly concerning changing glycoside hydrolases to catalyze relevant transglycosylations. The protein engineering strategies for these enzymes range from rationally modifying specific catalytic residues, over targeted subsite −1 mutations, to unique and novel transplantations of designed peptide sequences near the active site, so-called loop engineering. These strategies have proven useful to foster enhanced transglycosylation to promote different types of HMO synthesis reactions. The rationale of subsite −1 modification, acceptor binding site matching, and loop engineering, including changes that may alter the spatial arrangement of water in the enzyme active site region, may prove useful for novel enzyme-catalyzed carbohydrate design in general. [ABSTRACT FROM AUTHOR]

The early-lineage, aerobic, zoosporic fungi from the Chytridiomycota constitute less than 1% of the described fungi and can use diverse sources of nutrition from plant or animal products. One of the ancestral sources of fungal nutrition could be products following enzymatic degradation of plant material. However, carbohydrate-active enzymes from these ancient fungi have been less studied. A GH11 xylanase (RrXyn11A) (EC 3.2.1.8) and a GH43 xylosidase (RrXyl43A) (EC 3.2.1.37) were identified from an early-lineage aerobic zoosporic fungus, Rhizophlyctis rosea NBRC 105426. Both genes were heterologously expressed in Pichia pastoris and the recombinant enzymes were purified and characterized. The optimal pH for recombinant RrXyn11A and RrXyl43A was pH 7. RrXyn11A had high stability over a wide range of pH (4-8) and temperature (25-70 °C). RrXyn11A also showed high substrate specificity on both azurine-cross-linked (AZCL) arabinoxylan and AZCL xylan. RrXyl43A had β-xylosidase and minor α-L-arabinofuranosidase activity. This enzyme showed low product inhibition and retained 51% activity in the presence of 100 mM xylose. A combination of RrXyn11A and RrXyl43A exhibited significantly higher hydrolytic and polymer degradation capability and xylose release on wheat bran and beechwood xylan compared to treatment with commercial enzymes. This study was the first to heterologously express and characterize the GH11 xylanase (RrXyn11A) and GH43 xylosidase (RrXyl43A) from the ancient fungus, R. rosea. Meanwhile, this study also demonstrated that the enzymes from the ancient fungus R. rosea can be easily handled and heterologously expressed in Pichia, which presents a promising path to a new source of enzymes for biomass degradation. [ABSTRACT FROM AUTHOR]

The α-1,3/4- l -fucosidases (EC 3.2.1.111; GH29) Bb AfcB from Bifidobacterium bifidum and Cp Afc2 from Clostridium perfringens can catalyse formation of the human milk oligosaccharide (HMO) lacto- N -fucopentaose II (LNFP II) through regioselective transfucosylation of lacto- N- tetraose (LNT) with 3-fucosyllactose (3FL) as donor substrate. The current work exploits structural differences between the two enzymes with the aim of engineering Bb AfcB into a more efficient transfucosidase and approaches an understanding of structure-function relations of hydrolytic activity vs. transfucosylation activity in GH29. Replacement of a 23 amino acids long α-helical loop close to the active site of Bb AfcB with the corresponding 17-aminoacid α-helical loop of Cp Afc2 resulted in almost complete abolishment of the hydrolytic activity on 3FL (6000 times lower hydrolytic activity than WT Bb AfcB), while the transfucosylation activity was lowered only one order of magnitude. In turn, the loop engineering resulted in an α-1,3/4- l -fucosidase with transfucosylation activity reaching molar yields of LNFP II of 39 ± 2% on 3FL and negligible product hydrolysis. This was almost 3 times higher than the yield obtained with WT Bb AfcB (14 ± 0.3%) and comparable to that obtained with Cp Afc2 (50 ± 8%). The obtained transfucosylation activity may expand the options for HMO production: mixtures of 3FL and LNT could be enriched with LNFP II, while mixtures of 3FL and lacto- N -neotetraose (LNnT) could be enriched with LNFP III. [ABSTRACT FROM AUTHOR]