Observation of substrate diffusion and ligand binding in enzyme crystals using high-repetition-rate mix-and-inject serial crystallography

Here, we illustrate what happens inside the catalytic cleft of an enzyme when substrate or ligand binds on single-millisecond timescales. The initial phase of the enzymatic cycle is observed with near-atomic resolution using the most advanced X-ray source currently available: the European XFEL (EuXFEL). The high repetition rate of the EuXFEL combined with our mix-and-inject technology enables the initial phase of ceftriaxone binding to the Mycobacterium tuberculosis β-lactamase to be followed using time-resolved crystallography in real time. It is shown how a diffusion coefficient in enzyme crystals can be derived directly from the X-ray data, enabling the determination of ligand and enzyme–ligand concentrations at any position in the crystal volume as a function of time. In addition, the structure of the irreversible inhibitor sulbactam bound to the enzyme at a 66 ms time delay after mixing is described. This demonstrates that the EuXFEL can be used as an important tool for biomedically relevant research.

Preparation of ZIF@ADH/NAD-MSN/LDH Core Shell Nanocomposites for the Enhancement of Coenzyme Catalyzed Double Enzyme Cascade

The field of enzyme cascades in limited microscale or nanoscale environments has undergone a quick growth and attracted increasing interests in the field of rapid development of systems chemistry. In this study, alcohol dehydrogenase (ADH), lactate dehydrogenase (LDH), and mesoporous silica nanoparticles (MSN) immobilized nicotinamide adenine dinucleotide (NAD+) were successfully immobilized on the zeolitic imidazolate frameworks (ZIFs). This immobilized product was named ZIF@ADH/NAD-MSN/LDH, and the effect of the multi-enzyme cascade was studied by measuring the catalytic synthesis of lactic acid. The loading efficiency of the enzyme in the in-situ co-immobilization method reached 92.65%. The synthesis rate of lactic acid was increased to 70.10%, which was about 2.82 times that of the free enzyme under the optimal conditions (40 °C, pH = 8). Additionally, ZIF@ADH/NAD-MSN/LDH had experimental stability (71.67% relative activity after four experiments) and storage stability (93.45% relative activity after three weeks of storage at 4 °C; 76.89% relative activity after incubation in acetonitrile-aqueous solution for 1 h; 27.42% relative activity after incubation in 15% N, N-Dimethylformamide (DMF) solution for 1 h). In summary, in this paper, the cyclic regeneration of coenzymes was achieved, and the reaction efficiency of the multi-enzyme biocatalytic cascade was improved due to the reduction of substrate diffusion.

In-depth sequence-function characterization reveals multiple paths to enhance phenylalanine ammonia-lyase (PAL) activity.

Phenylalanine ammonia-lyases (PALs) deaminate L-phenylalanine to trans-cinnamic acid and ammonium and have widespread application in chemo-enzymatic synthesis, agriculture, and medicine. In particular, the PAL from Anabaena variabilis has garnered significant attention as the active ingredient in Pegvaliase®, the only FDA-approved drug treating classical phenylketonuria (PKU). Although an extensive body of literature exists on structure, substrate-specificity, and catalytic mechanism, protein-wide sequence determinants of function remain unknown, which limits the ability to rationally engineer these enzymes. Previously, we developed a high-throughput screen (HTS) for PAL, and here, we leverage it to create a detailed sequence-function landscape of PAL by performing deep mutational scanning (DMS). Our method revealed 79 hotspots that affected a positive change in enzyme fitness, many of which have not been reported previously. Using fitness values and structure-function analysis, we picked a subset of residues for comprehensive single- and multi-site saturation mutagenesis to improve the catalytic activity of PAL and identified combinations of mutations that led to improvement in reaction kinetics in cell-free and cellular contexts. To understand the mechanistic role of the most beneficial mutations, we performed QM/MM and MD and observed that different mutants confer improved catalytic activity via different mechanisms, including stabilizing first transition and intermediate states and improving substrate diffusion into the active site, and decreased product inhibition. Thus, this work provides a comprehensive sequence-function relationship for PAL, identifies positions that improve PAL activity when mutated and assess their mechanisms of action.

Encapsulated NOLA™ Fit 5500 Lactase—An Economically Beneficial Way to Obtain Lactose-Free Milk at Low Temperature

The current requirements of industrial biocatalysis are related to economically beneficial and environmentally friendly processes. Such a strategy engages low-temperature reactions. The presented approach is essential, especially in food processes, where temperature affects the quality and nutritional value foodstuffs. The subject of the study is the hydrolysis of lactose with the commercial lactase NOLA™ Fit 5500 (NOLA). The complete decomposition of lactose into two monosaccharides gives a sweeter product, recommended for lactose intolerant people and those controlling a product’s caloric content. The hydrolysis reaction was performed at 15 °C, which is related to milk transportation and storage temperature. The enzyme showed activity over the entire range of substrate concentrations (up to 55 g/L lactose). For reusability and easy isolation, the enzyme was encapsulated in a sodium alginate network. Its stability allows carrying out six cycles of the complete hydrolysis of lactose to monosaccharides, lasting from two to four hours. During the study, the kinetic description of native and encapsulated NOLA was conducted. As a result, the model of competitive galactose inhibition and glucose mixed influence (competitive inhibition and activation) was proposed. The capsule size does not influence the reaction rate; thus, the substrate diffusion into capsules can be omitted from the process description. The prepared 4 mm capsules are easy to separate between cycles, e.g., using sieves.

Composites of Crosslinked Aggregates of Eversa® Transform and Magnetic Nanoparticles. Performance in the Ethanolysis of Soybean Oil

Eversa® Transform 2.0 has been launched to be used in free form, but its immobilization may improve its performance. This work aimed to optimize the immobilization of Eversa® Transform 2.0 by the crosslinked enzyme aggregates (CLEAs) technique, using almost all the available tools to improve its performance. Several variables in the CLEA preparation were optimized to improve the recovered activity, such as precipitant nature and crosslinker concentration. Moreover, some feeders were co-precipitated to improve the crosslinking step, such as bovine serum albumin, soy protein, or polyethyleneimine. Starch (later enzymatically degraded) was utilized as a porogenic agent to decrease the substrate diffusion limitations. Silica magnetic nanoparticles were also utilized to simplify the CLEA handling, but it was found that a large percentage of the Eversa activity could be immobilized on these nanoparticles before aggregation. The best CLEA protocol gave a 98.9% immobilization yield and 30.1% recovered activity, exhibited a porous structure, and an excellent performance in the transesterification of soybean oil with ethanol: 89.8 wt% of fatty acid ethyl esters (FAEEs) yield after 12 h of reaction, while the free enzyme required a 48 h reaction to give the same yield. A caustic polishing step of the product yielded a biodiesel containing 98.9 wt% of FAEEs and a free fatty acids content lower than 0.25%, thus the final product met the international standards for biodiesel. The immobilized biocatalyst could be reused for at least five 12 h-batches maintaining 89.6% of the first-batch yield, showing the efficient catalyst recovery by applying an external magnetic field.

Carbohydrate binding domains facilitate efficient oligosaccharides synthesis by enhancing mutant catalytic domain transglycosylation activity

Chemoenzymatic approaches using carbohydrate-active enzymes (CAZymes) offer a promising avenue for synthesis of glycans like oligosaccharides. Here, we report a novel chemoenzymatic route for cellodextrins synthesis employed by chimeric CAZymes, akin to native glycosyltransferases, involving the unprecedented participation of a ‘non-catalytic’ lectin-like or carbohydrate-binding domains (CBMs) in the catalytic step for glycosidic bond synthesis using β-cellobiosyl donor sugars as activated substrates. CBMs are often thought to play a passive substrate targeting role in enzymatic glycosylation reactions mostly via overcoming substrate diffusion limitations for tethered catalytic domains (CDs) but are not known to participate directly in any nucleophilic substitution mechanisms that impact the actual glycosyl transfer step. Our study provides evidence for the direct participation of CBMs in the catalytic reaction step for β-glucan glycosidic bonds synthesis enhancing activity for CBM-based CAZyme chimeras by >140-fold over CDs alone. Dynamic intra-domain interactions that facilitate this poorly understood reaction mechanism were further revealed by small-angle X-ray scattering structural analysis along with detailed mutagenesis studies to shed light on our current limited understanding of similar transglycosylation-type reaction mechanisms. In summary, our study provides a novel strategy for engineering similar CBM-based CAZyme chimeras for synthesis of bespoke oligosaccharides using simple activated sugar monomers.

Increasing the Enzyme Loading Capacity of Porous Supports by a Layer-by-Layer Immobilization Strategy Using PEI as Glue

A new strategy to increase the enzyme-loading capacity of porous supports was investigated. Lipase from Pseudomonas fluorescens (PFL) was immobilized on octyl-agarose (OA) beads and treated with polyethyleneimine (PEI). Then, PFL was immobilized on the previous PFL layer. Next, the biocatalyst was coated with PEI and a third layer of PFL was added. Sodium dodecyl sulfate polyacrylamide electrophoresis showed that the amount of PFL proportionally increased with each enzyme layer; however, the effects on biocatalyst activity were not as clear. Hydrolyzing 50 mM of triacetin at 25 °C, the activity of the three-layer biocatalyst was even lower than that of the bi-layer one; on the contrary its activity was higher when the activity was measured at 4 °C in the presence of 30% acetonitrile (that reduced the activity and thus the relevance of the substrate diffusion limitations). That is, the advantage of the multilayer formation depends on the specific activity of the enzyme and on the diffusion limitations of the substrate. When octyl agarose (OA)-PFL-PEI-PFL preparation was treated with glutaraldehyde, the activity was reduced, although the enzyme stability increased and the immobilization of the last PFL layer offered results similar to the one obtained using the three-layer preparation without glutaraldehyde modification (90%).