Diána Weiser

Immobilization of Phenylalanine Ammonia‐Lyase on Single‐Walled Carbon Nanotubes for Stereoselective Biotransformations in Batch and Continuous‐Flow Modes

Carboxylated single‐walled carbon nanotubes (SwCNTCOOH) were used as a support for the covalent immobilization of phenylalanine ammonia‐lyase (PAL) from parsley by two different methods. The nanostructured biocatalysts (SwCNTCOOH‐PALI and SwCNTCOOH‐PALII) with low diffusional limitation were tested in the batch‐mode kinetic resolution of racemic 2‐amino‐3‐(thiophen‐2‐yl)propanoic acid (1) to yield a mixture of (R)‐1 and (E)‐3‐(thiophen‐2‐yl)acrylic acid (2) and in ammonia addition to 2 to yield enantiopure (S)‐1. SwCNTCOOH‐PALII was a stable biocatalyst (>90 % of the original activity remained after six cycles with 1 and after three cycles in 6 M NH3 with 2). The study of ammonia addition to 2 in a continuous‐flow microreactor filled with SwCNTCOOH‐PALII (2 M NH3, pH 10.0, 15 bar) between 30–80 °C indicated no significant loss of activity over 72 h up to 60 °C. SwCNTCOOH‐PALII in the continuous‐flow system at 30 °C was more productive (specific reaction rate, rflow=2.39 μmol min−1 g−1) than in the batch reaction (rbatch=1.34 μmol min−1 g−1).

Electrospun polylactic acid and polyvinyl alcohol fibers as efficient and stable nanomaterials for immobilization of lipases.

Electrospinning was applied to create easy-to-handle and high-surface-area membranes from continuous nanofibers of polyvinyl alcohol (PVA) or polylactic acid (PLA). Lipase PS from Burkholderia cepacia and Lipase B from Candida antarctica (CaLB) could be immobilized effectively by adsorption onto the fibrous material as well as by entrapment within the electrospun nanofibers. The biocatalytic performance of the resulting membrane biocatalysts was evaluated in the kinetic resolution of racemic 1-phenylethanol (rac-1) and 1-phenylethyl acetate (rac-2). Fine dispersion of the enzymes in the polymer matrix and large surface area of the nanofibers resulted in an enormous increase in the activity of the membrane biocatalyst compared to the non-immobilized crude powder forms of the lipases. PLA as fiber-forming polymer for lipase immobilization performed better than PVA in all aspects. Recycling studies with the various forms of electrospun membrane biocatalysts in ten cycles of the acylation and hydrolysis reactions indicated excellent stability of this forms of immobilized lipases. PLA-entrapped lipases could preserve lipase activity and enantiomer selectivity much better than the PVA-entrapped forms. The electrospun membrane forms of CaLB showed high mechanical stability in the repeated acylations and hydrolyses than commercial forms of CaLB immobilized on polyacrylamide beads (Novozyme 435 and IMMCALB-T2-150).

Bisepoxide Cross‐Linked Enzyme Aggregates—New Immobilized Biocatalysts for Selective Biotransformations

Glycerol diglycidyl ether (GDE) is a convenient and inexpensive bisepoxide cross‐linker as demonstrated by the preparation of cross‐linked enzyme aggregates (CLEAs) from two enzyme classes. The GDE CLEAs of lipase from Pseudomonas fluorescens (AK), lipase from Burkholderia cepacia (PS), and lipase B from Candida antarctica (CaL B) as well as of phenylalanine ammonia‐lyase (PAL) from Petroselinum crispum demonstrated improved properties as compared with their glutaraldehyde (GA) cross‐linked counterparts. Ultrasonication studies indicated that the GDE CLEAs of lipase PS and PAL were mechanically more stable than the GA CLEAs. In the kinetic resolution of rac‐1‐phenylethanol, the catalytic activity of the GDE–lipase CLEAs (U=69.6, 134.8, and 127.4 U g−1 for AK, CaL B, and PS prepared at 22 °C, respectively) surpassed that of the corresponding GA–lipase CLEAs (U=24.4, 131.0, and 119.2 U g−1 for AK, CaL B, and PS prepared at 22 °C, respectively). The GDE co‐CLEAs from PAL and bovine serum albumin (BSA) could be recycled at least three times if used for the stereoselective ammonia addition in 6 M ammonia to (E)‐3‐(thiophen‐2‐yl)acrylic acid, whereas the recycling of the conventional GA–PAL CLEAs from this medium failed.

Phenylalanine Ammonia-Lyase-Catalyzed Deamination of an Acyclic Amino Acid: Enzyme Mechanistic Studies Aided by a Novel Microreactor Filled with Magnetic Nanoparticles

Phenylalanine ammonia‐lyase (PAL), found in many organisms, catalyzes the deamination of l‐phenylalanine (Phe) to (E)‐cinnamate by the aid of its MIO prosthetic group. By using PAL immobilized on magnetic nanoparticles and fixed in a microfluidic reactor with an in‐line UV detector, we demonstrated that PAL can catalyze ammonia elimination from the acyclic propargylglycine (PG) to yield (E)‐pent‐2‐ene‐4‐ynoate. This highlights new opportunities to extend MIO enzymes towards acyclic substrates. As PG is acyclic, its deamination cannot involve a Friedel–Crafts‐type attack at an aromatic ring. The reversibility of the PAL reaction, demonstrated by the ammonia addition to (E)‐pent‐2‐ene‐4‐ynoate yielding enantiopure l‐PG, contradicts the proposed highly exothermic single‐step mechanism. Computations with the QM/MM models of the N‐MIO intermediates from l‐PG and l‐Phe in PAL show similar arrangements within the active site, thus supporting a mechanism via the N‐MIO intermediate.

Microfluidic multiple cell chip reactor filled with enzyme-coated magnetic nanoparticles — An efficient and flexible novel tool for enzyme catalyzed biotransformations

Biotransformation of L-phenylalanine (L-1a) and five unnatural substrates (rac-1b–f) by phenylalanine ammonia-lyase (PAL) was investigated in a novel microfluidic device (Magne-Chip) that comprises microliter volume reaction cells filled with PAL-coated magnetic nanoparticles (MNPs). Experiments proved the excellent reproducibility of enzymecatalyzed biotransformation in the chip and the excellent reusability of the enzyme layer during 14 h continuous measurement (>98% over 7 repetitive measurements with L-1a). The platform also enabled fully automatic multiparameter measurements with a single biocatalyst loading of about 1 mg PAL-MNP. Computational fluid dynamics (CFD) calculations were used to study the flow field in the chambers and the effect of unintended bubble formation. Optimal flow rate for L-1a reaction and specific activities for rac-1b–f under these conditions were determined.

Creating an Efficient Methanol-Stable Biocatalyst by Protein and Immobilization Engineering Steps towards Efficient Biosynthesis of Biodiesel

Two ternary sol-gel matrices, an octyltriethoxysilane-based aliphatic matrix and a phenyltriethoxysilane (PTEOS)-based aromatic matrix, were used to immobilize a methanol-stable variant of lipase from Geobacillus stearothermophilus T6 for the synthesis of biodiesel from waste oil. Superior thermal stability of the mutant versus the wildtype in methanol was confirmed by intrinsic protein fluorescence measurements. The influence of skim milk and soluble E. coli lysate proteins as bulking and stabilizing agents in conjunction with sol-gel entrapment were investigated. E. coli lysate proteins were better stabilizing agents of the purified lipase mutant than skim milk, as evidenced by reverse engineering of the aromatic-based system. This was also shown for commercial Candida antarctica lipase B (CaLB) and Thermomyces lanuginosus lipase (TLL). Uniform, dense, and nonaggregated particles imaged by scanning electron microscopy and a small particle size of 13 μm pertaining to the system comprising PTEOS and E. coli lysate proteins correlated well with high esterification activity. Combining protein and immobilization engineering resulted in a durable biocatalyst with efficient recycling ability and high biodiesel conversion rates.

Immobilization engineering – How to design advanced sol–gel systems for biocatalysis?

An immobilization engineering approach using bioinformatics and experimental design tools was applied to improve the sol–gel enzyme entrapment methodology. This strategy was used for the immobilization of lipase B from Candida antarctica (CaLB), a versatile enzyme widely used even on the industrial scale. The optimized entrapment of CaLB in sol–gel matrices is reported by the response-surface methodology enabling efficient process development. The immobilized CaLBs characterized by functional efficiency and enhanced recovery provided economical and green options for flow chemistry. Various ternary mixtures of sol–gel precursors allowed the creation of tailored entrapment matrices best suited for the enzyme and its targeted substrate. The sol–gel-entrapped forms of CaLB were excellent biocatalysts in the kinetic resolutions of secondary alcohols and secondary amines with aromatic or aliphatic substituents both in batch and continuous-flow biotransformations.

Microfluidic Multiple Chamber Chip Reactor Filled with Enzyme-Coated Magnetic Nanoparticles

In this chapter, a novel microfluidic device (MagneChip) is described which comprises microliter volume reaction chambers filled with magnetically fixed enzyme-coated magnetic nanoparticles (ecMNPs) and with an in-line UV detector. In the experiments, MNPs with phenylalanine ammonia-lyase (PAL)—an enzyme which catalyzes the deamination of l-phenylalanine (Phe) to (E)-cinnamate in many organisms—immobi‐ lized on the surface were applied as biocatalyst to study the characteristics of the MagneChip device. In the reaction chambers of this microfluidic device, the accurate in situ quantization of the entrapped MNPs was possible using a resonant coil magneto‐ meter integrated below the chambers. Computational fluid dynamics (CFD) calcula‐ tions were used to simulate the flow field in the chambers. The enzyme-catalyzed biotransformations could be performed in the chip with excellent reproducibility and of repeatability. The platform enabled fully automatic multiparameter measurements with a single biocatalyst loading of about 1 mg PAL-ecMNP in the chip. A study on the effect of particle size and arrangement on the catalytic activity revealed that the mass of ecMNPs fixed in the chamber is independent of the particle diameter. Decreasing the particle size resulted in increasing catalytic activity due to the increased area to volume ratio. A binary mixture of particles with two different particle sizes could increase the entrapped particle mass and further the catalytic activity compared to the best uniform packing. The platform enabled a study of biotransformation of L-phenylalanine and five unnatural substrates by consecutive reactions using same PAL-ecMNP loading. With the aid of the platform, we first demonstrated that PAL can catalyze the ammonia elimination from the noncyclic propargylglycine as substrate.

Sensitivity enhancement for mycotoxin determination by optical waveguide lightmode spectroscopy using gold nanoparticles of different size and origin

Mycotoxins, present in a wide range of food and feed commodities, are toxic secondary metabolites produced by a number of different fungi. Certain mycotoxins do not readily degrade at high temperatures, therefore are resistant to food processing, and consequently are present in the human and animal food supply. Optical waveguide lightmode spectroscopy (OWLS) was applied for the detection of aflatoxin B1, in a competitive immunoassay format, to compare the analytical sensitivity achieved with an immunosensor design allowing signal enhancement by increasing the sensor surface through immobilization of gold nanoparticles (AuNPs) of different size and origin (obtained by chemical or biotechnological synthesis). The effects of AuNPs median size, the methods of sensitization and the biochemical parameters on immunosensor performace were examined. After optimization of the sensitized sensor surface, an immunosensing method was developed for the analysis of aflatoxin in paprika matrix and the results were compared with HPLC reference measurements.

Biomimetic Synthesis of Drug Metabolites in Batch and Continuous-Flow Reactors

A medium-throughput screening (MTS) of biomimetic drug metabolite synthesis is developed by using an iron porphyrin catalyst. The microplate method, in combination with HPLC-MS analysis, was shown to be a useful tool for process development and parameter optimization in the production of targeted metabolites and/or oxidation products of forty-three different drug substances. In the case of the biomimetic oxidation of amiodarone, the high quantity and purity of the isolated products enabled detailed HRMS and NMR spectroscopic studies. In addition to identification of known metabolites, several new oxidation products of the drug that was studied were characterized. Fast degradation and poor recovery of the catalyst under batch conditions was overcome by immobilization of 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrin iron(III) chloride (FeTSPP) on the surface of 3-aminopropyl-functionalized silica by electrostatic interaction. The supported catalyst was successfully applied in a packed-bed reactor under continuous-flow reaction conditions for the large-scale synthesis of amiodarone metabolites.