Marrit Eckstein

Enzyme catalysis in ionic liquids.

Ionic liquids offer new possibilities for the application of solvent engineering to biocatalytic reactions. Although in many cases ionic liquids have simply been used to replace organic solvents, they have often led to improved process performance. Unlike conventional organic solvents, ionic liquids possess no vapor pressure, are able to dissolve many compounds, and can be used to form two-phase systems with many solvents. To date, reactions involving lipases have benefited most from the use of ionic liquids, but the use of ionic liquids with other enzymes and in whole-cell processes has also been described. In some cases, remarkable results with respect to yield, (enantio)selectivity or enzyme stability were observed.

At low water activity alpha-chymotrypsin is more active in an ionic liquid than in non-ionic organic solvents

The kinetics of the α-chymotrypsin catalysed transesterification of N-acetyl-l-phenylalanine ethyl ester with 1-butanol and the competing hydrolysis were evaluated at fixed water activity in two ionic liquids and two non-ionic organic solvents. In most respects the four solvents behaved similarly. However, at a water activity of 0.33, higher catalytic activity was observed in the ionic liquid, 1-butyl-3-methylimidazolium bis[(trifluoromethyl)sulfonyl]amide, than in the other solvents, and at aw=0.11 catalysis was only observed in this solvent.

Use of an ionic liquid in a two-phase system to improve an alcohol dehydrogenase catalysed reductionElectronic supplementary information (ESI) available: experimental section. See http://www.rsc.org/suppdata/cc/b4/b401065e/

Due to favourable partition coefficients the highly enantioselective reduction of 2-octanone, catalysed by an alcohol dehydrogenase from Lactobacillus brevis, is faster in a biphasic system containing buffer and the ionic liquid [BMIM][(CF(3)SO(2))(2)N] compared to the reduction in a biphasic system containing buffer and methyl tert-butyl ether.

Enhanced enantioselectivity of lipase from Pseudomonas sp. at high temperatures and fixed water activity in the ionic liquid, 1-butyl-3-methylimidazolium bis[(trifluoromethyl)sulfonyl]amide

The water activity equilibration over saturated salt solutions was monitored in the ionic liquid 1-butyl-3-methylimidazolium bis[(trifluoromethyl)sulfonyl]amide and in two organic solvents (methyl tert-butylether and n-hexane). Water uptake by the ionic liquid was comparable to that in polar organic solvents. Furthermore, the kinetic resolution of (R,S)-1-phenylethanol catalysed by a lipase from Pseudomonas sp. had a higher selectivity in the ionic liquid at low water activities (aW<0.53) than in methyl tert-butylether. At 60–90 °C E-values of about E∼150 occured in the ionic liquid, while the enantioselectivity drops to values of 4 in methyl tert-butylether at the same temperatures.

Recent Developments in NAD(P)H Regeneration for Enzymatic Reductions in One- and Two-Phase Systems

This review discusses recent achievements in the field of cofactor regeneration for the nicotinamide cofactors NADH and NADPH. The examples discussed include alcohol dehydrogenases, formate dehydrogenase, glucose dehydrogenase and a hydrogenase. For the reaction either one-phase systems or two-phase systems in combination with an organic solvent are discussed. For the enantioselective reduction of 2-octanone to (R)-2-octanol it could be shown that enzyme coupled NADPH regeneration with glucose dehydrogenase and glucose results in shorter reaction times and higher yields when compared to the substrate coupled regeneration with 2-propanol. ADH: alcohol dehydrogenase; LDH: Lactose dehydrogenase; GDH: Glucose dehydrogenase; FDH: Formate dehydrogenase; LB-ADH: alcohol dehydrogenase from Lactobacillus brevis; HL-ADH: alcohol dehydrogenase from horse liver; TB-ADH: alcohol dehydrogenase from Thermoanaerobicum brockii; PS-GDH: Glucose dehydrogenase from Pseudomonas species; [BMIM][PF6]: Butyl-methyl-imidazoliumhexafluorophosphate

The Metagenome-Derived Enzymes LipS and LipT Increase the Diversity of Known Lipases

Triacylglycerol lipases (EC 3.1.1.3) catalyze both hydrolysis and synthesis reactions with a broad spectrum of substrates rendering them especially suitable for many biotechnological applications. Most lipases used today originate from mesophilic organisms and are susceptible to thermal denaturation whereas only few possess high thermotolerance. Here, we report on the identification and characterization of two novel thermostable bacterial lipases identified by functional metagenomic screenings. Metagenomic libraries were constructed from enrichment cultures maintained at 65 to 75°C and screened resulting in the identification of initially 10 clones with lipolytic activities. Subsequently, two ORFs were identified encoding lipases, LipS and LipT. Comparative sequence analyses suggested that both enzymes are members of novel lipase families. LipS is a 30.2 kDa protein and revealed a half-life of 48 h at 70°C. The lipT gene encoded for a multimeric enzyme with a half-life of 3 h at 70°C. LipS had an optimum temperature at 70°C and LipT at 75°C. Both enzymes catalyzed hydrolysis of long-chain (C12 and C14) fatty acid esters and additionally hydrolyzed a number of industry-relevant substrates. LipS was highly specific for (R)-ibuprofen-phenyl ester with an enantiomeric excess (ee) of 99%. Furthermore, LipS was able to synthesize 1-propyl laurate and 1-tetradecyl myristate at 70°C with rates similar to those of the lipase CalB from Candida antarctica. LipS represents the first example of a thermostable metagenome-derived lipase with significant synthesis activities. Its X-ray structure was solved with a resolution of 1.99 Å revealing an unusually compact lid structure.

Composite Particles of Novozyme 435 and Silicone: Advancing Technical Applicability of Macroporous Enzyme Carriers

The mechanical and leaching stability of enzymes adsorbed on macroporous carriers is an important issue for the technical applicability of such biocatalysts. Both can considerably benefit from the deposition of silicone coating on the carrier surface. The coating of the immobilized lipase Novozyme 435 (NZ435), as a model enzyme preparation, with different silicone loadings was studied in detail by scanning electron microscopy (SEM) and transmission electron microscopy (TEM), as well as by energy‐dispersive X‐ray spectroscopy (EDX) and BET isotherms, and offers explanations and prerequisites for its stabilizing effects. The deposition of silicone on the poly(methyl methacrylate) (PMMA) carrier was found to form an interpenetrating network composite rather than the anticipated core‐shell structure. The silicone precursors homogeneously wet the carrier surface including all inner pores and gradually fill the complete carrier. In parallel, the surface area of NZ435 decreases from an initial value of 89 m2g−1to 0.2 m2g−1after silicone loading. A visible layer of silicone on the outer surface of the carrier was only observed at a silicone concentration of 54 % w/w and more. Maximum leaching stability corresponds to the formation of this layer. The mechanical stability increases with the amount of deposited silicone. It can be expected that stabilization against leaching and/or mechanical stress by formation of silicone composites can easily be transferred to a whole range of alternative biocatalytic systems. This should considerably advance their general technical applicability and overall implementation of biocatalysts in chemical synthesis.

Online monitoring of biotransformations in high viscous multiphase systems by means of FT-IR and chemometrics.

In unstable emulsion systems, the determination of concentrations is a challenge. The use of standard methods like GC, HPLC, or titration is highly inaccurate and makes the acquisition of precise data for these systems complex. In addition, the handicap of high viscosity often comes into play. To overcome these fundamental limitations, the online FT-IR technique was identified in combination with chemometric modeling in order to improve accuracy. The reactor type used in this study is a bubble column reactor with up to four dispersed phases (solid catalyst, two liquid immiscible substrates, and a gaseous phase). The investigated reactions are solvent free enzymatic esterifications yielding myristyl myristate (10 mPa s) and high viscous polyglycerol-3-laurate (300-1500 mPa s), representative industrial products for cosmetic applications. For both reactions, chemometric models were successfully set up and reproducibly applied in the prediction of progress curves of a new set of experiments. This allows the automated determination of sensitive kinetic and thermodynamic data as well as reaction velocities in high viscous multiphase (bio)chemical systems.

Simultaneous Determination of Mono-, Di-, and Triglycerides in Multiphase Systems by Online Fourier Transform Infrared Spectroscopy

Glycerides are of significant value for industry as ingredients with different purposes in food or cosmetics. The analysis of glycerides is mainly performed by gas chromatography (GC) or high-pressure liquid chromatography (HPLC), which demonstrate limitations in dealing with multiphase systems. In this article, an in situ differentiation between mono-, di-, and triglycerides in multiphase systems by Fourier transform infrared (FT-IR) spectroscopy is demonstrated. The enzymatic esterification of glycerol with lauric acid was analyzed as a model system. The reaction was carried out in a bubble column reactor containing four phases (two liquid phases of glycerol and lauric acid, air as gaseous phase, and a heterogeneous catalyst as solid phase). As a feasibility study, a chemometric model was generated for the pure components only. The quantities of lauric acid and the three products (mono-, di-, and trilaurin) were simultaneously determined over the course of the reaction with acceptable errors (1.8-12.5%) with regard to the calibration effort. This technology has the potential to give accurate results, particularly in unstable emulsion systems containing fats, oils, or emulsifiers, which are currently afflicted by analytical errors caused by the challenge of accurate sampling.

In Situ Microscopy for In-line Monitoring of the Enzymatic Hydrolysis of Cellulose

A new in-line method for the monitoring of enzymatic hydrolysis of cellulose is described. Using a new in situ microscope prototype, the noninvasive determination of particle size distributions was possible. For the automated analysis of the acquired images, a new processing algorithm called CelluloseAnalyzer was developed. It enabled tracking of the number of particles and moreover allowed monitoring of the proportions of particle size fractions during the course of enzymatic hydrolysis reactions. Using this technique, significant differences between hydrolysis with endoglucanases and cellulase mixtures were observed. Furthermore, the in situ microscopy results were compared with results from off-line measurements with laser diffraction spectroscopy and gel permeation chromatography.