Lutz Hilterhaus

Evaluation of immobilized enzymes for industrial applications

In contrast to the application of soluble enzymes in industry, immobilized enzymes often offer advantages in view of stability, volume specific biocatalyst loading, recyclability as well as simplified downstream processing. In this tutorial review the focus is set on the evaluation of immobilized enzymes in respect to mass transport limitations, immobilization yield and stability, to enable industrial applications.

Practical application of different enzymes immobilized on sepabeads

The immobilization of an endoglucanase, benzoylformate decarboxylase (BFD) from Pseudomonas putida, as well as of lipase B from Candida antarctica (CALB) onto the carrier supports Sepabeads EC-EP, Sepabeads EC-EA, and Sepabeads EC-BU was accomplished. It is shown that via these immobilized biocatalysts the synthesis of both fine and bulk chemicals is possible. This is illustrated by the syntheses of polyglycerol esters and (S)-hydroxy phenyl propanone. The benefit of immobilization is illustrated by repetitive use in a bubble column reactor as well as in a stirred tank reactor. High stability of two biocatalysts was achieved and reusability up to eight times was demonstrated. The comparison of CALB immobilized on Sepabeads EC-EP to Novozym 435 shows similar activity.

Kinetic investigation of a solvent-free, chemoenzymatic reaction sequence towards enantioselective synthesis of a β-amino acid ester.

A solvent‐free, chemoenzymatic reaction sequence for the enantioselective synthesis of β‐amino acid esters has been kinetically and thermodynamically characterized. The coupled sequence comprises a thermal aza‐Michael addition of cheap starting materials and a lipase catalyzed aminolysis for the kinetic resolution of the racemic ester. Excellent ee values of >99% were obtained for the β‐amino acid ester at 60% conversion. Kinetic constants for the aza‐Michael addition were obtained by straightforward numerical integration of second‐order rate equations and nonlinear fitting of the progress curves. A different strategy had to be devised for the biocatalytic reaction. Initially, a simplified Michaelis–Menten model including product inhibition was developed for the reaction running in THF as an organic solvent. Activity based parameters were used instead of concentrations in order to facilitate the transfer of the kinetic model to the solvent‐free system. Observed solvent effects not accounted for by the use of thermodynamic activities were incorporated into the kinetic model. Enzyme deactivation was observed to depend on the ratio of the applied substrates and also included in the kinetic model. The developed simple model is in very good agreement with the experimental data and allows the simulation and optimization of the solvent‐free process. Biotechnol. Bioeng. 2012; 109:1479–1489.

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.

Label-free photonic biosensors fabricated with low-loss hydrogenated amorphous silicon resonators

Abstract. The precise detection of chemicals and biomolecules is of great interest in the areas of biotechnology and medical diagnostics. Thus, there is a need for highly sensitive, small area, and low-cost sensors. We fabricated and optically characterized hydrogenated amorphous silicon photonic resonators for label-free lab-on-chip biosensors. The sensing was performed with small-footprint microdisk and microring resonators that detect a refractive-index change via the evanescent electric field. Homogeneous sensing with NaCl and surface-sensing experiments with immobilized bovine serum albumin (BSA) were carried out. A sensitivity as high as 460  nm/RIU was measured for NaCl dissolved in deionized water for the disk, whereas about 50  nm/RIU was determined for the ring resonator. The intrinsic limits of detection were calculated to be 3.3×10−4 and 3.2×10−3 at 1550-nm wavelength. We measured the binding of BSA to functionalized ring resonators and found that molecular masses can be detected down to the clinically relevant femtogram regime. The detection and quantification of related analytes with hydrogenated amorphous silicon photonic sensors can be used in medical healthcare diagnostics like point-of-care-testing and biotechnological screening.

Site directed immobilization of glucose-6-phosphate dehydrogenase via thiol-disulfide interchange: Influence on catalytic activity of cysteines introduced at different positions

This study shows the effect of site-directed enzyme immobilization upon the enzyme activity of covalently bound glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides. Immobilization points were introduced at sterically accessible sites in order to control the proteins orientation and twice as much activity was recovered in comparison to conventionally immobilized enzyme. Immobilization of G6PDH via genetically engineered cysteine provided a simple, but effective method to control the immobilization process. G6PDH variants with cysteine close to the active center (L218C), close to the dimer interface (D205C) as well as far from the active center (D453C) showed changes in activity and the efficacy of immobilization.

Arylmalonate Decarboxylase-Catalyzed Asymmetric Synthesis of Both Enantiomers of Optically Pure Flurbiprofen

The bacterial decarboxylase (AMDase) catalyzes the enantioselective decarboxylation of prochiral arylmalonates with high enantioselectivity. Although this reaction would provide a highly sustainable synthesis of active pharmaceutical compounds such as flurbiprofen or naproxen, competing spontaneous decarboxylation has so far prevented the catalytic application of AMDase. Here, we report on reaction engineering and an alternate protection group strategy for the synthesis of these compounds that successfully suppresses the side reaction and provides pure arylmalonic acids for subsequent enzymatic conversion. Protein engineering increased the activity of the synthesis of the (S)‐ and (R)‐enantiomers of flurbiprofen. These results demonstrated the importance of synergistic effects in the optimization of this decarboxylase. The asymmetric synthesis of both enantiomers in high optical purity (>99 %) and yield (>90 %) can be easily integrated into existing industrial syntheses of flurbiprofen, thus providing a sustainable method for the production of this important pharmaceutical ingredient.

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.

Pseudomonas aeruginosa Biofilm Growth Inhibition on Medical Plastic Materials by Immobilized Esterases and Acylase

Biofilms are matrix‐encapsulated cell aggregates that cause problems in technical and health‐related areas; for example, 65 % of all human infections are biofilm associated. This is mainly due to their ameliorated resistance against antimicrobials and immune systems. Pseudomonas aeruginosa, a biofilm‐forming organism, is commonly responsible for nosocomial infections. Biofilm development is partly mediated by signal molecules, such as acyl‐homoserine lactones (AHLs) in Gram‐negative bacteria. We applied horse liver esterase, porcine kidney acylase, and porcine liver esterase; these can hydrolyze AHLs, thereby inhibiting biofilm formation. As biofilm infections are often related to foreign material introduced into the human body, we immobilized the enzymes on medical plastic materials. Biofilm formation was quantified by Crystal Violet staining and confocal laser scanning microscopy, revealing up to 97 % (on silicone), 54 % (on polyvinyl chloride), and 77 % (on polyurethane) reduced biomass after 68 h growth.