Oliver Thum

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.

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.

Increasing the synthesis/hydrolysis ratio of aminoacylase 1 by site-directed mutagenesis.

Aminoacylase-1 from pig kidney (pAcy1) catalyzes the highly stereoselective acylation of amino acids, a useful conversion for the preparation of optically pure N-acyl-l-amino acids. The kinetic of this thermodynamically controlled conversion is determined by maximal velocities for synthesis (V(mS)) and hydrolysis (V(mH)) of the N-acyl-l-amino acid. To investigate which parameter affects maximal velocities, we focused on the proton acceptor potential of the catalytic base, E146, and studied the influence of the active site architecture on its contribution to the pKa of residue E146. The modeled structure of pAcy1 identified residue D346 as having the strongest impact on the electrostatic features of the catalytic base. Substitutions of D346 generally decreased enzymatic activities but also altered both the pH-dependency of hydrolytic activity and the V(mS)/V(mH) ratio of pAcy1. A reduced theoretical pKa value and a lowered experimental pH optimum of hydrolytic rates for the D346A mutant were associated with a 9-fold increase in V(mS)/V(mH). This supports the importance of electrostatic contributions of D346 to the acid-base properties of E146 and demonstrates for the first time the possibility of engineering the V(mS)/V(mH) ratio of pAcy1.

The Power of Biocatalysis: A One-Pot Total Synthesis of Rhamnolipids from Butane as the Sole Carbon and Energy Source

Abstract Microbially derived surfactants, so‐called biosurfactants, have drawn much attention in recent years and are expected to replace current petrochemical surfactants, owing to their environmental and toxicological benefits. One strategy to support that goal is to reduce production costs by replacing relatively expensive sugars with cheaper raw materials, such as short‐chain alkanes. Herein, we report the successful one‐pot total synthesis of rhamnolipids, a class of biosurfactants with 12 stereocenters, from butane as sole carbon and energy source through the design of a tailored whole‐cell biocatalyst.

Scalable preparation of silCoat‐biocatalysts by use of a fluidized‐bed reactor

SilCoat‐biocatalysts are immobilized enzyme preparations with an outstanding robustness against leaching and mechanical stress and therefore promising tools for technical synthesis. They consist of a composite material made from a solid enzyme carrier and silicone. In this study, a method has been found to enable provision of these catalysts in large scale. It makes use of easily scalable fluidized‐bed technology and, in contrast to the original method, works in almost complete absence of organic solvent. Thus, it is both a fast and safe method. When the Pt‐catalyst required for silicone formation is cast on the solid enzyme carrier before coating, resulting composites resemble the original preparations in morphology, catalytic activity, and stability against leaching and mechanical forces. Only the maximum total content of silicone in the composites lies about 10% w/w lower resulting in an overall leaching stability below the theoretical maximum. When the Pt‐catalyst is mixed with cooled siloxane solution before coating, surficial coating of the enzyme carriers is achieved, which provides maximum leaching stability at very low silicone consumption. Thus, the technology offers the possibility to produce both composite and for the first time also core‐shell silCoat‐particles, and optimize leaching stability over mechanical strength according to process requirements.

Robuste, langzeitstabile Enzympräparate

ZusammenfassungEin innovatives Beschichtungsverfahren unter Verwendung von maßgeschneiderten Silikonbausteinen liefert Enzymimmobilisate, die bei hohen Enzymaktivitäten hervorragende Langzeitstabilitäten aufweisen. Hierdurch erschließen sich neue Anwendungsfelder in der industriellen Biokatalyse.AbstractAn innovative coating process based on tailored siloxane building blocks yields enzyme immobilisates with outstanding long time stabilities and high enzyme activities, thus giving access to new application areas for industrial biotechnology.