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.
Evaluation of immobilized enzymes in respect to mass transport limitations, immobilization yield and stability, to enable industrial applications.
To overcome limitations due to the high viscosity in the solvent free esterification of polyglycerol-3 and related polyols, such as poly(ethylene glycol)s, an alternative reactor concept was ...developed. The new reactor comprises a bubble column that prevents mechanical erosion of Novozym 435 (lipase B from Candida antarctica) found by mechanical stirring of the mixture. That way polyglycerol-3 laurate was synthesized at a space time yield (sty) of 3042 g/L/d and PEG-55-propylene glycol dioleate at a sty of 738 g/L/d. To proof the broad application range of this system, low-viscosity myristyl myristate was synthesized at a sty of 6731 g/L/d, thus outperforming conventional methods such as stirred tank or fixed bed. The newly developed reactor concept is universally applicable to esterification reactions and can be advantageously applied in the synthesis of a broad range of high quality surfactants.
The downscale of different unit operations for the biocatalytic carboligation of benzaldehyde and acetaldehyde catalyzed by benzoylformate decarboxylase from Pseudomonas putida was investigated. The ...reactor volume was reduced to 115 μl thus enabling a substrate and enzyme saving by a factor of 52 in comparison to standard laboratory techniques. Additionally, the successful downscale of membrane based liquid-liquid contactors was shown, which allows, for example, the screening of solvents for extraction as well as the feed of a substrate. Here, comparable volumes as well as residence times were realized, enabling the integration of all three unit operations.
•Cofactor regeneration system using glucose dehydrogenase successfully implemented.•Addition of low surfactant and co-solvent amounts enhanced reaction performance.•Oxygen supply had major influence ...on STY and enzymatic process stability.•Coupling efficiency of ∼15% determined during the process under varied conditions.•Limited total turnover character of the P450 constituted main process restriction.
Cytochrome P450 monooxygenases are a unique family of enzymes that are able to catalyze regio- and stereospecific oxidations for a broad substrate range. However, due to limited enzyme activities and stabilities, hydrophobicity of substrates, as well as the necessity of a continuous electron and oxygen supply the implementation of P450s for industrial processes remains challenging. Aim of this study was to point out key aspects for the development of an efficient synthesis concept for cytochrome P450 catalyzed oxidations. In order to regenerate the natural cofactor NADPH, a glucose dehydrogenase was applied. The low water soluble terpene α-ionone was used as substrate for the model reaction system. The studies reveal that an addition of surfactants in combination with low volumetric amounts of co-solvent can significantly increase substrate availability and reaction rates. Furthermore, these additives facilitated a reliable sampling procedure during the process. Another key factor for the process design was the oxygen supply. Based on various investigations, a bubble-aerated stirred tank reactor in batch mode represents a promising reactor concept for P450 oxidations. Main restriction of the investigated reaction system was the low process stability of the P450 monooxygenase, characterized by maximum total turnover numbers of ∼4100molα‐ionone/molP450.
Non-specific adsorption and specific interaction between a chimeric green fluorescent protein (GFP) carrying metal-binding region and the immobilized zinc ions on artificial solid-supported lipid ...membranes was investigated using the quartz crystal microbalance technique and the atomic force microscopy (AFM). Supported lipid bilayer, composed of octanethiol and 1,2-dipalmitoyl-
sn-glycero-3-phosphocholine/1,2-dioleoyl-
sn-glycero-3-
N-(5-amino-1-carboxypentyl iminodiacetic acid)succinyl (NTA-DOGS)-Zn
2+, was formed on the gold electrode of quartz resonator (5
MHz). Binding of the chimeric GFP to zinc ions resulted in a rapid decrease of resonance frequency. Reversibility of the process was demonstrated via the removal of metal ions by EDTA. Nanoscale structural orientation of the chimeric GFP on the membrane was imaged by AFM. Association constant of the specific binding to metal ions was 2- to 3-fold higher than that of the non-specific adsorption, which was caused by the fluidization effect of the metal-chelating lipid molecules as well as the steric hindrance effect. This infers a possibility for a further development of biofunctionalized membrane. However, maximization is needed in order to attain closer advancement to a membrane-based sensor device.
Levulinic acid is a feasible platform chemical derived from acid-catalyzed hydrolysis of lignocellulose. The conversion of this substrate to (
S
)-γ-valerolactone ((
S
)-GVL) was investigated in a ...chemo-enzymatic reaction sequence that benefits from mild reaction conditions and excellent enantiomeric excess of the desired (
S
)-GVL. For that purpose, levulinic acid was chemically esterified over the ion exchange resin Amberlyst 15 to yield ethyl levulinate (LaOEt). The keto ester was successfully reduced by (
S
)-specific carbonyl reductase from
Candida parapsilosis
(CPCR2) in a substrate-coupled cofactor regeneration system utilizing isopropanol as cosubstrate. In classical batch experiments, a maximum conversion of 95 % was achieved using a 20-fold excess of isopropanol. Continuous reduction of LaOEt was carried out for 24 h, and a productivity of more than 5 mg (
S
)-ethyl-4-hydroxypentanoate (4HPOEt) per μg CPCR2 was achieved. Afterwards (
S
)-4HPOEt (>99%
ee
) was substituted to lipase-catalyzed lactonization using immobilized lipase B from
Candida antarctica
to yield (
S
)-GVL in 90 % overall yield and >99%
ee
.
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.
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.
Optically pure flurbiprofen: A novel deprotection strategy for the preparation of the starting material combined with decarboxylase (AMDase) variants optimized by enzyme engineering allowed the asymmetric synthesis of both enantiomers of the non‐steroidal anti‐inflammatory drug (NSAID) flurbiprofen in excellent yield and optical purity.
A sequential, chemoenzymatic process for a continuously operating production of the chiral β‐amino acid ester ethyl (S)‐3‐(benzylamino)butanoate was developed. The reactor set‐up combined a plug‐flow ...reactor for the thermal aza‐Michael addition of benzylamine to trans‐ethyl crotonate coupled to a subsequent packed‐bed reactor for the lipase (Novozym 435)‐catalyzed kinetic resolution of the racemic intermediate product, which was formed in the initial step. The coupled reactors were operated continuously for a time period of 4 days without significant loss of enzyme activity. The target β‐amino acid ester was obtained with 92% conversion in the plug‐flow reactor and 59% conversion in the packed bed reactor at high enantiomeric excess of >98%. A space‐time yield of 0.4 kg L−1 d−1 was calculated for the total reactor system and 1.8 kg L−1 d−1 based solely on the volume of the packed bed reactor. A total turnover number of 158,000 was calculated for the biocatalyst assuming the same deactivation rate as observed in batch experiments. The continuously operating, solvent‐free process thus represents an efficient method for the enantioselective production of a value added (S)‐β‐amino acid ester starting from cheap substrates.
Abbreviations: CALB=Candida antarctica lipase B; CSTR=continuously stirred tank reactor; di=inner diameter mm; ee=enantiomeric excess; ε=porosity; kcat,obs=apparent turnover number h−1; kdeact=enzyme deactivation constant h−1; L=length of reactor m; m̄p=average mass of a single particle g; N435=Novozym 435 (Candida antarctica lipase B immobilized on acrylic resin); PBR=packed‐bed reactor; PFR=plug‐flow reactor; PTFE=polytetrafluoroethylene; ρbp=bulk particle density kg m−3; ρp=density of carrier material kg m−3; Q=productivity kg ${{\rm kg}{{- 1\hfill \atop {\rm N435}\hfill}}}$ d−1; R=channel radius of curvature cm; ttn=total turnover number mol mol−1; T=temperature °C; τ=residence time h; uf=fluid velocity cm min−1; $\dot \nu $=volumetric flow rate mL min−1; νι=flow rate of substance mmol min−1; V=volume mL; χi=mole fraction of compound i; Xi=conversion of compound i; STY=space‐time yield kg L−1 d−1.
