Enzymatic synthesis of unsaturated fatty acid glucoside esters for dermo-cosmetic applications.

Unsaturated fatty acid alpha-butylglucoside esters were prepared by enzymatic esterification of alpha-butylglucoside in nonaqueous media. Conditions were firstly optimized using oleic acid as acyl group. Synthesis was possible in several solvents but the presence of water co-product in the medium limited the reaction to a thermodynamic equilibrium corresponding to a maximal conversion yield of 62%. In pure molten substrates, the removal of water under reduced pressure enabled yields superior to 95% to be obtained. Product profiles depended on enzyme origin : whatever the support, immobilized lipase B from Candida antarctica proved to be far more regioselective for the primary hydroxyl group of glucose than immobilized lipase from Rhizomucor miehei. Results obtained could be easily transposed to the acylation of alpha-butylglucoside with a commercial mixture of unsaturated fatty acids containing more than 60% of linoleic acid. The biocatalyst could be recycled more than ten times without any significant activity loss.     

Enzymatic synthesis of alpha-butylglucoside lactate: a new alpha-hydroxy acid derivative

An alpha-hydroxy acid derivative, alpha-butylglucoside lactate, was successfully prepared by enzymatic transesterification of alpha-butylglucoside with a lactate alkyl ester in a non-aqueous medium using immobilized lipase as biocatalyst. Ester synthesis in organic solvent was optimized. Solvent choice was made on the basis of substrate solubility and enzyme stability in the medium. A solvent-free reaction using butyllactate as lactate donor led to the highest yields. In the presence of 0.5M alphabutylglucoside and 100 g/L Novozym(R), a 67 % yield could be obtained within 40 h at 50 degrees C. However, the presence of butanol by-product limited the reaction to a maximum that could not be exceeded in closed systems. The elimination of the alcohol under reduced pressure resulted in the complete equilibrium shift of the transesterification reaction in favor of synthesis; below 15 mbars, more than 95% of 0.5M alpha-butylglucoside could be converted within 30 h. Moreover, simultaneous evaporation of water allowed hydrolysis of butyllactate to be eliminated. Consequently, a very high alpha-butylglucoside lactate concentration (170 g/) could be obtained in a single batch reaction. A single purification procedure, consisting of butyllactate extraction with hexane, enabled the product to be obtained at a purity above 95% (w/w). 1H and 13C NMR analysis later demonstrated that lactic acid was exclusively grafted onto the primary hydroxyl group of alphabutylglucoside.     

The lipase-catalyzed hydrolysis of lutein diesters in non-aqueous media is favored at extremely low water activities

The enzymatic hydrolysis of a mixture of lutein diesters from Marigold flower (Tagetes erecta) was performed both in organic solvents and supercritical CO(2) (SC-CO(2)) using two commercial lipases: lipase B from Candida antarctica (Novozym 435) and the lipase from Mucor miehei (Lipozyme RM IM). Both lipases showed an unexpected dependence of initial reaction rate with the initial water activity (a(wi)) in hexane, with the highest rates of hydrolysis taking place at the lowest a(wi) of the biocatalyst particles. The same result was observed using isooctane, toluene, or SC-CO(2). It is proposed that an increase in a(wi) generates a hydrophilic microenvironment that prevents efficient partitioning of the highly hydrophobic lutein diesters to the enzyme. The critical role of water in this system has not been reported for other hydrolytic reactions in low water media. Calculations of water available for hydrolysis from isotherm analysis, Karl-Fischer titration, and substrate conversion at a(wi) = 0.13, indicate that the extent of reaction is not limited by the amount of available water. Accordingly, the enzyme that holds the largest amount of water after prehydration at the same a(wi) (0.13) will yield the greatest substrate conversion and concentration of the free lutein product. The highest conversion occurred in SC-CO(2), which opens up new opportunities to develop a combined extraction-reaction process for the environmentally benign synthesis of lutein, an important nutraceutical compound.     

Substrate selectivity of lipases in the esterification of oleic acid, linoleic acid, linolenic acid and their all-trans-isomers and in the transesterification of cis/trans-isomers of linoleic acid methyl ester

The substrate selectivity of several microbial lipases has been examined in the esterification of oleic acid, linoleic acid, linolenic acid and their all-trans-isomers and in the alcoholysis of isomeric linoleic acid methyl esters with n-butanol. Lipases from Candida cylindracea and Mucor miehei preferred fatty acids and methyl esters with a (first) cis double bond in 9-position, while Chirazyme L-5, a Candida antarctica lipase A, had a preference for trans-9 unsaturated substrates.     

Selection of CalB immobilization method to be used in continuous oil transesterification: analysis of the economical impact

Enzymatic transesterification of triglycerides in a continuous way is always a great challenge with a large field of applications for biodiesel, bio-lubricant, bio-surfactant, etc. productions. The lipase B from Candida antarctica (CalB) is the most appreciated enzyme because of its high activity and its non-regio-selectivity toward positions of fatty acid residues on glycerol backbone of triglycerides. Nevertheless, in the field of heterogeneous catalysis, we demonstrated that the medium hydrophilic nature of the support used for its commercial form (Lewatit VPOC1600) is a limitation. Glycerol is adsorbed onto support inducing drastic decrease in enzyme activity. Glycerol would form a hydrophilic layer around the enzyme resulting in diffusional limitations during triglyceride transfer to the enzyme. Accurel MP, a very hydrophobic macroporous polymer of propylene, was found not to adsorb glycerol. Immobilization conditions using this support were optimized. The best support was Accurel MP1001 (particle size<1000 μm) and a pre-treatment of the support with acetone instead of ethanol enables the adsorption rate and the immobilized enzyme quantity to be maximized. An economical approach (maximization of the process net present value) was expanded in order to explore the impact of immobilization on development of an industrial packed bed reactor. The crucial ratio between the quantity of lipase and the quantity of support, taking into account enzyme, support and equipped packed bed reactor costs was optimized in this sense. The biocatalyst cost was found as largely the main cost centre (2-10 times higher than the investments for the reactor vessel). In consequence, optimal conditions for immobilization were a compromise between this immobilization yield (90% of lipase immobilized), biocatalyst activity, reactor volume and total investments.     

Lipase-catalysed synthesis of glucose fatty acid esters in tert-butanol

Synthesis of 6-O-acylate--d-glycopyranose from underivatised substrates in anhydrous tert-butanol was achieved using immobilised lipases from Candida antarctica and Mucor miehei. Except for acetic acid, the initial reaction rates with the C. antarctica lipase were independent of acyl donor chain lengths and in a range of 3.9±0.4 mol glucose converted min–1 g enzyme preparation. The catalytic activity of the M. miehei lipase increased with increasing acyl donor chain length with a maximum for stearic acid of 0.45 mol min–1 g. Using maltose as substrate, the catalytic activity decreased by a factor of 48 and 20 with the lipase from C. antarctica and M. miehei, respectively, while with maltotriose no reaction was observed.     

Enzymatic conversion of sunflower oil to biodiesel in a solvent-free system: Process optimization and the immobilized system stability

The feasibility of using the commercial immobilized lipase from Candida antarctica (Novozyme 435) to synthesize biodiesel from sunflower oil in a solvent-free system has been proved. Using methanol as an acyl acceptor and the response surface methodology as an optimization technique, the optimal conditions for the transesterification has been found to be: 45 ^oC, 3% of enzyme based on oil weight, 3:1 methanol to oil molar ratio and with no added water in the system. Under these conditions, >99% of oil conversion to fatty acid methyl ester (FAME) has been achieved after 50 h of reaction, but the activity of the immobilized lipase decreased markedly over the course of repeated runs. In order to improve the enzyme stability, several alternative acyl acceptors have been tested for biodiesel production under solvent-free conditions. The use of methyl acetate seems to be of great interest, resulting in high FAME yield (95.65%) and increasing the half-life of the immobilized lipase by about 20.1 times as compared to methanol. The reaction has also been verified in the industrially feasible reaction system including both a batch stirred tank reactor and a packed bed reactor. Although satisfactory performance in the batch stirred tank reactor has been achieved, the kinetics in a packed bed reactor system seems to have a slightly better profile (93.6 ± 3.75% FAME yield after 8–10 h), corresponding to the volumetric productivity of 48.5 g/(dm³ h). The packed bed reactor has operated for up to 72 h with almost no loss in productivity, implying that the proposed process and the immobilized system could provide a promising solution for the biodiesel synthesis at the industrial scale.     

Continuous enantioselective esterification of trans-2-phenyl-1-cyclohexanol using a new Candida rugosa lipase in a packed bed bioreactor

Enantioselective resolution of trans-2-phenyl-1-cyclohexanol (TPCH) by a Candida rugosa lipase, obtained by fermentation in the laboratory, and immobilised on EP100 polypropylene powder has been carried out using isooctane as solvent and propionic acid as esterifying agent. The study have included the utilisation of this biocatalyst in a batch process and the optimisation of the esterification conditions by means of a Box-Hunter-based experimental design. The main variables controlling the process, concentration of acid and alcohol, have been numerically optimised using initial esterification rate as objective function. The optimal concentrations for the batch process were 50 mM for the alcohol and 71 mM for the acid. This esterification reaction kinetics corresponded to a reversible Michaelis-Menten kinetic law for the optimal conditions, which has permitted to select a plug-flow packed bed bioreactor as the most appropriate configuration to minimise the residence time and to avoid shear stress effect on the biocatalyst. The behaviour of the continuous packed bed bioreactor at two different residence times (302 and 582 min) was in accordance with predictions from batch experiments, with slightly deviations (less than 10%). Continuous experiments maintained high values of enantioselectivity (enantiomeric factor was practically 1) and conversion near equilibrium value (35%) when long-time operation was carried out. Besides, long-time stability of biocatalyst has permitted to scale-up the production of enantioenriched (1R,2S)-TPCH propionate to yield gram quantities.     

Production of flavor esters catalyzed by CALB-displaying Pichia pastoris whole-cells in a batch reactor

Candida antarctica lipase B (CALB) has been employed as an efficient catalyst in the preparation of many flavor esters. A CALB-displaying yeast whole-cell biocatalyst could be an attractive alternative to commercial immobilized CALB because of its low-cost preparation and high enzymatic activity. We investigated the potential application of CALB-displaying Pichia pastoris cells for the production of flavor esters. The optimal conditions for flavor esters synthesis by this biocatalyst were determined in 50-ml shake flasks. Under optimized conditions, the synthesis of 12 kinds flavor esters were scaled up in a 5-l batch stirred reactor. Among these, the mole conversions of 10 exceeded 95% after reactions for 4h. In addition, this biocatalyst showed good tolerance for high substrates concentration and excellent operational stability. Repeated use of the cells in 10 batches resulted in an activity loss of less than 10%. Thus, CALB-displaying P. pastoris whole cells are robust biocatalysts with potential commercial application in the large-scale production of flavor esters in non-aqueous media.     

Lipase-catalyzed esterification of conjugated linoleic acid with sorbitol: a kinetic study

The kinetics of esterification of conjugated linoleic acid (CLA) with sorbitol in acetone was investigated. An immobilized lipase from Candida antarctica (Chirazyme L-2) was used as the biocatalyst. A 2(2) x 3 factorial design was employed to find an experimental region in which one obtains a high rate of formation of the diester product. Best results were obtained at 10 degrees C using a CLA to sorbitol molar ratio of 5 and a biocatalyst loading of 150 mg/mL of acetone. Under these conditions, in 72 h one obtains a nearly quantitative yield (ca. 98%) of the diester of sorbitol with CLA. To minimize formation of products with degrees of esterification greater than two, the reaction should be carried out at 10 degrees C. A kinetic model developed using the King-Altman method was employed to fit the data. Use of the steady-state approximation for the monoester and an assumption that the concentration of sorbitol was constant and equal to its solubility limit permit one to minimize the number of parameters necessary to model the reaction network. Nonlinear regression analysis based on either two or three parameters provides very good fits of the multiresponse data in the presence or absence of triesters, respectively.     

Six‐membered cyclic carbonates from trimethylolpropane: Lipase‐mediated synthesis in a flow reactor and in silico evaluation of the reaction

Six‐membered cyclic carbonates with hydroxyl and methoxycarbonyloxy functional groups were prepared by transesterification of trimethylolpropane (TMP) with dimethylcarbonate (DMC) by solvent‐free lipase‐mediated flow reaction followed by thermal cyclization. The flow reaction efficiency was evaluated using different configurations of reactor consisting of packed beds of Novozym®435 (immobilized Candida antarctica lipase B—CalB—a.k.a. N435) and molecular sieves, flowrate, and biocatalyst loads. The mixed column of the biocatalyst and molecular sieves, allowing rapid and efficient removal of the by‐product—methanol—was the most efficient setup. Higher conversion (81.6%) in the flow reaction compared to batch process (72%) was obtained using same amount of N435 (20% (w/w) N435:TMP) at 12 h, and the undesirable dimer and oligomer formation were suppressed. Moreover, the product was recovered easily without extra separation steps, and the biocatalyst and the molecular sieves remained intact for subsequent regeneration and recycling. The reaction of CalB with DMC and the primary transesterification product, monocarbonated TMP, respectively, as acyl donors was evaluated by in silico modeling and empirically to determine the role of the enzyme in the formation of cyclic carbonates and other side products. DMC was shown to be the preferred acyl donor, suggesting that TMP and its carbonated derivatives serve only as acyl acceptors in the lipase‐catalyzed reaction. Subsequent cyclization to cyclic carbonate is catalyzed at increased temperature and not by the enzyme. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:375–382, 2017     

Regioselective synthesis of ethoxylated glycoside esters using beta-glucosidase in supersaturated solutions and lipases in organic solvents

Three ethoxylated glycosides, tetraethylene glycol beta-D-glucoside, tetraethylene glycol beta-D-xyloside, and methoxy triethyleneglycol beta-D-glucoside, were prepared via almond beta-glucoside-catalyzed (trans)glycosylation carried out in supersaturated solutions of glucose or p-nitrophenyl beta-D-xyloside and the respective polyethylene glycols. The products were isolated and further modified by enzymatic esterification with Candida antarctica and Mucor miehei lipases. The latter enzyme showed a much greater selectivity for the primary hydroxyl group on the polyethylene glycol chain of the glucoside substrate, thus enabling us to obtain exclusively the corresponding monoester, omega-O-oleoyl tetraethylene glycol beta-D-glucoside. Novozyme was used for the preparative synthesis of two other monoesters, 6-O-oleoyl (methoxy triethyleneglycol) beta-D-glucoside and omega-O-oleoyl tetraethylene glycol beta-D-xyloside. Two diesters, di-oleoyl tetraethylene glycol beta-D-glucoside and tetraethylene-bis(6-0-oleoyl glucoside) were also synthesized in good yields using this lipase. Copyright 1998 John Wiley & Sons, Inc.     

Effect of water activity and immobilization on fatty acid selectivity for esterification reactions mediated by lipases

The effect of water activity (a(w)) and immobilization on fatty acid (FA) selectivity of Burkholderia (formerly Pseudomonas) cepacia, Rhizomucor miehei, Candida antarctica (type B), and Candida rugosa lipases in esterification reactions was determined. Studies were based on measuring ester formation in multicompetitive reaction mixtures containing either the homologous series of even carbon number n-chain saturated FA (C4-C18) or a series of n-chain (un)saturated FA (C18:X, where X = 0-3 double bonds) as cosubstrates with 1,3-propanediol in ter-butyl methyl ether at a(w) of 0.19, 0.69, and 0.90. Activity and FA selectively patterns were similar for free and Celite-adsorbed lipases in response to changes in a(w'), although specific effects were observed for selectivity of B. cepacia and C. rugosa lipases toward C16 and C4/C6 FA, respectively. Also, selectivity toward unsaturated C18:X FA as a group was modulated by changes in a(w) for three of the four lipase studied. Resin-fixed lipases from R. miehei and C. antarctica exhibited profound differences in activity and FA selectively in response to changes in a(w'), relative to free and Celite-bound forms. These findings suggest that FA selectivity for lipid modification is influenced by a(w) and immobilization, but that each lipase has a characteristic response to these factors in a manner that cannot be predicted.     

Highly enantioselective esterification of racemic ibuprofen in a packed bed reactor using immobilised Rhizomucor miehei lipase

A systematic study of the enantioselective resolution of ibuprofen by commercial Rhizomucor miehei lipase (Lipozyme(R) IM20) has been carried out using isooctane as solvent and butanol as esterificating agent. The main variables controlling the process (temperature, ibuprofen concentration, ratio butanol:ibuprofen) have been studied using an orthogonal full factorial experimental design, in which the selected objective function was enantioselectivity. This strategy has resulted in a polynomial function that describes the process. By optimizing this function, optimal conditions for carrying out the esterification of racemic ibuprofen have been determined. Under these conditions, enantiomeric excess and total conversion values were 93.8% and 49.9%, respectively, and the enantioselectivity was 113 after 112 h of reaction. These conditions have been considered in the design of a continuous reactor to scale up the process. The esterification of ibuprofen was properly described by pseudo first-order kinetics. Thus, a packed bed reactor operating as a plug-flow reactor (PFR) is the most appropriate in terms of minimizing the residence time compared with a continuous stirred tank reactor (CSTR) to achieve the same final conversion. This reactor shows a similar behavior in terms of enantioselectivity, enantiomeric excess, and conversion when compared with batch reactors. A residence-time distribution (RTD) shows that the flow model is essentially a plug flow with a slight nonsymmetrical axial dispersion (Peclet number = 43), which was also corroborated by the model of CSTR in series. The stability of the system (up to 100 h) and the possibility of reutilization of the enzyme (up to four times) lead to consider this reactor as a suitable configuration for scale up of the process.     

Comparative fatty acid selectivity of lipases in esterification reactions with glycerol and diol analogues in organic media

Reaction selectivity of Pseudomonas cepacia, Rhizomucor miehei, and Candida antarctica B lipases was assessed in multicompetitive esterification reaction mixtures containing an homologous series of n-chain even carbon number fatty acid (FA; C4-C18) substrates and a single alcohol cosubstrate (glycerol, 1,2-propanediol (1,2-PD), or 1, 3-propanediol (1,3-PD)) in tert-butyl methyl ether at water activity of 0.69 or 0.90 and a reaction temperature of 35 degrees C. For P. cepacia lipase, the ordinal patterns of FA selectivities observed were, with glycerol, C8 > C10, C6, C16 > other FA; with 1,2-PD and 1, 3-PD, C16 > C8 > C14 > other FA. For R. miehei lipase, the ordinal patterns of FA selectivities observed were, with glycerol, C8 > C12 > C10, C14 > other FA; with 1,2-PD and 1,3-PD, C8 > C12 > other FA. For C. antarctica B lipase, the ordinal patterns of FA selectivities observed were, with glycerol, C8 > C10, C6, C12 > other FA; with 1, 2-PD, C8 > C10, C6 > other FA; and with 1,3-PD, C8 > C10 > C6 > other FA. The differences in selectivity among FA ranged up to 16-fold, depending upon the lipase and alcohol cosubstrate used. These findings represent intrinsic and substrate-modulated features of FA selectivities that are of particular relevance to the use of lipases for acylglycerol synthesis reactions.     

Enzymatic resolution to (-)-ormeloxifene intermediates from their racemates using immobilized Candida rugosa lipase

In the synthesis of (-)-ormeloxifene, a drug candidate recently under development, enzymatic resolution of potential intermediates can be carried out using a simple, practical method. Five commercially available lipases, Candida rugosa lipase, Candida antarctica lipase B, Mucor miehei lipase, Pseudomonas cepacia lipase, and Humicola lanuginosa lipase, all immobilized on Accurel(R), were initially screened in combination with four different substrates belonging to the class of phenyl esters. Excellent stereoselectivity was observed using C. rugosa lipase with an acetate as substrate, but low reaction rates were observed in scale-up experiments. However, by changing the acyl part of the ester into a hexanoyl moiety and subjecting this substrate to enzymatic hydrolysis in aqueous acetonitrile at room temperature by C. rugosa lipase, it became possible to run the reaction to a 50% conversion on a 10 g scale within a period of 4 h, obtaining a phenolic product of more than 95% ee that could be converted to the target molecule, (-)-ormeloxifene, in two synthetic steps. Simple recovery of the immobilized enzyme by filtration allowed multiple recycling of the catalyst without significant loss of enzymatic activity. Capillary electrophoresis with sulfobutyl ether beta-cyclodextrin as a chiral buffer additive and acetonitrile as an organic modifier was demonstrated to provide an excellent chiral analytical tool for monitoring the enzymatic reactions.     

Lipase-mediated hydrolysis of blackcurrant oil

Four commercially available lipases, both free and immobilized, were tested for their ability to catalyze hydrolysis of blackcurrant (Ribes nigrum) oil using two different approaches. The lipase from Mucor miehei was studied free and immobilized in two different ways. The former series of enzymic reactions were performed in tap water at 40 degrees C, but the latter series of enzymic processes were carried out in mixtures of isooctane and phosphate buffer (in a typical 2/1 ratio of the components) at 30 degrees C. These conditions were optimized to increase and/or to maximize the yields of the products, which were priority targets in this study. A rate of hydrolysis and a selective preference of the hydrolytic enzymes towards fatty acids, with a special focus on enrichment of alpha-linolenic acid and/or gamma-linolenic acid, were studied. Higher rates of hydrolysis of the blackcurrant oil in the former series of reactions were observed with the immobilized lipase from Pseudomonas cepacia used as biocatalyst. In the latter approach, the most favorable results of the rate of hydrolysis of the target blackcurrant oil were achieved with the immobilized lipase from Mucor miehei employed as biocatalyst. Only three lipases, selected from a series of lipases tested during this investigation, displayed specificity towards alpha-linolenic acid and gamma-linolenic acid, i.e. the immobilized lipase from P. cepacia, lipase from M. miehei and lipase from P. fluorescens.     

General trend of lipase to self-assemble giving bimolecular aggregates greatly modifies the enzyme functionality

Three microbial lipases (those from Candida rugosa, Humicola lanuginosa, and Mucor miehei) have been found to exhibit a tendency to form bimolecular aggregates in solution even at very low enzyme concentrations (44 microg/mL) in the absence of a detergent, as detected by gel filtration. The monomolecular form of the enzymes was found as unique only at low enzyme concentration and in the presence of detergents. However, in the case of the lipase B from Candida antarctica, no bimolecular form could be identified even at enzyme concentrations as high as 1.2 mg/mL in the absence of detergent. It has been stated that bimolecular and monomolecular structures display very different functional properties: (i) the enzyme specific activity decreased when the lipase concentration increased; (ii) the bimolecular form was much more stable than the monomeric one yielding a higher optimal T (increasing between 5 and 10 degrees C) and higher stability in inactivation experiments (the dimer half-life became several orders of magnitude higher than that of the monomer); (iii) the enantioselectivity depended on the enzyme concentration even after immobilization. For example, with use of the lipase from H. lanuginosa, the enantiomeric excess of the remaining ester in the hydrolysis of fully soluble ethyl ester of (R,S)-2-hydroxy-4-phenylbutanoic acid varied from 4 to 57 when the concentrated or diluted enzyme immobilized on PEI support, respectively, was used. It seems that the bimolecular structure of lipases might be formed by two open lipase molecules (interfacially activating each other) in very close contact and hence with a very altered active center.     

Enrichment of butteroil with conjugated linoleic acid via enzymatic interesterification (acidolysis) reactions

Microbial lipases from Candida cylindracea, Pseudomonas sp., Mucor miehei and Candida antarctica were screened for their ability to incorporate conjugated linoleic acid into butteroil triacylglycerides. The lipase from Candida antarctica was employed in a substrates-only medium to increase the conjugated linoleic acid content of the acylglycerides from 0.6 to 15 g/100 g fat.     

A continuous single organic phase process for the lipase catalyzed synthesis of peroxy acids increases productivity

The design of an optimal process is particularly crucial when the reactants deactivate the biocatalyst. The reaction cascades of the chemo‐enzymatic epoxidation where the intermediate peroxy acid is produced by an enzyme are still limited by enzyme inhibition and deactivation by hydrogen peroxide. To avoid additional effects caused by interfaces (aq/org) and to reduce the process limiting deactivation by the substrate hydrogen peroxide, a single‐phase concept was applied in a fed‐batch and a continuous process (stirred tank), without the commonly applied addition of a carrier solvent. The synthesis of peroxyoctanoic acid catalyzed by Candida antarctica lipase B was chosen as the model reaction. Here, the feasibility of this biocatalytic reaction in a single‐phase system was shown for the first time. The work shows the economic superiority of the continuous process compared to the fed‐batch process. Employing the fed‐batch process reaction rates up to 36 mmol h^?1 per gram_cat, and a maximum yield of 96 % was achieved, but activity dropped quickly. In contrast, continuous operation can maintain long‐term enzyme activity. For the first time, the continuous enzymatic reaction could be performed for 55 h without any loss of activity and with a space‐time yield of 154 mmol L^?1 h^?1, which is three times higher than in the fed‐batch process. The higher catalytic productivity compared to the fed‐batch process (34 vs. 18 g_Prod g^?1_cat) shows the increased enzyme stability in the continuous process.