Juan M. Bolivar

Biotransformations in microstructured reactors: more than flowing with the stream?

The state of the art in the application of microstructured flow reactors for biocatalytic process research is reviewed. A microstructured reactor that is fully automated and analytically equipped presents a powerful screening tool with which to perform biocatalyst selection and optimization of process conditions at intermediary stages of process development. Enhanced mass transfer provided by the microstructured reactor can be exploited for process intensification, particularly during multiphase biocatalytic processing where mass transfer across phase boundaries is often limiting. Reversible immobilization of enzymes in microchannels remains a challenge for flexible realization of biotransformations in microstructured reactors. Compartmentalization in microstructured reactors could be useful in performing multistep chemoenzymatic conversions.

Positively Charged Mini-Protein Zbasic2 As a Highly Efficient Silica Binding Module: Opportunities for Enzyme Immobilization on Unmodified Silica Supports

Silica is a highly attractive support material for protein immobilization in a wide range of biotechnological and biomedical-analytical applications. Without suitable derivatization, however, the silica surface is not generally usable for attachment of proteins. We show here that Z(basic2) (a three α-helix bundle mini-protein of 7 kDa size that exposes clustered positive charges from multiple arginine residues on one side) functions as highly efficient silica binding module (SBM), allowing chimeras of target protein with SBM to become very tightly attached to underivatized glass at physiological pH conditions. We used two enzymes, d-amino acid oxidase and sucrose phosphorylase, to demonstrate direct immobilization of Z(basic2) protein from complex biological samples with extremely high selectivity. Immobilized enzymes displayed full biological activity, suggesting that their binding to the glass surface had occurred in a preferred orientation via the SBM. We also show that charge complementarity was the main principle of affinity between SBM and glass surface, and Z(basic2) proteins were bound in a very strong, yet fully reversible manner, presumably through multipoint noncovalent interactions. Z(basic2) proteins were immobilized on porous glass in a loading of 30 mg protein/g support or higher, showing that attachment via the SBM combines excellent binding selectivity with a technically useful binding capacity. Therefore, Z(basic2) and silica constitute a fully orthogonal pair of binding module and insoluble support for oriented protein immobilization, and this opens up new opportunities for the application of silica-based materials in the development of supported heterogeneous biocatalysts.

Oriented and selective enzyme immobilization on functionalized silica carrier using the cationic binding module Zbasic2: Design of a heterogeneous D‐amino acid oxidase catalyst on porous glass

D‐Amino acid oxidase from Trigonopsis variabilis (TvDAO) is applied in industry for the synthesis of pharmaceutical intermediates. Because free TvDAO is extremely sensitive to exposure to gas–liquid interfaces, biocatalytic processing is usually performed with enzyme immobilizates that offer enhanced stability under bubble aeration. We herein present an “Immobilization by Design” approach that exploits engineered charge complementarity between enzyme and carrier to optimize key features of the immobilization of TvDAO. A fusion protein between TvDAO and the positively charged module Zbasic2 was generated, and a corresponding oppositely charged carrier was obtained by derivatization of mesoporous glass with 3‐(trihydroxysilyl)‐1‐propane‐sulfonic acid. Using 250 mM NaCl for charge screening at pH 7.0, the Zbasic2 fusion of TvDAO was immobilized directly from E. coli cell extract with almost absolute selectivity and full retention of catalytic effectiveness of the isolated enzyme in solution. Attachment of the homodimeric enzyme to the carrier was quasi‐permanent in low‐salt buffer but fully reversible upon elution with 5 M NaCl. Immobilized TvDAO was not sensitive to bubble aeration and received substantial (≥tenfold) stabilization of the activity at 45°C as compared to free enzyme, suggesting immobilization via multisubunit oriented interaction of enzyme with the insoluble carrier. The Zbasic2 enzyme immobilizate was demonstrated to serve as re‐usable heterogeneous catalyst for D‐amino acid oxidation. Zbasic2‐mediated binding on a sulfonic acid group‐containing glass carrier constitutes a generally useful strategy of enzyme immobilization that supports transition from case‐specific empirical development to rational design. Biotechnol. Bioeng. 2012; 109:1490–1498.

Dual-lifetime referencing (DLR): a powerful method for on-line measurement of internal pH in carrier-bound immobilized biocatalysts

BackgroundIndustrial-scale biocatalytic synthesis of fine chemicals occurs preferentially as continuous processes employing immobilized enzymes on insoluble porous carriers. Diffusional effects in these systems often create substrate and product concentration gradients between bulk liquid and the carrier. Moreover, some widely-used biotransformation processes induce changes in proton concentration. Unlike the bulk pH, which is usually controlled at a suitable value, the intraparticle pH of immobilized enzymes may deviate significantly from its activity and stability optima. The magnitude of the resulting pH gradient depends on the ratio of characteristic times for enzymatic reaction and on mass transfer (the latter is strongly influenced by geometrical features of the porous carrier). Design and selection of optimally performing enzyme immobilizates would therefore benefit largely from experimental studies of the intraparticle pH environment. Here, a simple and non-invasive method based on dual-lifetime referencing (DLR) for pH determination in immobilized enzymes is introduced. The technique is applicable to other systems in which particles are kept in suspension by agitation.ResultsThe DLR method employs fluorescein as pH-sensitive luminophore and Ru(II) tris(4,7-diphenyl-1,10-phenantroline), abbreviated Ru(dpp), as the reference luminophore. Luminescence intensities of the two luminophores are converted into an overall phase shift suitable for pH determination in the range 5.0-8.0. Sepabeads EC-EP were labeled by physically incorporating lipophilic variants of the two luminophores into their polymeric matrix. These beads were employed as carriers for immobilization of cephalosporin C amidase (a model enzyme of industrial relevance). The luminophores did not interfere with the enzyme immobilization characteristics. Analytical intraparticle pH determination was optimized for sensitivity, reproducibility and signal stability under conditions of continuous measurement. During hydrolysis of cephalosporin C by the immobilizate in a stirred reactor with bulk pH maintained at 8.0, the intraparticle pH dropped initially by about 1 pH unit and gradually returned to the bulk pH, reflecting the depletion of substrate from solution. These results support measurement of intraparticle pH as a potential analytical processing tool for proton-forming/consuming biotransformations catalyzed by carrier-bound immobilized enzymes.ConclusionsFluorescein and Ru(dpp) constitute a useful pair of luminophores in by DLR-based intraparticle pH monitoring. The pH range accessible by the chosen DLR system overlaps favorably with the pH ranges at which enzymes are optimally active and stable. DLR removes the restriction of working with static immobilized enzyme particles, enabling suspensions of particles to be characterized also. The pH gradient developed between particle and bulk liquid during reaction steady state is an important carrier selection parameter for enzyme immobilization and optimization of biocatalytic conversion processes. Determination of this parameter was rendered possible by the presented DLR method.

Oriented Immobilization of Enzymes Made Fit for Applied Biocatalysis: Non‐Covalent Attachment to Anionic Supports using Zbasic2 Module

Immobilization of enzymes on insoluble carriers is a key technology for biocatalytic process development. The main advantage of immobilized enzymes is that they are readily separated from solution and, therefore, support continuous processing in combination with an integrated re-use of the catalyst. Full realization of the benefit of immobilization is often seen upon moving from the laboratoryto a larger-scale process operation, and the majority of enzymatic transformations performed on a multiton-per-year manufacturing scale employ carrier-bound catalysts. 3] There is usually a significant cost contribution of immobilization to the total costs of the catalyst. Therefore, the maximum amount of enzyme activity that can be loaded on the unit mass of a carrier is a clear target for optimization. Available methods for enzyme immobilization can be categorized according to whether positioning of the protein on the carrier surface is specific or random in orientation. Specific positioning facilitates the design of the immobilization for optimum retention of the activity of the free enzyme in the carrier-bound catalyst. 5] However, specific positioning usually falls short, often by orders of magnitude, of the high protein loading capacity of common techniques of random immobilization, which represents the current industrial standard. Therefore, a broadly applicable method that combines the advantages of specific and random modes of protein binding to achieve immobilization of enzymes on carriers of industrial use would present a major advancement. The strategy presented herein was originally developed for an application in protein purification and exploits charge complementarity between the cationic “binding module” Zbasic2 and the anionic supports. Zbasic2 fusion partners that display a negative net charge at the applied pH, will experience charge repulsion from the carrier surface (Scheme 1). Adsorption of Zbasic2 fusion proteins should thus occur in a highly directed manner, driven almost exclusively by Zbasic2. By using two industrially applied enzymes, for example the d-amino acid oxidase from Trigonopsis variabilis (TvDAO, EC 1.4.3.3) 5b, 7] and sucrose phosphorylase from Leuconostoc mesenteroides (LmSPase, EC 2.4.1.7), we show that protein chimeras harboring Zbasic2 at their respective N-terminus are bound in high density ( 200 mgprotein gdry carrier ) and yield ( 95 % of free-enzyme activity) on common porous resins displaying anionic sulfoalkyl surface groups. Non-covalent immobilization of each Zbasic2 protein was highly selective from crude protein mixtures, showed useful resistance to leaching, and, because it was largely reversible upon applying a high salt concentration, allowed easy regeneration of the carrier material for multiple rounds of immobilization. For each of the two enzymes chosen, fusion to the Zbasic2 module did not compromise recombinant protein production in Escherichia coli (E. coli) and was fully compatible with the catalytic function in as-isolated preparations. Note, that TvDAO is a functional homodimer, whereas LmSPase is a monomer, implying that the proposed method is in principle applicable to proteins having a quarternary structural organization. Preparation and characterization of Zbasic2 enzymes. Zbasic2 is an engineered, arginine (Arg)-rich variant of the Z domain, a 58-amino acid (7 kDa), three-helix bundle obtained from the B domain of staphylococcal protein A. We used subcloning in the previously described plasmid vector pT7ZbQGKlenow to achieve fusion of Zbasic2 (including the 3C protease cleavage site) to the N-terminus of TvDAO and LmSPase (see the Supporting Information). Chimeric enzymes were produced in E. coli BL21 (DE3) by using conditions known to result in useful overexpression of the respective “native” protein. Zbasic2 fusions were purified without further optimization in an approximately 30 % (LmSPase) and 15 % (TvDAO) yield by using cation exchange chromatography (Figures S1 and S2), and enzyme preparations, which were pure by the criterion of a single protein band in SDS PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis, Figures S1 and S2), were characterized biochemically (see the Supporting Information for the methods used). The specific activities of the Zbasic2 forms of TvDAO and LmSPase were identical, within the limits of experimental error, Scheme 1. Strategy for oriented immobilization of chimeric enzymes through the Zbasic2 binding module. A structural model of sucrose phosphorylase from Leuconostoc mesenteroides fused to Zbasic2 was generated by using Modeller9v8. Surface charge calculation and visualization were done by using UCSF Chimera 1.5.

Shine a light on immobilized enzymes: real-time sensing in solid supported biocatalysts

Enzyme immobilization on solid supports has been key to biotransformation development. Although technologies for immobilization have largely reached maturity, the resulting biocatalysts are not well understood mechanistically. One limitation is that their internal environment is usually inferred from external data. Therefore, biological consequences of the immobilization remain masked by physical effects of mass transfer, obstructing further development. Work reviewed herein shows that opto-chemical sensing performed directly within the solid support enables the biocatalysts internal environment to be uncovered quantitatively and in real time. Non-invasive methods of intraparticle pH and O2 determination are presented, and their use as process analytical tools for development of heterogeneous biocatalysts is described. Method diversification to other analytes remains a challenging task for the future.

Quantitating intraparticle O2 gradients in solid supported enzyme immobilizates: Experimental determination of their role in limiting the catalytic effectiveness of immobilized glucose oxidase†

Enzymatic O2‐dependent oxidations are receiving increased attention for use in fine chemicals synthesis. Solid supported oxidation catalysts often show poor efficiency due to pronounced O2 diffusion restriction. Internal O2 supply therefore constitutes a key parameter for optimizing the enzyme immobilization. We herein describe an optical sensing method for quantitation of space‐averaged intraparticle O2 concentrations in porous Sepabeads carriers. The method applies phosphorescence lifetime measurements on Sepabeads labeled with an O2 sensitive indicator dye. Using glucose oxidase immobilized at different loadings (0.005–12 mg/g) on labeled Sepabeads, we analyzed in real time during the enzymatic reaction the formation of O2 concentration differences between bulk liquid and the intraparticle environment. We show that the O2 gradient at apparent steady state increased with increasing enzyme loading, so that O2 eventually became totally depleted from inside the highly loaded carriers. We also show that the residual intraparticle O2 concentration was correlated with the catalytic effectiveness factor (η) of the enzyme immobilizate used, thus providing a direct measure of the magnitude of O2 diffusion limitation. Once corrected for diffusional effect, η was no longer dependent on enzyme loading and its constant value now described the intrinsic activity of immobilized glucose oxidase. Three common procedures of enzyme immobilization, involving adsorption, cross‐linking, and covalent attachment, are shown to differ widely concerning the obtained intrinsic activity. Therefore, intraparticle O2 concentration data enable distinction between diffusional restriction and activity loss as the two principal factors limiting the effectiveness of immobilized O2 dependent enzymes, and thus they inform rational design of an optimally active oxidation biocatalyst on solid support. Biotechnol. Bioeng. 2013; 110: 2086–2095.

Let the substrate flow, not the enzyme: Practical immobilization of d-amino acid oxidase in a glass microreactor for effective biocatalytic conversions

Exploiting enzymes for chemical synthesis in flow microreactors necessitates their reuse for multiple rounds of conversion. To achieve this goal, immobilizing the enzymes on microchannel walls is a promising approach, but practical methods for it are lacking. Using fusion to a silica‐binding module to engineer enzyme adsorption to glass surfaces, we show convenient immobilization of d‐amino acid oxidase on borosilicate microchannel plates. In confocal laser scanning microscopy, channel walls appeared uniformly coated with target protein. The immobilized enzyme activity was in the range expected for monolayer coverage of the plain surface with oxidase (2.37 × 10−5 nmol/mm2). Surface attachment of the enzyme was completely stable under flow. The operational half‐life of the immobilized oxidase (25°C, pH 8.0; soluble catalase added) was 40 h. Enzymatic oxidation of d‐Met into α‐keto‐γ‐(methylthio)butyric acid was characterized in single‐pass and recycle reactor configurations, employing in‐line measurement of dissolved O2, and off‐line determination of the keto‐acid product. Reaction‐diffusion time‐scale analysis for different flow conditions showed that the heterogeneously catalyzed reaction was always slower than diffusion of O2 to the solid surface (DaII ≤ 0.3). Potential of the microreactor for intensifying O2‐dependent biotransformations restricted by mass transfer in conventional reactors is thus revealed. Biotechnol. Bioeng. 2016;113: 2342–2349.

Development of a fully integrated falling film microreactor for gas-liquid-solid biotransformation with surface immobilized O2 -dependent enzyme.

Microstructured flow reactors are powerful tools for the development of multiphase biocatalytic transformations. To expand their current application also to O2‐dependent enzymatic conversions, we have implemented a fully integrated falling film microreactor that provides controllable countercurrent gas–liquid phase contacting in a multi‐channel microstructured reaction plate. Advanced non‐invasive optical sensing is applied to measure liquid‐phase oxygen concentrations in both in‐ and out‐flow as well as directly in the microchannels (width: 600 μm; depth: 200 μm). Protein–surface interactions are designed for direct immobilization of catalyst on microchannel walls. Target enzyme (here: d‐amino acid oxidase) is fused to the positively charged mini‐protein Zbasic2 and the channel surface contains a negatively charged γ‐Al2O3 wash‐coat layer. Non‐covalent wall attachment of the chimeric Zbasic2_oxidase resulted in fully reversible enzyme immobilization with fairly uniform surface coverage and near complete retention of biological activity. The falling film at different gas and liquid flow rates as well as reactor inclination angles was shown to be mostly wavy laminar. The calculated film thickness was in the range 0.5–1.3 × 10−4 m. Direct O2 concentration measurements at the channel surface demonstrated that the liquid side mass transfer coefficient (KL) for O2 governed the overall gas/liquid/solid mass transfer and that the O2 transfer rate (≥0.75 mM · s−1) vastly exceeded the maximum enzymatic reaction rate in a wide range of conditions. A value of 7.5 (±0.5) s−1 was determined for the overall mass transfer coefficient KLa, comprising a KL of about 7 × 10−5 m · s−1 and a specific surface area of up to 105 m−1. Biotechnol. Bioeng. 2016;113: 1862–1872.

Multiphase biotransformations in microstructured reactors: opportunities for biocatalytic process intensification and smart flow processing

Abstract Enzymes are gaining increased importance as highly selective catalysts for green chemical synthesis. Multiphase microreaction systems are emerging tools for the development of enzyme-catalysed transformations involving two or more partly immiscible fluids in continuous flow. Mass transfer intensification due to miniaturisation of the flow dimensions and the associated enlargement of the interfacial area presents a powerful approach of effective reaction rate enhancement and thus reactor productivity increase for smart flow bioprocessing. Use of microstructured flow reactors for the study of multiphase (gas-liquid, liquid-liquid) biocatalytic conversions is reviewed. Multiphase flow characterisation based on dimensionless scaling parameters and flow-regime categorisation is presented with emphasis on the different flows applied to experimental studies of enzymatic reactions. Development of instrumented microsystems, flow instabilities, fast inactivation of biocatalysts and low conversion rates are problems often encountered with biotransformation under multiphase flow conditions. Key parameters controlling reaction performance are discussed along with some guidelines for design of scalable multiphase biocatalytic microreactors. Opportunities for biocatalytic process intensification are revealed in examples from fine chemical and materials synthesis.