Johanna Wiesbauer

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.

Renewal of the air-water interface as a critical system parameter of protein stability: aggregation of the human growth hormone and its prevention by surface-active compounds.

Soluble proteins are often highly unstable under mixing conditions that involve dynamic contacting between the main liquid phase and a gas phase. The recombinant human growth hormone (rhGH) was recently shown to undergo aggregation into micrometer-sized solid particles composed of non-native (mis- or unfolded) protein, once its solutions were stirred or shaken to generate a continuously renewed air-water interface. To gain deepened understanding and improved quantification of the air-water interface effect on rhGH stability, we analyzed the proteins aggregation rate (r(agg)) at controlled specific air-water surface areas (a(G/L)) established by stirring or bubble aeration. We show that in spite of comparable time-averaged values for a(G/L) (≈ 100 m(2)/m(3)), aeration gave a 40-fold higher r(agg) than stirring. The enhanced r(agg) under aeration was ascribed to faster macroscopic regeneration of free a(G/L) during aeration as compared to stirring. We also show that r(agg) was independent of the rhGH concentration in the range 0.67 - 6.7 mg/mL, and that it increased linearly dependent on the available a(G/L). The nonionic surfactant Pluronic F-68, added in 1.6-fold molar excess over rhGH present, resulted in complete suppression of r(agg). Foam formation was not a factor influencing r(agg). Using analysis by circular dichroism spectroscopy and small-angle X-ray scattering, we show that in the presence of Pluronic F-68 under both stirring and aeration, the soluble protein retained its original fold, featuring native-like relative composition of secondary structural elements. We further provide evidence that the efficacy of Pluronic F-68 resulted from direct, probably hydrophobic protein-surfactant interactions that prevented rhGH from becoming attached to the air-water interface. Surface-induced aggregation of rhGH is suggested to involve desorption of non-native protein from the air-water interface as the key limiting step. Proteins or protein aggregates released back into the bulk liquid appear to be essentially insoluble.

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.

Non-native aggregation of recombinant human granulocyte-colony stimulating factor under simulated process stress conditions

Effective inhibition of protein aggregation is a major goal in biopharmaceutical production processes optimized for product quality. To examine the characteristics of process-stress-dependent aggregation of human granulocyte colony-stimulating factor (G-CSF), we applied controlled stirring and bubble aeration to a recombinant non-glycosylated preparation of the protein produced in Escherichia coli. We characterized the resulting denaturation in a time-resolved manner using probes for G-CSF conformation and size in both solution and the precipitate. G-CSF was precipitated rapidly from solutions that were aerated or stirred; only small amounts of soluble aggregates were found. Exposed hydrophobic surfaces were a characteristic of both soluble and insoluble G-CSF aggregates. Using confocal laser scanning microscopy, the aggregates presented mainly a circular shape. Their size varied according to incubation time and stress applied. The native intramolecular disulfide bonds in the insoluble G-CSF aggregates were largely disrupted as shown by mass spectrometry. New disulfide bonds formed during aggregation. All involved Cys(18) , which is the only free cysteine in G-CSF; one of them had an intermolecular Cys(18(A)) -Cys(18(B)) crosslink. Stabilization strategies can involve external addition of thiols and extensive reduction of surface exposition during processing.