Baeyer-Villiger monooxygenases (BVMOs) are a very well-known and intensively studied class of flavin-dependent enzymes. Their substrate promiscuity, high chemo-, regio-, and enantioselectivity are prerequisites for the use in synthetic chemistry and should pave the way for successful industrial processes. Nonetheless, only a very limited number of industrial relevant transformations are known, mainly due to the lack of BVMOs stability and cofactor dependency. In this review, we focus on novel BVMO-mediated transformations, BVMOs in cascade type reactions, potential industrial applications, and how limitations have been tackled by the community. Special attention will be put on whole-cell immobilization strategies. We emphasize to bridge recent developments in fundamental research to industrial applications.
BackgroundPhysiological aggregation of a recombinant enzyme into enzymatically active inclusion bodies could be an excellent strategy to obtain immobilized enzymes for industrial biotransformation processes. However, it is not convenient to recycle “gelatinous masses” of protein inclusion bodies from one reaction cycle to another, as high centrifugation forces are needed in large volumes. The magnetization of inclusion bodies is a smart solution for large-scale applications, enabling an easier separation process using a magnetic field.ResultsMagnetically modified inclusion bodies of UDP–glucose pyrophosphorylase were recycled 50 times, in comparison, inclusion bodies of the same enzyme were inactivated during ten reaction cycles if they were recycled by centrifugation. Inclusion bodies of sialic acid aldolase also showed good performance and operational stability after the magnetization procedure.ConclusionsIt is demonstrated here that inclusion bodies can be easily magnetically modified by magnetic iron oxide particles prepared by microwave-assisted synthesis from ferrous sulphate. The magnetic particles stabilize the repetitive use of the inclusion bodies .Electronic supplementary materialThe online version of this article (10.1186/s12934-018-0987-7) contains supplementary material, which is available to authorized users.
A novel immobilization matrix for the entrapment of viable whole-cell Baeyer-Villiger monooxygenase was developed. Viable recombinant Escherichia coli cells overexpressing cyclohexanone monooxygenase were entrapped in polyelectrolyte complex beads prepared by a two-step reaction of oppositely-charged polymers including highly defined cellulose sulphate. Immobilized cells exhibited higher operational stability than free cells during 10 repeated cycles of Baeyer-Villiger biooxidations of rac-bicyclo[3.2.0]hept-2-en-6-one to the corresponding lactones (1R,5S)-3-oxabicyclo-[3.3.0]oct-6-en-3-one and (1S,5R)-2-oxabicyclo-[3.3.0]oct-6-en-3-one. The morphology of polyelectrolyte complex beads was characterised by environmental scanning electron microscopy; the spatial distribution of polymers in the beads and cell viability were examined using confocal laser scanning microscopy, and the texture was characterised by the mechanical resistance measurements.
Development of new polyelectrolyte complex (PEC) capsules/beads for biotechnological applications such as the immunoisolation of Langerhans islets for the treatment of diabetes and the stabilization and reuse of enzymes and bacterial cells as biocatalysts is very important [1]. Morphological characterization and study of PEC beads properties represents important challenge for electron microscopy. These very beam-sensitive bio-polymer capsules used for immobilization of cells are laboratory produced as a uniform with a controlled shape, size, membrane thickness, permeability and mechanical resistance [1]. Owing to importance to study above mentioned parameters, samples must be inspected in their fully native and functional state. It means free of freezing, chemical contamination or preparation, shape distortion and in thermodynamically stabile and fully wet state, precisely reached after very slow changing of conditions in the specimen chamber of ESEM. Violation of these conditions leads to deformation and burst of thin semipermeable membrane surrounding liquid core, containing live bacterial cells, for example E. coli. The aim of this work is to prove ability to use our non-commercial ESEM AQUASEM II for inspection and developmental support of this type of samples and demonstrate their sensitivity on two types of PEC beads prepared from alternative materials.PEC beads has been produced by air-stripping nozzle via polyelectrolyte complexation (20 min) of sodium alginate and cellulose sulphate (CS) as polyanions, poly(methylene-co-guanidine) as a polycation, CaCl 2 as a gelling agent and NaCl as an antigelling agent [1] without the use of a multiloop reactor. Two different sources of CS have been tested for preparation of PEC beads (CS from Across Organics, N.J., USA; Fig. 1) and alternative PEC beads (tailor-made CS from Senova, Weimar, Germany; Fig 2). Due to the relatively big size of samples (800m in diameter) and their beam sensitivity, a combination of our newly published method [2] and special improvement of our ionization detector of SE were used. The gentle and slow sample chamber pumping procedure [2] and our ionization detector of SEs [3] (beam current up to 40 pA) enhanced for larger field of view (850 μm) were combined. Samples were observed in small droplet (approximately 10 l) of distilled water. Both types of matrices were observed at sample to second pressure limiting aperture distance 4 mm. For thermodynamic equilibrium adjustment control, Pfeiffer pressure gauges CMR 261 and CMR 263, a custom build Peltier stage and a hydration system were used.
A novel, high performance, and scalable immobilization protocol using a laminar jet break-up technique was developed for the production of polyelectrolyte complex beads with entrapped viable Escherichia coli cells expressing an enzyme cascade of alcohol dehydrogenase, enoate reductase, and cyclohexanone monooxygenase. A significant improvement of operational stability was achieved by cell immobilization, which was manifested as an almost two-fold higher summative product yield of 63% after five cascade reaction cycles as compared to the yield using free cells of 36% after the maximum achievable number of three cycles. Correspondingly, increased metabolic activity was observed by multimodal optical imaging in entrapped cells, which was in contrast to a complete suppression of cell metabolism in free cells after five reaction cycles. Additionally, a high density of cells entrapped in beads had a negligible effect on bead permeability for low molecular weight substrates and products of cascade reaction.
Polyelectrolyte complex (PEC) capsules/beads are very important for biotechnological applications such as drug delivery and bacterial whole‐cell biocatalyst development. The very beam‐sensitive bio‐polymer capsules are laboratory produced as a uniform with a controlled shape, size, membrane thickness, permeability and mechanical resistance [1]. PEC capsules are very sensitive to any treatment and samples could be inspected in their fully native and functional state to prevent any misinterpretation. Characterization and study of PEC capsules properties is possible using thermodynamically stabile and fully wet state, precisely reached after very slow changing of conditions in the specimen chamber of ESEM. The morphological study using low current ESEM was already presented [2]. The internal structure can be in solvent, semisolid or solid state, depend on capsule type and manufacturing process [3], nevertheless it was not described in its native state yet. Study of inner part as well as surface morphology of PEC capsules using classical SEM or cryo‐SEM can be misleading due to requirement of dry resp. freeze sample. The aim of this work is in‐situ study of internal structure of PEC capsules in fully wet state and demonstration of state of matter of PEC capsules core. PEC capsules has been produced by air‐stripping nozzle via polyelectrolyte complexation (20 min) of sodium alginate and cellulose sulphate (CS) as polyanions, poly(methylene‐co‐guanidine) as a polycation, CaCl 2 as a gelling agent and NaCl as an antigelling agent [1] without the use of a multiloop reactor. Due to the high beam sensitivity of samples and its relatively big size (800 μm in diameter), a combination of our published method [4] and special improvement of our ionization detector of SE were used. The gentle and slow sample chamber pumping procedure [4] and our ionization detector of SEs [2] (beam current up to 40 pA) enhanced for larger field of view (850 μm) were combined Samples were observed in conditions of vapor pressure 684 Pa, stage temperature 2°C, humidity 97%, acc. voltage 20 kV and probe current 35 pA. Fully wet and well preserved PEC capsule with visible surface microstructure is presented in Fig. 1A. PEC capsules are very sensitive to beam impact which was used to in‐situ disruption of outer shell. Afterwards the liquid core slowly rose by capillary action on the PEC capsules wall simultaneously with capsule collapsing due to its emptying, see Fig. 1B. Due to different temperatures between the sample and the Peltier cooling stage, the liquid core was dried and crystalized on the PEC capsule surface, see Fig. 1C. First results provide promising information leading to statement that the inner structure of this type of PEC capsules is viscous liquid.
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