Process intensification for O2 -dependent enzymatic transformations in continuous single-phase pressurized flow.

Oxidative O2 -dependent biotransformations are promising for chemical synthesis, but their development to an efficiency required in fine chemical manufacturing has proven difficult. General problem for process engineering of these systems is that thermodynamic and kinetic limitations on supplying O2 to the enzymatic reaction combine to create a complex bottleneck on conversion efficiency. We show here that continuous-flow microreactor technology offers a comprehensive solution. It does so by expanding the process window to the medium pressure range (here, ≤34 bar) and thus enables biotransformations to be conducted in a single liquid phase at boosted concentrations of the dissolved O2 (here, up to 43 mM). We take reactions of glucose oxidase and d-amino acid oxidase as exemplary cases to demonstrate that the pressurized microreactor presents a powerful engineering tool uniquely apt to overcome restrictions inherent to the individual O2 -dependent transformation considered. Using soluble enzymes in liquid flow, we show reaction rate enhancement (up to six-fold) due to the effect of elevated O2 concentrations on the oxidase kinetics. When additional catalase was used to recycle dissolved O2 from the H2 O2 released in the oxidase reaction, product formation was doubled compared to the O2 supplied, in the absence of transfer from a gas phase. A packed-bed reactor containing oxidase and catalase coimmobilized on porous beads was implemented to demonstrate catalyst recyclability and operational stability during continuous high-pressure conversion. Product concentrations of up to 80 mM were obtained at low residence times (1-4 min). Up to 360 reactor cycles were performed at constant product release and near-theoretical utilization of the O2 supplied. Therefore, we show that the pressurized microreactor is practical embodiment of a general reaction-engineering concept for process intensification in enzymatic conversions requiring O2 as the cosubstrate.

Enzymatic synthesis of beta-glucosylglycerol using a continuous-flow microreactor containing thermostable beta-glycoside hydrolase CelB immobilized on coated microchannel walls.

beta-Glucosylglycerol (betaGG) has potential applications as a moisturizing agent in cosmetic products. A stereochemically selective method of its synthesis is kinetically controlled enzymatic transglucosylation from a suitable donor substrate to glycerol as acceptor. Here, the thermostable beta-glycosidase CelB from Pyrococcus furiosus was used to develop a microstructured immobilized enzyme reactor for production of betaGG under conditions of continuous flow at 70 degrees C. Using CelB covalently attached onto coated microchannel walls to give an effective enzyme activity of 30 U per total reactor working volume of 25 microL, substrate conversion and formation of transglucosylation product was monitored in dependence of glucosyl donor (2-nitrophenyl-beta-D-glucoside (oNPGlc), 3.0 or 15 mM; cellobiose, 250 mM), the concentration of glycerol (0.25-1.0 M), and the average residence time (0.2-90 s). Glycerol caused a concentration-dependent decrease in the conversion of the glucosyl donor via hydrolysis and strongly suppressed participation of the substrate in the reaction as glucosyl acceptor. The yields of betaGG were > or =80% and approximately 60% based on oNPGlc and cellobiose converted, respectively, and maintained up to near exhaustion of substrate (> or =80%), giving about 120 mM (30 g/L) of betaGG from the reaction of cellobiose and 1 M glycerol. The structure of the transglucosylation products, 1-O-beta-D-glucopyranosyl-rac-glycerol (79%) and 2-O-beta-D-glucopyranosyl-sn-glycerol (21%), was derived from NMR analysis of the product mixture of cellobiose conversion. The microstructured reactor showed conversion characteristics similar to those for a batchwise operated stirred reactor employing soluble CelB. The advantage of miniaturization to the microfluidic format lies in the fast characterization of full reaction time courses for a range of process conditions using only a minimum amount of enzyme.

Coated-wall microreactor for continuous biocatalytic transformations using immobilized enzymes.

Microstructured flow reactors are emerging tools for biocatalytic process development. A compelling design is that of the coated-wall reactor where enzyme is present as a surface layer attached to microchannel walls. However, preparation of a highly active wall biocatalyst remains a problem. Here, a stainless steel microreactor was developed where covalent immobilization of the enzyme in multiple linear flow channels of the reaction plate was supported by a macroporous wash-coat layer of gamma-aluminum oxide. Using surface functionalization with aminopropyl triethoxysilane followed by activation with glutardialdehyde, the thermophilic beta-glycosidase CelB from Pyrococcus furiosus was bound with retention of half of the specific activity of the free enzyme (800 U/mg), yielding a high catalyst loading of about 500 U/mL. This microreactor was employed for the continuous hydrolysis of lactose (100 mM) at 80 degrees C, providing a space-time yield of 500 mg glucose/(mL h) at a stable conversion of > or =70%. The immobilized enzyme displayed a half-life of 15 days under the operational conditions. Due to the absence of hydrophobic solute-material interactions, which limit the scope of microstructures fabricated from poly(dimethylsiloxane) for biocatalytic applications, the new microreactor was fully compatible with the alternate enzyme substrate 2-nitro-phenyl-beta-D-galactoside and the 2-nitro-phenol product resulting from its hydrolysis catalyzed by CelB.

Development of a microfluidic immobilised enzyme reactor.

A microfluidic immobilised enzyme reactor consisting of a catalytically functionalised microstructure fabricated from silicone rubber material was used for steady-state kinetic characterisation of a thermophilic beta-glycosidase under pressure-driven flow conditions and continuous conversion of lactose by this enzyme at 80 degrees C.

Interactions of the basic N-terminal and the acidic C-terminal domains of the maize chromosomal HMGB1 protein.

Maize HMGB1 is a typical member of the family of plant chromosomal HMGB proteins, which have a central high-mobility group (HMG)-box DNA-binding domain that is flanked by a basic N-terminal region and a highly acidic C-terminal domain. The basic N-terminal domain positively influences various DNA interactions of the protein, while the acidic C-terminal domain has the opposite effect. Using DNA-cellulose binding and electrophoretic mobility shift assays, we demonstrate that the N-terminal basic domain binds DNA by itself, consistent with its positive effects on the DNA interactions of HMGB1. To examine whether the negative effect of the acidic C-terminal domain is brought about by interactions with the basic part of HMGB1 (N-terminal region, HMG-box domain), intramolecular cross-linking in combination with formic acid cleavage of the protein was used. These experiments revealed that the acidic C-terminal domain interacts with the basic N-terminal domain. The intramolecular interaction between the two oppositely charged termini of the protein is enhanced when serine residues in the acidic tail of HMGB1 are phosphorylated by protein kinase CK2, which can explain the negative effect of the phosphorylation on certain DNA interactions. In line with that, covalent cross-linking of the two terminal domains resulted in a reduced affinity of HMGB1 for linear DNA. Comparable to the finding with maize HMGB1, the basic N-terminal and the acidic C-terminal domains of the Arabidopsis HMGB1 and HMGB4 proteins interact, indicating that these intramolecular interactions, which can modulate HMGB protein function, generally occur in plant HMGB proteins.

HMGB6 from Arabidopsis thaliana specifies a novel type of plant chromosomal HMGB protein.

The high-mobility group (HMG) proteins of the HMGB family are chromatin-associated proteins that act as architectural factors in various nucleoprotein structures, which regulate DNA-dependent processes such as transcription and recombination. Database analyses revealed that in addition to the previously identified HMGB1-HMGB5 proteins, the Arabidopsis genome encodes at least three other family members having the typical overall structure of a central HMG-box DNA binding domain, which is flanked by basic and acidic regions. These novel HMGB proteins display some structural differences, when compared to HMGB1-HMGB5. Therefore, a representative of the identified proteins, now termed HMGB6, was further analyzed. The HMGB6 protein of approximately 27 kDa is the largest plant HMGB protein identified so far. This is essentially due to its unusually extended N-terminal domain of 109 amino acid residues. Subcellular localization experiments demonstrate that it is a nuclear protein. According to CD measurements, HMGB6 has an alpha-helical HMG-box domain. HMGB6 can bind DNA structure-specifically, and it is a substrate for the protein kinase CK2alpha. Because of these features, HMGB6, and presumably its relatives, can be considered members of the plant HMGB protein family. Hence, eight different chromosomal HMGB proteins are expressed in Arabidopsis, and they may serve specialized architectural functions assisting various DNA-dependent processes.