•Advanced characterization of immobilized enzymes is important for heterogeneous biocatalyst development.•Structural and in-operando studies contribute to identification of factors governing the ...activity of immobilized enzymes.•Protein visualization in solid supports is used to facilitate enzyme loading in high quantity and quality.•Elucidation of structural features of immobilized enzymes on solid support remains challenging.•In-operando opto-chemical internal sensing is prominently used to characterize the biocatalyst's internal environment.
Like in chemical catalysis, there is a clear trend in biocatalysis to carry out synthetic transformations at the manufacturing scale heterogeneously catalyzed. Recycling of insoluble catalysts is simplified, and continuous reactor development thus promoted. Heterogeneous biocatalysis usually involves enzymes immobilized on mesoporous solid supports that offer a large internal surface area. Unraveling enzyme behavior under the confinement of a solid surface and its effect on the catalytic reaction in heterogeneous environment present longstanding core problems of biocatalysis with immobilized enzymes. Progress in deepening the mechanistic understanding of heterogeneous biocatalytic conversions is often restrained by severe limitations in methodology applicable to a direct characterization of solid-supported enzymes. Here we highlight recent evidence from the analysis of protein distribution on porous solid support using microscopic imaging methods with spatiotemporal resolution capability. We also show advance in the use of spectroscopic methods for the analysis of protein conformation on solid support. Methods of direct characterization of activity and stability of immobilized enzymes as heterogeneous biocatalysts are described and their important roles in promoting rational biocatalyst design as well as optimization and control of heterogeneously catalyzed processes are emphasized.
The liquid milieu in which enzymes operate when they are immobilized in solid materials can be quite different from the milieu in bulk solution. Important differences are in the substrate and product ...concentration but also in pH and ionic strength. The internal milieu for immobilized enzymes is affected by the chemical properties of the solid material and by the interplay of reaction and diffusion. Enzyme performance is influenced by the internal milieu in terms of catalytic rate ("activity") and stability. Elucidation, through direct measurement of differences in the internal as compared to the bulk milieu is, therefore, fundamentally important in the mechanistic characterization of immobilized enzymes. The deepened understanding thus acquired is critical for the rational development of immobilized enzyme preparations with optimized properties. Herein we review approaches by opto-chemical sensing to determine the internal milieu of enzymes immobilized in porous particles. We describe analytical principles applied to immobilized enzymes and focus on the determination of pH and the O
concentration. We show measurements of pH and O
with spatiotemporal resolution, using in operando analysis for immobilized preparations of industrially important enzymes. The effect of concentration gradients between solid particle and liquid bulk on enzyme performance is made evident and quantified. Besides its use in enzyme characterization, the method can be applied to the development of process control strategies.
The integration of enzymes with solid materials is important in many biotechnological applications, including the use of immobilized enzymes for biocatalytic synthesis. The development of functional ...enzyme-material composites is restrained by the lack of molecular-level insight into the behavior of enzymes in confined, surface-near environments. Here, we review recent advances in surface-sensitive spectroscopic techniques that push boundaries for the determination of enzyme structure and orientation at the solid-liquid interface. We discuss recent evidence from single-molecule studies showing that analyses sensitive to the temporal and spatial heterogeneities in immobilized enzymes can succeed in disentangling the effects of conformational stability and active-site accessibility on activity. Different immobilization methods involve distinct trade-off between these effects, thus emphasizing the need for a holistic (systems) view of immobilized enzymes for the rational development of practical biocatalysts.
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•Advances in methodology for structural analysis of immobilized enzymes.•Relationships between structure and activity in immobilized enzymes.•Enzyme orientation on solid surface.•Single-molecule analysis of immobilized enzymes.•Single-molecule characterization of stability and activity of immobilized enzymes.
The D746E variant of Bifidobacterium bifidum β‐N‐acetyl‐hexosaminidase is a promising glycosynthase (engineered glycosidase deficient in hydrolase activity) for the synthesis of lacto‐N‐triose II ...(LNT II), a core structural unit of human milk oligosaccharides. Here, we develop a flow process for the glycosynthase reaction, which is the regioselective β‐1,3‐glycosylation of lactose from a d‐glucosamine 1,2‐oxazoline donor. Using the glycosynthase immobilized on agarose beads (∼30 mg/g) packed into a fixed bed (1 ml), we show stable continuous production of LNT II (145–200 mM) at quantitative yield from the donor substrate. The wild‐type β‐N‐acetyl‐hexosaminidase used under exactly comparable conditions gives primarily (∼85%) the hydrolysis product d‐glucosamine. By enabling short residence times (2 min) that are challenging for mixed‐vessel types of reactor to establish, the glycosynthase flow reactor succeeds in an effective uncoupling of the LNT II formation (∼80–100 mM/min) from the slower side reactions (decomposition of donor substrate, enzymatic hydrolysis of LNT II) to obtain optimum synthetic efficiency. Our study thus provides a strong case for the application of flow chemistry principles to glycosynthase reactions and by that, it reveals the important synergy between enzyme and reaction engineering for biocatalytic synthesis of oligosaccharides.
Glycosynthases are engineered glycoside hydrolases for oligosaccharide synthesis. The D746E glycosynthase of Bifidobacterium bifidum β‐N‐acetyl‐hexosaminidase is promising for synthesis of lacto‐N‐triose II, a core structural unit of human milk oligosaccharides. Here, the authors developed an efficient flow process for the glycosynthase reaction, which is regioselective β‐1,3‐glycosylation of lactose from d‐glucosamine 1,2‐oxazoline. By enabling residence times of just 2 min, the flow reactor obtains optimum synthetic efficiency. Important synergy between enzyme and reaction engineering for oligosaccharide synthesis is thus demonstrated.
Enzymes incorporated into hydrogen‐bonded organic frameworks (HOFs) via bottom‐up synthesis are promising biocomposites for applications in catalysis and sensing. Here, we explored synthetic ...incorporation of d‐amino acid oxidase (DAAO) with the metal‐free tetraamidine/tetracarboxylate‐based BioHOF‐1 in water. N‐terminal enzyme fusion with the positively charged module Zbasic2 strongly boosted the loading (2.5‐fold; ≈500 mg enzyme gmaterial−1) and the specific activity (6.5‐fold; 23 U mg−1). The DAAO@BioHOF‐1 composites showed superior activity with respect to every reported carrier for the same enzyme and excellent stability during catalyst recycling. Further, extension to other enzymes, including cytochrome P450 BM3 (used in the production of high‐value oxyfunctionalized compounds), points to the versatility of genetic engineering as a strategy for the preparation of biohybrid systems with unprecedented properties.
Immobilization of d‐amino acid oxidase in crystalline frameworks at high protein loading is reported. A biocomposite of hydrogen‐bonded organic framework (BioHOF‐1) shows excellent retention of activity. Fusion to the positively charged module Zbasic2 (Z‐DAAO) promotes incorporation of the active enzyme. Protein engineering can facilitate development of framework‐based enzyme composites, as shown with three further examples of industrial enzymes.
While O2 substrate for bio‐transformations in bulk liquid is routinely provided from entrained air or O2 gas, tailored solutions of O2 supply are required when the bio‐catalysis happens spatially ...confined to the microstructure of a solid support. Release of soluble O2 from H2O2 by catalase is promising, but spatiotemporal control of the process is challenging to achieve. Here, we show monitoring and control by optical sensing within a porous carrier of the soluble O2 formed by an immobilized catalase upon feeding of H2O2. The internally released O2 is used to drive the reaction of d‐amino acid oxidase (oxidation of d‐methionine) that is co‐immobilized with the catalase in the same carrier. The H2O2 is supplied in portions at properly timed intervals, or continuously at controlled flow rate, to balance the O2 production and consumption inside the carrier so as to maintain the internal O2 concentration in the range of 100–500 µM. Thus, enzyme inactivation by excess H2O2 is prevented and gas formation from the released O2 is avoided at the same time. The reaction rate of the co‐immobilized enzyme preparation is shown to depend linearly on the internal O2 concentration up to the air‐saturated level. Conversions at a 200 ml scale using varied H2O2 feed rate (0.04–0.18 mmol/min) give the equivalent production rate from d‐methionine (200 mM) and achieve rate enhancement by ∼1.55‐fold compared to the same oxidase reaction under bubble aeration. Collectively, these results show an integrated strategy of biomolecular engineering for tightly controlled supply of O2 substrate from H2O2 into carrier‐immobilized enzymes. By addressing limitations of O2 supply via gas‐liquid transfer, especially at the microscale, this can be generally useful to develop specialized process strategies for O2‐dependent biocatalytic reactions.
Supply of O2 substrate to biotransformations in porous carriers requires tailored solutions beyond bubble aeration. Here, the authors used immobilized catalase (CAT) to produce O2 from H2O2 inside the carrier to generate O2 for reaction of a co‐immobilized D‐amino acid oxidase (DAAO). O2 measurement in liquid and inside particle (based on a co‐immobilized luminescence dye) guided development of a H2O2 feeding strategy for tightly controlled O2 supply. This enabled rate enhancement compared to the same oxidase reaction under bubble aeration.
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.
While O
substrate for bio-transformations in bulk liquid is routinely provided from entrained air or O
gas, tailored solutions of O
supply are required when the bio-catalysis happens spatially ...confined to the microstructure of a solid support. Release of soluble O
from H
O
by catalase is promising, but spatiotemporal control of the process is challenging to achieve. Here, we show monitoring and control by optical sensing within a porous carrier of the soluble O
formed by an immobilized catalase upon feeding of H
O
. The internally released O
is used to drive the reaction of d-amino acid oxidase (oxidation of d-methionine) that is co-immobilized with the catalase in the same carrier. The H
O
is supplied in portions at properly timed intervals, or continuously at controlled flow rate, to balance the O
production and consumption inside the carrier so as to maintain the internal O
concentration in the range of 100-500 µM. Thus, enzyme inactivation by excess H
O
is prevented and gas formation from the released O
is avoided at the same time. The reaction rate of the co-immobilized enzyme preparation is shown to depend linearly on the internal O
concentration up to the air-saturated level. Conversions at a 200 ml scale using varied H
O
feed rate (0.04-0.18 mmol/min) give the equivalent production rate from d-methionine (200 mM) and achieve rate enhancement by ∼1.55-fold compared to the same oxidase reaction under bubble aeration. Collectively, these results show an integrated strategy of biomolecular engineering for tightly controlled supply of O
substrate from H
O
into carrier-immobilized enzymes. By addressing limitations of O
supply via gas-liquid transfer, especially at the microscale, this can be generally useful to develop specialized process strategies for O
-dependent biocatalytic reactions.
The Science of Enzyme Immobilization Guisan, Jose M; López-Gallego, Fernando; Bolivar, Juan M ...
Methods in molecular biology (Clifton, N.J.),
01/2020, Letnik:
2100
Journal Article
Protocols for simple immobilization of unstable enzymes are plenty, but the vast majority of them, unfortunately, have not reached their massive implementation for the preparation of improved ...heterogeneous biocatalyst. In this context, the science of enzyme immobilization demands new protocols capable of fabricating heterogeneous biocatalysts with better properties than the soluble enzymes. The preparation of very stable immobilized biocatalysts enables the following: (1) higher operational times of enzyme, increasing their total turnover numbers; (2) the use of enzymes under non-conventional media (temperatures, solvents, etc.) in order to increase the concentrations of substrates for intensification of processes or in order to shift reaction equilibria; (3) the design of solvent-free reaction systems; and (4) the prevention of microbial contaminations. These benefits gained with the immobilization are critical to scale up chemical processes like the synthesis of biodiesel, synthesis of food additives or soil decontamination, where the cost of the catalysts has an enormous impact on their economic feasibility. The science of enzyme immobilization requires a multidisciplinary focus that involves several areas of knowledge such as, material science, surface chemistry, protein chemistry, biophysics, molecular biology, biocatalysis, and chemical engineering. In this chapter, we will discuss the most relevant aspects to do "the science of enzyme immobilization." We will emphasize the immobilization techniques that promote multivalent attachments between the surface of the enzymes and the porous carriers. Finally, we will discuss the effect that the structural rigidification promotes at different protein regions on the functional properties of the immobilized enzymes.
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