Enzyme catalysis on solid surfaces

Enzyme-catalysed reactions in which substrates are bound (immobilised) to solid surfaces are becoming increasingly important in biotechnology. There is a general drive for miniaturisation and automation in chemistry and biology, and immobilisation of the reaction intermediates and substrates, for example on microarrays or nanoparticles, helps to address technical challenges in this area. In bionanotechnology, enzyme catalysis can provide highly selective and biocompatible tools for the modification of surfaces on the nano-scale. Here, we review the range of enzyme-catalysed reactions that have been successfully performed on the solid phase and discuss their application in biotechnology.     

The one-bead two-compound assay for solid phase screening of combinatorial libraries

Fluorescent quenched substrate libraries are a very powerful tool for investigation of protease activity and specificity. Particularly, libraries where the fluorescent resonance energy transfer (FRET) pair is 3-nitrotyrosine and 2-amino-benzamide are easy to prepare by split and combine synthesis to yield a one-bead one-compound library format. The solid support is critical for the successful hydrolysis of the resin-bound substrates. For this purpose, a range of highly porous poly(ethylene glycol) (PEG)-based resins have been developed. Active substrates yield highly fluorescent beads and these are selected under a fluorescence microscope or isolated on a bead sorter. Edman sequence analysis yields the substrate sequence, the cleavage point, and the degree of conversion. The method gives a complete map of the substrate specificity, and substrates with high affinity for the active site can be selected. These may in turn be used as inhibition indicators in a second solid phase library assay for enzyme inhibition where each single bead is transformed into an assay container. The substrate is attached to temporarily shielded functional groups after completion of inhibitor library synthesis. By using a photolabile linker and ladder synthesis, the active inhibitors may be rapidly identified by mass spectrometry. In each bead, the putative inhibitor competes with the substrate attached for binding to the enzyme, and when the inhibitor binds strongly, the substrate remains intact and quenched. Thus dark beads indicate inhibitors, and these may be isolated using a bead-sorter and the structure determined by mass spectrometry. A selection of the best substrates and inhibitors should always be resynthesized for solution kinetics and confirmation of the results obtained on solid support. The inhibitor assay is almost free from false positives, which is a consequence of combining the binding of the protease to the inhibitor with observation of activity toward a FRET substrate. The K(i) of the identified inhibitors are typically in the nM range.     

Immobilisation of the Thermostable l -aminoacylase from Thermococcus litoralis to Generate a Reusable Industrial Biocatalyst

A thermostable archaeal l -aminoacylase from Thermococcus litoralis has been used in immobilisation trials to optimise its application in industrial biotransformation reactions. Immobilisation techniques used included direct adsorption and crosslinking of the enzyme onto solid supports, bioencapsulation, and covalent bonding onto a variety of activated matrices. The most successful immobilisation methods were covalent binding of the enzyme onto glyoxyl-Sepharose and Amberlite XAD7. These methods yielded an average of 15 and 80 mg of protein bound per gram of support (wet weight for glyoxyl-Sepharose), respectively, with nearly 80% activity recovery in both cases. Enzyme immobilised onto glyoxyl-agarose was stabilised 106-fold under aqueous conditions and 142-fold in 100% acetonitrile when activity was measured after 24 h at 90°C. A column bioreactor containing the recombinant l -aminoacylase immobilised onto Sepharose beads was constructed with the substrate, N -acetyl- dl -Trp, continuously flowing at 60°C for 10 days. No loss of activity was detected over five days, with 32% activity remaining after 40 days at 60°C. These results show the potential of the use of immobilised l -aminoacylase in biotransformation reactions for the production of fine chemicals.     

Immobilisation of the Thermostable l -aminoacylase from Thermococcus litoralis to Generate a Reusable Industrial Biocatalyst

A thermostable archaeal l -aminoacylase from Thermococcus litoralis has been used in immobilisation trials to optimise its application in industrial biotransformation reactions. Immobilisation techniques used included direct adsorption and crosslinking of the enzyme onto solid supports, bioencapsulation, and covalent bonding onto a variety of activated matrices. The most successful immobilisation methods were covalent binding of the enzyme onto glyoxyl-Sepharose and Amberlite XAD7. These methods yielded an average of 15 and 80 mg of protein bound per gram of support (wet weight for glyoxyl-Sepharose), respectively, with nearly 80% activity recovery in both cases. Enzyme immobilised onto glyoxyl-agarose was stabilised 106-fold under aqueous conditions and 142-fold in 100% acetonitrile when activity was measured after 24 h at 90°C. A column bioreactor containing the recombinant l -aminoacylase immobilised onto Sepharose beads was constructed with the substrate, N -acetyl- dl -Trp, continuously flowing at 60°C for 10 days. No loss of activity was detected over five days, with 32% activity remaining after 40 days at 60°C. These results show the potential of the use of immobilised l -aminoacylase in biotransformation reactions for the production of fine chemicals.     

Estimation of cell surface associated protease activity and its application to lymphocytes

A new method has been developed to estimate proteolytic activity available at the cell surface. Radioiodinated protein substrates are covalently linked to modified polystyrene-divinylbenzene beads with various diameters. These beads are presented to viable cells. Secreted enzyme activity is estimated when no contact occurs between beads and cells. Surface associated proteolytic activity is estimated by the increased rate of iodinated peptide release due to a contact between beads and cells. This method was applied to various lymphocyte preparations. In the absence of serum, mouse spleen lymphocytes produce three- to fourfold higher proteolytic activity than lymph node cells. This activity is completely inhibited by serum diluted 1:10. Since the proteolysis is so marked in the case of spleen cells, one must conclude that lymphocytes removed from the serum and treated in buffered mediums at 37° C have enzymatically altered surface properties. Cell surface associated enzyme activity was measured using rat lymph node lymphocytes with less than 0.1% contamination by granulocytes. This predominantly thymus derived, T cell population had 30% increase in proteolysis due to contact between cells and solid-phase localized substrate of casein. The released enzymatic activity was inhibited by diisopropylfluorophosphate, but its effect on the surface associated enzyme activity remains questionable since it perturbs several membrane functions.     

Autodisplay of functional CYP106A2 in Escherichia coli

Cytochrome P450 enzymes catalyse a wide variety of reactions, including the hydroxylation and epoxidation of CC bonds, and dealkylation reactions. There is high interest in these reactions for biotechnology and pharmaceutical processes. Many P450s require membrane surroundings and have substrates that do not cross biological membranes. To circumvent these obstacles, CYP106A2 from Bacillus megaterium was expressed on the outer membrane of Escherichia coli cells by Autodisplay. Exposure on the surface was confirmed by a protease accessibility test and flow cytometry after immunolabelling. HPLC assays showed that 0.5 ml of cells displaying the enzyme (OD??? = 6) converted 9.13 μmol of deoxycorticosterone to 15β-OH-deoxycorticosterone within 1h. Imipramine and abietic acid were also accepted as substrates. The number of active enzyme molecules per cell was calculated to be 20,000. Surprisingly, surface-exposed CYP106A2 was active in E. coli BL21 without the external addition of the heme group. However, when CYP106A2 was expressed on the surface of an E. coli strain lacking the TolC channel protein (JW5503), enzymatic activity was almost completely abolished. The activity of CYP106A2 on the surface of E. coli JW5503 could be restored by the external addition of the heme group. This suggests, as has been reported before, that E. coli uses a TolC-dependent mechanism to export heme into the growth media, where it can be scavenged by a surface-displayed apoenzyme. Our results indicate that Autodisplay enables the functional surface display of P450 enzymes and provides a new platform to access their synthetic potential.     

A standardized spectrophotometric assay of endoglycanase activities using dyed, amorphous polysaccharides

This is a new technique to assay virtually any endoglycanase activity where enough polysaccharide material is available to allow for production of the amorphous, dyed beads used as substrates. It allows for a direct comparison of endoglycanase activities between laboratories since dyed beads from at least the most common polysaccharides such as cellulose, xylan, mannan, and chitin are now under development and will soon be commercially available; cellulose beads already are. It is a very sensitive technique and enzyme activities can be measured using a nonsophisticated spectrophotometer.     

A microreactor for the study of biotransformations by a cross-linked γ-lactamase enzyme

A (+)-γ-lactamase was precipitated, cross-linked and the resulting solid crushed prior to immobilisation within a capillary column microreactor. The microreactor was subsequently used to study enzyme stability, activity, kinetics and substrate specificity. The thermophilic (+)-γ-lactamase retained 100% of its initial activity at the assay temperature, 80°C, for 6 h and retained 52% activity after 10 h, indicating the advantage of immobilisation. This high stability of the immobilised enzyme provided the advantage that it could be utilised to screen many compounds in the microreactor system. This advantage overcame the fact that the immobilisation process affected enzyme kinetics and activity, which was reduced (by 70%) compared to the free enzyme. In general, the enzyme displayed similar substrate specificity to that found in a previous study for the free enzyme; however, enhanced activity was seen towards one substrate, acrylamide. The system developed correlates well with the free enzyme in batch assay and indicates the suitability of the system for enzyme substrate screening, allowing a significant reduction in cost, due to the reduced amounts of enzyme, substrates and other assay constituents required.     

A method for the determination of enzyme mass loading on an electrode surface through radioisotope labelling

A direct method has been developed for the quantitation of the amount of immobilised enzymes on biosensor surfaces. This quantity is of key importance in establishing the activity, kinetics and optimal immobilisation conditions in the construction of both amperometric and optical biosensors. Recombinant L-lactate dehydrogenase incorporating both a biosynthetically introduced radiolabel, 3H-leucine, and a hexahistidine peptide tag was immobilised on a poly(aniline) composite film and then quantitated by liquid scintillation counting. It was found that enzyme mass loading was proportional to the concentration of LDH in solution, and also depended on the morphology of the composite film. The LDH mass loading on the composite film doubled when a surface cysteine containing variant was used, possibly due to the covalent attachment of the cysteine to the diiminoquinoid rings of the poly(aniline).     

The use of optical spectroscopy in combinatorial chemistry.

Infrared and Raman spectroscopy allow direct spectral analysis of the solid-phase, thus avoiding the tedious cleavage of compounds from the solid support. With diagnostic bands in starting materials or products, infrared and Raman spectroscopy are efficient in monitoring each reaction step directly on the solid phase. Consequently, infrared and Raman spectroscopy have evolved as the premier analytical methodology for direct analysis on the solid support. While infrared transmission spectroscopy is a general analytical method for resin samples, internal reflection spectroscopy is especially suited for solid polymer substrates known as "pins" or "crowns." Single bead analysis is done best by infrared microspectroscopy, whereas photoacoustic spectroscopy allows totally nondestructive analysis of resin samples. With an automated accessory, diffuse reflection spectroscopy provides a method for high throughput on-bead monitoring of solid-phase reactions. Providing identification based on molecular structure, HPLC-FTIR is, therefore, complementary to LC-MS. Additionally, Raman spectroscopy as a complement to infrared spectroscopy can be applied to resin samples and-using a Raman microscope-to single beads. Fluorometry as an extremely sensitive spectroscopic detection method allows rapid quantification of organic reactions directly on the resin.     

Enzyme Kinetics for Complex System Enables Accurate Determination of Specificity Constants of Numerous Substrates in a Mixture by Proteomics Platform

Many important experiments in proteomics including protein digestion, enzyme substrate screening, enzymatic labeling, etc., involve the enzymatic reactions in a complex system where numerous substrates coexists with an enzyme. However, the enzyme kinetics in such a system remains unexplored and poorly understood. Herein, we derived and validated the kinetics equations for the enzymatic reactions in complex system. We developed an iteration approach to depict the enzymatic reactions in complex system. It was validated by 630 time-course points from 24 enzymatic reaction experiments and was demonstrated to be a powerful tool to simulate the reactions in the complex system. By applying this approach, we found that the ratio of substrate depletion is independent of other coexisted substrates under specific condition. This observation was then validated by experiments. Based on this striking observation, a simplified model was developed to determine the catalytic efficiencies of numerous competing substrates presented in the complex enzyme reaction system. When coupled with high-throughput quantitative proteomics technique, this simplified model enabled the accurate determination of catalytic efficiencies for 2369 peptide substrates of a protease by using only one enzymatic reaction experiment. Thus, this study provided, in the first time, a validated model for the large scale determination of specificity constants which could enable the enzyme substrate screening approach turned from a qualitative method of identifying substrates to a quantitative method of identifying and prioritizing substrates. Data are available via ProteomeXchange with identifier PXD004665.     

Evaluation of magnetic polymer micro-beads as carriers of immobilised biocatalysts for selective and stereoselective transformations

The kinetic, selective and stereoselective properties of enzyme immobilised on magnetic polymer beads with diameters in the range 1 microm was studied with penicillin amidase from E. coli. The enzyme was immobilised on epoxy and glutaraldehyde-activated poly(vinyl alcohol), poly(methylmetacrylate) and poly(vinyl acetate-divinylbenzene) magnetic beads. The amount of covalently bound active protein was dependent on the chemical modification of the matrix and increased at higher ionic strength of the immobilisation buffer. The small size of the magnetic beads, that reduces mass transfer limitations, and the decreased charge density in the electric double layer resulted in lower apparent Km values and higher efficiency for benzylpenicillin hydrolysis, higher stereoselectivity in condensation of R-phenylglycine amide with S- and R-Phe and in hydrolysis of racemic phenylacetyl-Phe and higher selectivity in kinetically controlled synthesis of cephalexin compared to the enzyme immobilised on larger and porous carriers.     

The use of optical spectroscopy in combinatorial chemistry

Infrared and Raman spectroscopy allow direct spectral analysis of the solid‐phase, thus avoiding the tedious cleavage of compounds from the solid support. With diagnostic bands in starting materials or products, infrared and Raman spectroscopy are efficient in monitoring each reaction step directly on the solid phase. Consequently, infrared and Raman spectroscopy have evolved as the premier analytical methodology for direct analysis on the solid support. While infrared transmission spectroscopy is a general analytical method for resin samples, internal reflection spectroscopy is especially suited for solid polymer substrates known as “pins” or “crowns.” Single bead analysis is done best by infrared microspectroscopy, whereas photoacoustic spectroscopy allows totally nondestructive analysis of resin samples. With an automated accessory, diffuse reflection spectroscopy provides a method for high throughput on‐bead monitoring of solid‐phase reactions. Providing identification based on molecular structure, HPLC‐FTIR is, therefore, complementary to LC‐MS. Additionally, Raman spectroscopy as a complement to infrared spectroscopy can be applied to resin samples and—using a Raman microscope—to single beads. Fluorometry as an extremely sensitive spectroscopic detection method allows rapid quantification of organic reactions directly on the resin. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng (Comb Chem) 61:179–187, 1998/1999.     

Picodroplet-deposition of enzymes on functionalized self-assembled monolayers as a basis for miniaturized multi-sensor structures

We are reporting on a novel approach for structured immobilisation of enzymes on gold surfaces modified with monolayers of functionalised alkylthiols. The formation of enzyme spots is achieved by shooting very small volumes of an appropriate enzyme solution (down to 100 pl) onto a thiol-monolayer modified gold surface using a micro-dispenser. Formation of enzyme patterns is obtained by moving the micro-dispenser relative to the modified gold surface using a micro-positioning device. Enzyme spots with typical lateral dimensions of 100 μm are obtained, but also, more complex structures, e.g. lines or meander structures, can be achieved by multiple droplets dispensed during the concomitant movement of the micro-dispenser. The first enzyme layer on top of the functionalised thiol-monolayer is subsequently covalently immobilised using either carbodiimide activation of carboxilic headgroups at the enzyme or via already introduced activated ester functions at the monolayer. Immobilised enzyme activities of glucose oxidase and lactate oxidase patterns have been characterised by means of scanning electrochemical microscopy. The product of the enzyme-catalysed reaction, H₂O₂, is detected with an micro-electrode in the presence of either or both substrates, glucose and lactate, leading to a visualisation of the corresponding enzyme pattern and the lateral enzymatic activity.     

Towards the protein phosphatase-based biosensor for microcystin detection

A colorimetric test for the detection of microcystins based on immobilised protein phosphatase (PP) has been developed. A PP2A produced by molecular engineering has been used and its performance has been compared to those of commercial PP2A and PP1. Covalent immobilisation of the enzyme using glutaraldehyde, encapsulation by sol-gel and entrapment with photocrosslinkable poly(vinyl alcohol) bearing styrylpyridinium groups (PVA-SbQ) have been compared, the latter method providing the highest immobilisation yields. Screen-printed carbon electrodes (SPEs), Maxisorp microtiter wells and Ultrabind modified polyethersulfone affinity membranes have been used as immobilisation supports. Whilst the highest immobilisation yields were obtained with microtiter wells, the highest operational and storage stabilities were achieved with carbon SPEs and membranes, respectively. The immobilisation of PP by PVA-SbQ provided a means to preserve the enzymatic activity, which decreased at fast rates when the enzyme was kept in solution. The colorimetric test using p-nitrophenyl phosphate has demonstrated that the immobilised enzyme is able to recognise both microcystin variants (MC-LR and MC-RR), although optimisation work should be performed to achieve appropriate limits of detection. With the purpose to develop an electrochemical biosensor, several phosphorylated substrates have been used. Promising results have been achieved with the commercial enzymes and alpha-naphtyl phosphate, p-aminophenol phosphate and catechol monophosphate as enzyme substrates, guaranteeing the viability of the electrochemical approach.     

One free sulfhydryl group of plasma fibronectin becomes titratable upon binding of the protein to solid substrates

The accessibility in human plasma fibronectin of the two free sulfhydryl groups per chain to sulfhydryl reagents 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and a maleimide derivative has been examined. For soluble fibronectin, the free sulfhydryl groups are not accessible to DTNB unless urea or guanidine hydrochloride is added [Smith et al. (1982) J. Biol. Chem. 257, 5831-5838]. Upon binding to polystyrene beads, 0.87 +/- 0.05 sulfhydryl group per chain becomes titratable to DTNB. Experiments using fibronectin fragments demonstrate that this newly exposed sulfhydryl group is located in a Type III homologous unit between the DNA-binding and the cell-binding domains. The results suggest that, upon adsorption to solid substrates, plasma fibronectin undergoes a conformational change, thereby exposing one buried sulfhydryl group. These findings have implications regarding the "surface activation" of adhesion activities of fibronectin.     

NMR studies of protein hydration and TEMPOL accessibility

Understanding the mechanisms of the interaction between a protein surface and its outer molecular environment is of primary relevance for the rational design of new drugs and engineered proteins. Protein surface accessibility is emerging as a new dimension of Structural Biology, since NMR methods have been developed to follow how molecules, even those different from physiological ligands, preferentially approach specific regions of the protein surface. Hen egg-white lysozyme, a paradigmatic example of the state of the art of protein structure and dynamics, has been selected as a model system to study protein surface accessibility. Bound water and soluble spin-labels have been used to investigate the interaction of this enzyme, both free and bound to the inhibitor (NAG)(3), with its molecular environment. No tightly bound water molecules were found inside the enzyme active site, which, conversely, appeared as the most exposed to visits from the soluble paramagnetic probe TEMPOL. From the presented set of data, an integrated view of lysozyme surface accessibility towards water and TEMPOL molecules is obtained.     

Magnetic-bead enzyme-linked immunosorbent assay verifies adsorption of ligand and epitope accessibility

Antigen- and antibody-coated magnetic beads, which are commercially available as activated particles and are readily coated with a variety of molecules, are excellent devices for selecting specific cell populations. We describe an enzyme-linked immunosorbent assay (ELISA)-based method that validates the adsorption of ligand to magnetic beads and verifies the surface accessibility of specific epitopes of the particular ligand. Accordingly, various ligands at a wide range of concentrations were incubated with magnetic beads and adsorption was then assessed using either a specific primary antibody and a secondary alkaline phosphatase-conjugated antibody or a specific primary alkaline phosphatase-conjugated antibody. The method is straightforward and fast and, due to the very low nonspecific background binding, it requires extremely small amounts of beads and ligand. It can be easily performed on newly prepared beads before using them for selection. Magnetic-bead ELISA also confirms the accessibility of specific epitopes on the surface of the beads, which corresponds to the primary antibody used in the assay.     

New substrates and enzyme assays for the detection of mucopolysaccharidosis III (Sanfilippo Syndrome) types A, B, C, and D by tandem mass spectrometry

The clinical phenotype of Sanfilippo Syndrome is caused by one of four enzyme deficiencies that are associated with a defect in mucopolysaccharide metabolism. The four subtypes (A, B, C, and D) are each caused by an enzyme deficiency involved in the degradation of heparan sulfate. We have developed a highly efficient synthesis of the substrates and internal standards required for the enzymatic assay of each of the four enzymes. The synthesis of the substrates involves chemical modification of a common intermediate. The substrates and internal standards allow the measurement of the enzymes relevant to heparan N-sulfatase (type A); N-acetyl-α-glucosaminidase (type B); acetyl-CoA:α-glucosamide N-acetyltransferase (type C); and N-acetylglucosamine 6-sulfatase (type D). The internal standards are similar to the substrates and allow for the accurate quantification of the enzyme assays using tandem mass spectrometry. The synthetic substrates incorporate a coumarin moiety and can also be used in fluorometric enzyme assays. We confirm that all four substrates can detect the appropriate Sanfilippo Syndrome in fibroblast lysates, and the measured enzyme activities are distinctly lower by a factor of 10 when compared to fibroblast lysates from unaffected persons.