Polymer-enzyme Conjugates Research Articles

Enzyme-Polymer Conjugates with Photocleavable Linkers for Control Over Protein Activity.

Reversible conjugation of polymers to proteins is important for a variety of applications, for example to control protein activity. Light is often employed as an external trigger to allow for spatio and temporal control over release of a payload. In this report, we demonstrate preparation of photocleavable poly(polyethylene glycol) acrylate)-lysozyme (pPEGA-Lys) conjugates via ortho-nitrobenzyl linkages. The conjugates were made by both grafting-to and grafting-from in order to compare and contrast the two synthetic approaches. First, a lysine-reactive ortho-nitrobenzyl atom transfer radical polymerization (ATRP) initiator was synthesized. For the grafting-to strategy, the initiator was employed in the ATRP of PEGA, and the subsequent polymer was conjugated to the lysine residues of lysozyme. For the grafting-from strategy, lysozyme was modified first with the photocleavable initiator, and the purified macroinitiator was then subjected to polymerization conditions to synthesize the protein-polymer conjugate. The polymer was cleaved from the protein via UV light, and activity before and after polymer removal was evaluated, showing 83% recovery. This work provides evidence that reversing conjugation is successful for activity modulation for ortho-nitrobenzyl linked protein-polymer conjugates.

Tuning Polymer Composition Leads to Activity-Stability Tradeoff in Enzyme-Polymer Conjugates.

Protein-polymer conjugation provides an opportune means to adjust the local environment of proteins and enhance protein stability, performance, and solubility. Although much attention has been focused on tuning protein-polymer interactions, the properties of polymer-modified proteins may also be altered by polymer-polymer interactions. Herein, we sought to better understand the influence of polymer-polymer interactions on Candida rugosa lipase, which was modified with random co-polymers composed of sulfobetaine methacrylate (SBMA) and poly(ethylene glycol) methacrylate (PEGMA). Our findings suggest that there is an apparent activity-stability tradeoff as a function of polymer composition. Specifically, as the ratio of SBMA to PEGMA increased, lipase stability was enhanced, whereas activity decreased. By tuning the monomer ratio, we showed that lipase productivity could be optimized. These findings are discussed in the context of complex enzyme-polymer and polymer-polymer interactions and ultimately may enable more informed conjugate designs and improved enzyme productivity in industrial biotransformations under harsh or extreme conditions.

Modeling of polymer-enzyme conjugates formation: Thermodynamic perturbation theory and computer simulations

A simple model for the formation of the polymer-enzyme conjugates has been proposed and described using corresponding modification of the Wertheim's first-order thermodynamic perturbation theory (TPT) for the system of associating chain molecules. A set of computer simulation data for the polymer chains containing various number of functional groups was generated and used to testify the accuracy of the theoretical results. Predictions of the present theoretical approach are more accurate than that of the conventional TPT and are in a very good agreement with the computer simulation data. In particular, the theory is able to account for the difference in the position of the polymer functional groups along its backbone.

Performance of a Flow-Through Enzyme Reactor Prepared from a Silica Monolith and an α-Poly(D-Lysine)-Enzyme Conjugate.

Horseradish peroxidase (HRP) is covalently bound in aqueous solution to polycationic α-poly(D-lysine) chains of ≈1000 repeating units length, PDL, via a bis-aryl hydrazone bond (BAH). Under the experimental conditions used, about 15 HRP molecules are bound along the PDL chain. The purified PDL-BAH-HRP conjugate is very stable when stored at micromolar HRP concentration in a pH 7.2 phosphate buffer solution at 4°C. When a defined volume of such a conjugate solution of desired HRP concentration (i.e., HRP activity) is added to a macro- and mesoporous silica monolith with pore sizes of 20-30µm as well as below 30nm, quantitative and stable noncovalent conjugate immobilization is achieved. The HRP-containing monolith can be used as flow-through enzyme reactor for bioanalytical applications at neutral or slightly alkaline pH, as demonstrated for the determination of hydrogen peroxide in diluted honey. The conjugate can be detached from the monolith by simple enzyme reactor washing with an aqueous solution of pH 5.0, enabling reloading with fresh conjugate solution at pH 7.2. Compared to previously investigated polycationic dendronized polymer-enzyme conjugates with approximately the same average polymer chain length, the PDL-BAH-HRP conjugate appears to be equally suitable for HRP immobilization on silica surfaces.

Biopolymeric Prodrug Systems as Potential Antineoplastic Therapy.

Nowadays, cancer represents a major public health issue, a substantial economic issue, and a burden for society. Limited by numerous disadvantages, conventional chemotherapy is being replaced by new strategies targeting tumor cells. In this context, therapies based on biopolymer prodrug systems represent a promising alternative for improving the pharmacokinetic and pharmacologic properties of drugs and reducing their toxicity. The polymer-directed enzyme prodrug therapy is based on tumor cell targeting and release of the drug using polymer–drug and polymer–enzyme conjugates. In addition, current trends are oriented towards natural sources. They are biocompatible, biodegradable, and represent a valuable and renewable source. Therefore, numerous antitumor molecules have been conjugated with natural polymers. The present manuscript highlights the latest research focused on polymer–drug conjugates containing natural polymers such as chitosan, hyaluronic acid, dextran, pullulan, silk fibroin, heparin, and polysaccharides from Auricularia auricula.

Controllable Enzyme Immobilization via Simple and Quantitative Adsorption of Dendronized Polymer-Enzyme Conjugates Inside a Silica Monolith for Enzymatic Flow-Through Reactor Applications.

Although many different methods are known for the immobilization of enzymes on solid supports for use in flow-through applications as enzyme reactors, the reproducible immobilization of predetermined amounts of catalytically active enzyme molecules remains challenging. This challenge was tackled using a macro- and mesoporous silica monolith as a support and dendronized polymer–enzyme conjugates. The conjugates were first prepared in an aqueous solution by covalently linking enzyme molecules and either horseradish peroxidase (HRP) or bovine carbonic anhydrase (BCA) along the chains of a water-soluble second-generation dendronized polymer using an established procedure. The obtained conjugates are stable biohybrid structures in which the linking unit between the dendronized polymer and each enzyme molecule is a bisaryl hydrazone (BAH) bond. Quantitative and reproducible enzyme immobilization inside the monolith is possible by simply adding a defined volume of a conjugate solution of a defined enzyme concentration to a dry monolith piece of the desired size. In that way, (i) the entire volume of the conjugate solution is taken up by the monolith piece due to capillary forces and (ii) all conjugates of the added conjugate solution remain stably adsorbed (immobilized) noncovalently without detectable leakage from the monolith piece. The observed flow-through activity of the resulting enzyme reactors was directly proportional to the amount of conjugate used for the reactor preparation. With conjugate solutions consisting of defined amounts of both types of conjugates, the controlled coimmobilization of the two enzymes, namely, BCA and HRP, was shown to be possible in a simple way. Different stability tests of the enzyme reactors were carried out. Finally, the enzyme reactors were applied to the catalysis of a two-enzyme cascade reaction in two types of enzymatic flow-through reactor systems with either coimmobilized or sequentially immobilized BCA and HRP. Depending on the composition of the substrate solution that was pumped through the two types of enzyme reactor systems, the coimmobilized enzymes performed significantly better than the sequentially immobilized ones. This difference, however, is not due to a molecular proximity effect with regard to the enzymes but rather originates from the kinetic features of the cascade reaction used. Overall, the method developed for the controllable and reproducible immobilization of enzymes in the macro- and mesoporous silica monolith offers many possibilities for systematic investigations of immobilized enzymes in enzymatic flow-through reactors, potentially for any type of enzyme.

Conjugation of a zwitterionic polymer with dimethyl chains to lipase significantly increases the enzyme activity and stability

Enzyme-polymer conjugates are complex molecules with great practical significance. This work was designed to develop a novel enzyme-polymer conjugate by covalently coupling a zwitterionic polymer with side dimethyl chains (pID) to Candida rugosa lipase (CRL) via the reaction between the anhydrides of polymer chains with the amino groups of the enzyme. The resulting two CRL-pID conjugates with different pID grafting densities were investigated in term of the catalytic activity, stability and structural changes. In comparison with native CRL, both the CRL conjugates displayed 2.2 times higher activity than the native enzyme, and showed an increase in the maximum reaction rate (Vmax) and a decrease in the Michaelis constant (Km), thus resulting in about three-fold increases in the catalytic efficiency (kcat/Km). These are mainly attributed to the activation of lipase by the hydrophobic alky side chains. Moreover, the thermostability and pH tolerance of the lipase conjugates were significantly enhanced due to the stabilizing effect of the zwitterion moieties. For instance, a five-fold increase of the enzyme half-life at 50 °C for the high-pID conjugated CRL was observed. Spectroscopic studies reveal that the pID conjugation protected the enzyme in the changes in its microenvironment and conformation, well correlating with enhanced activity and stability of lipase conjugates. The findings indicate that enzyme conjugation to the zwitterionic polymer is promising for improving enzyme performance and deserves further development.

Cooperative catalysis by a single-atom enzyme-metal complex

Anchoring single metal atoms on enzymes has great potential to generate hybrid catalysts with high activity and selectivity for reactions that cannot be driven by traditional metal catalysts. Herein, we develop a photochemical method to construct a stable single-atom enzyme-metal complex by binding single metal atoms to the carbon radicals generated on an enzyme-polymer conjugate. The metal mass loading of Pd-anchored enzyme is up to 4.0% while maintaining the atomic dispersion of Pd. The cooperative catalysis between lipase-active site and single Pd atom accelerates alkyl-alkyl cross-coupling reaction between 1-bromohexane and B-n-hexyl-9-BBN with high efficiency (TOF is 540 h−1), exceeding that of the traditional catalyst Pd(OAc)2 by a factor of 300 under ambient conditions.

The role of conformational dynamics in the activity of polymer-conjugated CalB in organic solvents

Perennial interest in enzyme catalysis has been expanding its applicability from aqueous phases where enzymes are naturally evolved to organic solvents in which the majority of industrial chemical syntheses are carried out. Although conjugating an enzyme with a soluble polymer has been attempted to enhance enzyme activity in organic solvents, the underlying mechanism remains poorly understood in terms of the conformational dynamics and enzyme activity. Herein, we combine LF-NMR measurements and MD simulations to investigate the effects of polymer grafting on the conformational dynamics of CalB in organic solvents and the consequential impacts on the catalytic kinetics, using the lipase-catalyzed transesterification reaction as a model system. LF-NMR measurements confirm that conjugation with a soluble polymer improves the enzyme flexibility in organic solvents, leading to an increase in the catalytic efficiency of up to two orders of magnitude. MD simulations suggest that the conjugated enzyme samples a larger conformational space, compared to its native counterpart, validating the hypothesis that polymer motion enhances enzyme dynamics. These experimental and simulation results provide new insights for enhancing enzyme conformational dynamics and thereby catalytic kinetics in organic solvents.

Synthesis of silver and gold nanoparticles-enzyme-polymer conjugate hybrids as dual-activity catalysts for chemoenzymatic cascade reactions.

Novel hybrids containing silver or gold nanoparticles have been synthesized in aqueous media and at room temperature using enzymes or tailor-made enzyme-polymer conjugates, which directly induced the formation of inorganic silver or gold species. The choice of pH, protein, or bioconjugate strongly affected the final metallic nanoparticles hybrid formation. Using Candida antarctica lipase (CALB) in a solution, nanobiohybrids containing Ag2O nanoparticles of 9 nm average diameter were obtained. The use of tailor-made bioconjugates, for example, the CALB modified with dextran-aspartic acid polymer (Dext6kDa), resulted in a nanobiohybrid containing smaller Ag(0)/Ag2O nanoparticles. In the case of nanobiohybrids based on gold, Au(0) species were found in all cases. The Au-CALB hybrid contained spherical nanoparticles with 18 nm average diameter size, with a minor range of larger ones (>100 nm) while the AuNPs-CALB-Dext6kDa hybrid was formed by much smaller nanoparticles (9 nm, minor range of 22 nm), and also nanorods of 20-30/40-50 nm length. Using Thermomyces lanuginosus lipase (TLL), apart from the nanoparticle formation, nanoflowers with a diameter range of 100-200 nm were obtained. All nanobiohybrids maintained (dual) enzymatic and metallic activities. For instance, these nanobiohybrids exhibited exquisite dual-activity for hydrolysis/cycloisomerization cascades starting from allenic acetates. By merging the transition metal reactivity with the inherent lipase catalysis, allenic acetates directly converted to the corresponding O-heterocycles in enantiopure form catalysed by AgNPs-CALB-Dext6kDa, taking advantage of a kinetic resolution/cyclization pathway. These results showed the high applicability of these novel hybrids, offering new opportunities for the design of novel reaction cascades.

W/W droplet-based microfluidic interfacial catalysis of xylanase-polymer conjugates for xylooligosaccharides production

To gain a high yield of xylooligosaccharides (XOS) at a low cost, W/W droplet-based microfluidic system combined with immobilized xylanase was constructed. Jetting-regime under the high viscosity of both two phases was correlated with the Ca and We numbers. W/W droplets were obtained with controllable sizes when the flow velocities of dispersed and continuous phases were in the range of 0.29–0.48 µL/min and 0.52–0.80 µL/min respectively. The droplet size could be expressed as a power-law relationship with the velocity ratio of two phases. The maximum yield of XOS was 75.0% within 10 min. Km value was reduced to a half compared with batch reactors. Xylanase-polymer conjugates were reused for 10 times, with a residual activity over 60%. The results confirmed that the combination of enzyme-polymer conjugates and W/W droplet-based microfluidic system is a low-cost and high catalytic efficiency method for biocatalysis.

Glucan Conversion and Membrane Recovery of Biomimetic Cellulosomes During Lignocellulosic Biomass Hydrolysis.

Enzyme immobilization has been identified as one way to recycle enzymes and reduce processing costs during enzymatic hydrolysis of lignocellulosic materials. However, most immobilization methods have not been attractive to lignocellulosic processing plants. In this study, cellulase enzymes were attached to a copolymer of glycidyl methacrylate (GMA) and poly(ethylene glycol) methyl ether methacrylate (PEGMA) to make polymer-enzyme conjugates (PECs) and facilitate recovery using a 50-kDa molecular weight cutoff membrane. Glucan conversion during biomass hydrolysis was investigated using new PECs and PECs recovered after an initial hydrolysis stage. Enzyme immobilization on PECs did not reduce effectiveness during the initial hydrolysis. Temperature and pH showed similar effects on free enzymes and PECs. PECs facilitated higher conversion rates than free enzymes at high biomass loadings. Recovered PECs were used to achieve approximately 100% glucan conversion in a subsequent hydrolysis step when supplemented with 40% of the free enzyme used in the first stage. The combination of PECs and membrane recovery has the potential to reduce hydrolysis cost during cellulosic bioprocessing.

Biomimetic Cellulosomes Assembled on Molecular Brush Scaffolds: Random Complexes vs Enzyme Mixtures

This work reports on the development of biomimetic polymeric cellulosomes—enzyme–polymer conjugates (EPCs) generated by covalent binding of cellulase enzymes onto a molecular brush polymer scaffold...

Activation and stabilization of lipase by grafting copolymer of hydrophobic and zwitterionic monomers onto the enzyme

We report herein new enzyme-polymer conjugates synthesized by grafting polymerization onto Candida rugosa lipase (CRL) with zwitterionic carboxyl betaine methacrylate (CBMA), hydrophobic tert-butyl methacrylate (TBMA) and their equimolar mixture, which respectively created CRL-pCBMA, CRL-pTBMA, and CRL-p(T50-C50). All the enzyme conjugates presented improved catalytic activity, thermostability and pH tolerance, and the kinetic studies indicated that the polymer-grafting resulted in significantly higher enzymatic reaction efficiency and enzyme-substrate affinity. CRL-pTBMA increased the catalytic activity by 2.26-fold and half-life by 43.1-fold at 50 °C as compared with the free enzyme due to the interfacial activation effect of TBMA. However, despite the lower activity of the copolymer conjugate (1.56 times of free enzyme), CRL-p(T50-C50), its half-life was 2.2-fold higher than CRL-pTBMA. This was due to the stabilization effect of the zwitterionic monomer component (CBMA) in the copolymer. This is evident from the similar stability of CRL-p(T50-C50) with CRL-pCBMA, but the latter showed lower activity than the former due to the lack of the interfacial activation effect of TBMA. Thus, a zwitterionic/hydrophobic balance is of vital importance to enhance the enzymatic performance. Spectroscopic characterizations revealed the changes in the microenvironment and secondary structures of the enzyme in CRL-polymer conjugates. Therefore, modification with zwitterionic-hydrophobic copolymer was found more effective in improving the catalytic performance of lipase.

Polymerization-Induced Coassembly of Enzyme-Polymer Conjugates into Comicelles with Tunable and Enhanced Cascade Activity.

Living organisms utilize spatially organized enzyme complexes to carry out signal transduction and metabolic pathways in an efficient and specific way. Herein, inspired by natural enzyme complexes, we report the polymerization-induced coassembly (PICA) of enzyme-polymer conjugates into comicelles with tunable and enhanced cascade activity by using the cascade reaction implemented by glucose oxidase (GOX) and horseradish peroxidase (HRP) as a model system. Notably, the cascade activity of GOX/HRP-polymer comicelles monotonically increases with the GOX/HRP ratio. The cascade activity of GOX/HRP-polymer comicelles is up to 4.9 times higher than that of free GOX and HRP mixtures at the same GOX/HRP ratio. We further demonstrate that our system can quickly detect glucose in contrast with a commercially available glucose assay kit. These findings provide a new and general method of PICA for the controlled construction of artificial enzyme complexes with tunable and enhanced activity in enzyme cascades for advanced biomedical applications.

Study on Water Swellable Enzyme-Polymer Conjugate for Biosensor

Water swellable enzyme-polymer conjugate prepared by polyacrylic acid and glucose oxidase(PAA-GOx) is suggested to enhance a performance of biosensor. According to results, PAA-GOx shows better electrochemical performance although the amount of enzyme in biocatalyst using polyethyleneimine and graphene oxide composite(PEI-GO) is lower than that of glutaraldehyde-GOx(GA-GOx) conjugate. The catalytic activity and sensitivity are 98.0 μA cm-2 with 20mM of glucose in PBS solution and 6.9 μA cm-2 mM-1, which are ca. 10 times higher value compare to GA-GOx due to its high swellability in water. Due to the reason, PAA-GOx conjugate show an excellent ability to absorb water based electrolyte and cause high response and sensitivity through smooth mass transfer of glucose substrate to active site of enzyme. Moreover, the long term stability of catalyst is also improved due to the amide bonds prepared by carboxylic acid of PAA and amine of GOx and PEI. This was supported by Korea National University of Transportation in 2019

Performance evaluation of enzymatic biofuel cells using a new cathodic catalyst containing hemin and poly acrylic acid promoting the oxygen reduction reaction

A new cathodic catalyst for EBCs is developed by adoption of a horseradish peroxidase cofactor mimetic supporter and enzyme–polymer conjugate.

Poly(2-oxazoline)s terminated with 2,2'-imino diacetic acid form noncovalent polymer-enzyme conjugates that are highly active in organic solvents.

A great limitation for the usability of free enzymes in organic solvents is their insolubility in these media. Some surfactants are capable of solubilizing enzymes in such media, but they are hard to remove. Covalent modification of enzymes with polymers has led to polymer-enzyme conjugates (PECs) that are soluble in organic solvents, but the process is quite elaborate. Poly(2-oxazoline)s (POx) with the end group 2,2'-imino diacetic acid were shown to form reversible, nano-sized noncovalent aggregates with enzymes. These PECs give clear solutions in organic solvents. The enzymes lysozyme, horseradish peroxidase (HRP), laccase, α-chymotrypsin (CT), catalase, and alcohol dehydrogenase could be solubilized in chloroform and toluene at concentrations of up to 2 mg protein/ml. Laccase, HRP, and CT were shown to survive the transfer into the organic medium and back to water in their active form. The distribution coefficient of the proteins between water and the organic solvent was shown to be dependent on the nature of the POx backbone. All three biocatalysts exhibit greatly enhanced activity in the respective organic solvent.

Cross-linked enzyme-polymer conjugates with excellent stability and detergent-enhanced activity for efficient organophosphate degradation

BackgroundEnzymatic biodegradation of organophosphate pesticides (OPs) is a promising technology to remove these toxic compounds. However, its application in industrial washing was restricted by the lack of efficient immobilized enzymes that can work at high temperatures and high pHs in the presence of various detergents. Therefore, it is necessary to develop a simple method to prepare a robust immobilized enzyme for efficient degradation of OPs.ResultsAn organophosphate hydrolase (OPH), PoOPHM9, was conjugated and immobilized with a commercially available polymer, Pluronic F127. The prepared cross-linked enzyme-polymer conjugate (CLEPC) displayed higher pH stability in the range from 7.0 to 11.0 and a higher optimal temperature (50 °C) than that of free PoOPHM9 (30 °C). Its half-life and apparent kcat/KM reached 12.8 h at 50 °C and 390.3 ± 7.8 mM−1 s−1, respectively, which were even better than that of the traditional cross-linked enzyme aggregates (CLEA, 7.2 h and 10.9 ± 1.7 mM−1 s−1). The activity of PoOPHM9 CLEPC was further enhanced up to 2.5-fold by the anionic, nonionic and biocompatible detergents, which was first observed. 0.15 mM Malathion was degraded completely by PoOPHM9 CLEPC after activation within 10 min in the presence of 0.1% (w/w) detergents of all types at pH 9.0 and 25 °C, demonstrating its capability in degrading OPs at practically relevant conditions.ConclusionThe conjugation of Pluronic F127 in enzyme immobilization could effectively reduce the activity loss of immobilized enzymes and enhance their stability and activity at high temperatures and high pHs. In addition, the activity of CLEPC can be even enhanced in the presence of various detergents. This technology can be extended easily to produce other immobilized polymer-enzyme conjugates due to its simplicity.

Enzyme–Polymer Conjugates as Robust Pickering Interfacial Biocatalysts for Efficient Biotransformations and One‐Pot Cascade Reactions

AbstractDespite the rapid development of Pickering interfacial catalysis (PIC) at liquid–liquid interfaces with chemocatalysts, the use of unstable biocatalysts at emulsion interfaces remains a technical challenge. Herein, we present a Pickering interfacial biocatalysis (PIB) platform based on robust and recyclable enzyme–polymer conjugates that act as both catalytic sites and stabilizers at the interface of Pickering emulsions. The conjugates were prepared by growing poly(N‐isopropylacrylamide) on a fragile enzyme, benzaldehyde lyase, under physiological conditions. The mild in situ conjugation process preserved the enzyme structure, and the conjugates were used to emulsify a water–organic two‐phase system into a stable Pickering emulsion, leading to a significantly larger interfacial area and a 270‐fold improvement in catalytic performance as compared to the unemulsified two‐phase system. The PIB system could be reused multiple times. Conjugates of other enzymes were also fabricated and applied for cascade reactions.