Engineering of enzyme biocatalysts with nano-sized polymers with upper critical solution temperature (UCST)

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INTRODUCTION
Immobilization of enzymes to polymers has become a research hotspot to empower enzymes with more extraordinary properties and broader usage. As compared with free enzymes, polymer-immobilized enzymes can improve operational and thermal stabilities in harsh environments such as extreme pH, concentration and temperature. Polymers possess underlying stimulus-responsive features, which allows them to undergo a conformational change or coil-to-globule transition upon a change in external stimuli such as temperature, counterion, pH, electricity and light (1-5).

 

Amid stimulus-responsive polymers, thermoresponsive polymers have been extensively studied in academic and applied polymer sciences over the last decades (2-8). A thermoresponsive polymer undergoes a change in solubility in a polymer-solvent system with varying temperature (9,10). As the temperature increases, the polymer dissolves in the solvent through an upper critical solution temperature (UCST) process, and/or precipitates from the solution through a lower critical solution temperature process. In Scheme 1 is shown the representative phase diagram pattern of UCST-type polymer-solvent systems, which depicts the solution behaviour of the polymer as a function of temperature versus polymer concentration.
In such a phase diagram, the maximum of the solubility curve is the UCST value (2,3,10).
Any point along the solubility curve is defined as a cloud point (Tcp).

Thermoresponsive polymers are expected to find “smart” applications that exploit the temperature responsivity. The “smartness” consists in integrating the advantage of homogeneous biocatalysis with the convenience of biocatalyst separation from a two-phase system via a thermoresponsive polymer phase transition, so as to meet the goal of high biocatalytic efficiency of enzymes. When a thermoresponsive polymer-immobilized enzyme is used to catalyze a chemical or biocatalytic reaction, the immobilized enzyme can be shifted from the solid to liquid phase by varying the temperature so that a homogeneously biocatalytic reaction can be carried out in high efficiency. After the reaction, the liquid-phase system can be shifted back to the two-phase system by changing the temperature for the purpose of separation and reuse of the immobilized enzyme. Such a reversible process for a biocatalytic reaction cycling with UCST-type polymer-immobilized enzymes is sketched in Scheme 2.

 

Nano-sized polymers are attractive supports to achieve higher specific enzyme loading, higher specific activity and less mass transfer limitations than their micro-sized counterparts in heterogeneous biocatalysis. This review presents an explicit overview of biocatalysis by nano-sized UCST-type polymer-immobilized enzymes to readers. We highlight the published studies with an outlook on the new engineering of enzymes.

 

APPLICATION OF NANO-SIZED UCST-TYPE POLYMERS IN BIOCATALYSIS BY IMMOBILIZED ENZYMES
Although the number of the published studies of nano-sized UCST-type polymers applied in immobilized enzyme biocatalysis is limited (11-17), these studies have a profound impact on the perspective of the developing UCST-type polymer-immobilized biocatalysis.

Zhu et al. communicated the first case of the nano-sized UCST-type polymers for engineering of enzyme biocatalysts, which related to conjugation of different enzymes to Pluronic F-127 and biocatalysis of the resultant enzyme-Pluronic nano-conjugates (11). Immobilization of enzymes to polymers is fulfilled by coupling the polymers to accessible amino acid side chains or end termini on the enzymes preferably via covalent linking. In the preparation of the enzyme-Pluronic nano-conjugates, the polymer was first aldehyde functionalized via the Dess-Martin oxidation of surface –OH, and subsequently the resultant functionalized polymer was bound to the enzymes via the reaction of –CHO with –NH2 forming the imines.

The phase transition of 20 % Pluronic F-127 in water was abrupt on turbidity curves, showing a Tcp of 23 oC (18,19). According to the electrophoretic and fluorometric analyses, high conjugation yields were achieved on all the enzymes including bovine serum albumin (BSA), Candida rugosa lipase (CRL), Candida antarctica lipase B (CALB) and cytochrome c (Cyt C). In the enzymatic hydrolysis of p-nitrophenyl butyrate at room temperature, the relative activities of the CRL-, CALB- and Cyt C-Pluronic conjugates to those of their respective free enzymes were found to be 58, 98 and 568 %, respectively. Such results can rule out the possibility of mass transfer limitations between the nano-sized conjugates and the substrate in water.

 

This work included the earliest examples of thermoresponsive enzyme-polymer conjugates that exhibit a higher activity than their native counterparts in organic solvents. At 40 oC, all the enzyme-Pluronic conjugates readily dissolved in most commonly used organic solvents including toluene, tetrahydrofuran, methanol, dichloromethane and chloroform, in which the free enzymes remained as precipitates. According to the UV-Vis absorbance measurements, a Tcp appeared around 12 oC with 10 % Pluronic F-127 in toluene while no UCST behaviour was observed with 1 % Pluronic F-127 in toluene. Nevertheless, the BSA-Pluronic conjugate as a representative nano-conjugate displayed a Tcp with a broad phase transition temperature interval of 23-43 oC with 1 % conjugate in toluene. The dramatic appearance of Tcp is definitely attributed to the positive effect of BSA conjugation on the conjugate-conjugate interaction in toluene, indicating that the conjugate-conjugate interaction is assumed to be between the hydrophobic segments of Pluronic in toluene.

 

Scheme 1. Phase diagram pattern of UCST-type polymer-solvent systems.

 

According to the dynamic light scattering measurements, the average particle size of the conjugates dissolved fell at 27-42 nm at room temperature with 0.01 % conjugate in toluene, in agreement with the TEM observations. In the enzymatic esterification of palmitic acid and n-octanol in toluene at 40 oC, the CRL-Pluronic conjugate displayed an increased activity by 58-fold compared to free CRL. In the enzymatic esterification of hexanoic acid and n-butanol in toluene at 40 oC, the CALB-Pluronic conjugate gave rise to an increased activity by 68-fold compared to free CALB. In the enzymatic oxidation of 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt by hydrogen peroxide in methanol at 40 oC, the peroxidase activity of Cyt C was 670-fold that of free Cyt C. The excellent relative activities can rule out the possibility of mass transfer limitations between the nano-sized conjugates and the substrates in the organic solvents. By cycling the temperature between 4 and 40 oC with 1 % conjugate in toluene, the CALB-Pluronic conjugate was successfully reused 9 times for the esterification of hexanoic acid and n-butyl alcohol, giving 98 % of the initial activity after 10 cycles. The reusability test is essential evidence that the recovery of the CALB-Pluronic conjugate proceeds smoothly at 4 oC and that the Pluronic-immobilized CALB via covalent linking is quite stable under the reaction conditions.

 

Cummings et al. published a paper on biocatalysis of nano-sized poly(3-dimethyl(methacryloyloxyethyl) ammonium propane sulfonate) (i.e., PDMAPS)-conjugated chymotrypsin (CT) for hydrolysis of N-succinyl-Ala-Ala-Pro-Phe p-nitroanilide (Suc-AAPF-pNA) (12). The conjugated CT was synthesized via the covalent attachment of the atom transfer radical polymerization initiator to accessible primary amines either on lysine residues or N-terminus of CT, followed by the growth of PDMAPS from the initiator-modified CT. In UV-Vis absorbance, the PDMAPS and the CT-PDMAPS conjugate in phosphate buffers responded to changes in temperature in a similar manner, giving their Tcp values of 12 and 13 oC, respectively. The slight rise in the Tcp is deemed to arise from more hydrophobic segments on the conjugate than on the PDMAPS. By dynamic light scattering, the CT-PDMAPS conjugate was found to have a maximal temperature-dependent hydrodynamic particle radius of 6.5 nm at 19 oC, corresponding to the one-phase state above the Tcp. Towards the enzymatic hydrolysis of Suc-AAPF-pNA, the conjugate and free CT showed close activities both at 25 and 40 oC in terms of the limiting reaction rate (Vm)/the Michaelis constant (Km). Such a relative activity of the conjugate can be attributed to no mass transfer limitations between the conjugate and the substrate. Although free CT had a higher Vm, the conjugate gained a lower Km because of an enhanced Suc-AAPF-pNA’s affinity with the conjugate mainly through the hydrophobic interaction between Suc-AAPF-pNA and PDMAPS. The lower Vm over the conjugate than over free CT implicates that the conjugation causes a conformational change of part of CT. The conjugate exhibited a higher enzyme thermal stability than free CT both at 25 and 40 oC, especially at 40 oC.
The conjugation led to a 3.1-fold enhancement in the half-life of CT at 1 mg mL-1
and 40 oC.

 

Scheme 2. Biocatalytic reaction cycling with UCST-type polymer-immobilized enzymes.

 

Limadinata et al. successfully synthesized nano-sized P(AAm-co-AAc) and developed good performance P(AAm-co-AAc)-immobilized cellulase and cellobiase nanoparticle biocatalysts for hydrolysis of insoluble celluloses such as filter paper and pretreated oil palm empty fruit bunch to glucose (14).
The enzymes including a cellulase (consisting of cellobiohydrolase, endoglucanase and cellobiase at a ratio of 20 : 80 : 1) and cellobiase (i.e., β-GS) were covalently immobilized onto the P(AAm-co-AAc). Functionalizing the P(AAm-co-AAc) nanoparticles with glycidyl methacrylate (GMA) gave an interpenetrating polymer network of PGMA-P(AAm-co-AAc) (i.e., IPN PGA) nanoparticles with a yield of 71 %. The nanoparticles had mean size of 109 nm and hydrodynamic size of 122 nm. Incorporating the IPN PGA nanoparticles with the cellulase or cellobiase resulted in IPN PGA-immobilized cellulase (IPN-Cellu) or cellobiase (IPN-Cello) nanoparticles with a yield of 100 %. In the enzymatic hydrolyses of carboxymethylcellullose as a water-soluble substrate filter paper and pretreated oil palm empty fruit bunch as water-insoluble substrates at 50 oC, the IPN-Cellu could retain 90, 86 and 63 % activities of the free cellulase, respectively after 12 h of reaction. In the enzymatic hydrolysis of cellobiose as a slightly water-soluble substrate at 50oC, the IPN-Cello could retain a 78 % activity of free cellobiase after 12 h of reaction. The relative activities of 63-90 % over the soluble nano-sized IPN-Cellu or IPN-Cello implies that there more or less exist mass transfer limitations between the conjugate and the substrate during the hydrolyses.

 

This group focused their work on the recyclability of the nano-sized IPN-Cellu and the nano-sized IPN-Cello towards the hydrolyses of the soluble and insoluble celluloses. The reactions were carried out at 50 oC. After each run, the insoluble substrate was removed by centrifugation at room temperature, and the supernatant was cooled down to 4 °C and was subjected to centrifugation at 4 °C to recover the conjugate for recycling. The supernatant was also used for product analysis.
No enzyme leaching from the conjugates in each cycle was detected by the Bradford assay, demonstrating a strong attachment of the enzymes to the nano-carrier. In the hydrolyses of carboxymethylcellullose, filter paper and pretreated oil palm empty fruit bunch, the IPN-Cellu with 1 % cellulase relative to substrate was able to produce glucose yields of 56, 42 and 31 %, respectively.
For the hydrolysis of carboxymethylcellullose, 72 % of the initial activity remained after 10 cycles, which represents one of the prominent recyclability results of immobilized cellulases (20-24). And for the hydrolysis of pretreated oil palm empty fruit bunch, 70 % of the initial activity remained after 10 cycles, which shows a strong biocatalyst recyclability. Due to the reason that the very low fraction of cellobiase in the cellulase used restricts the conversion efficiency of cellobiose (as the intermediate sugar) to glucose, a mixture of the IPN-Cellu and the IPN-Cello was investigated as a mixed biocatalyst for the purpose of increasing the glucose productivity from the hydrolysis of celluloses. As a result, the addition of the IPN-Cello obviously increased the glucose yields in the hydrolyses of filter paper and pretreated oil palm empty fruit bunch. The mixture of the IPN-Cellu and the IPN-Cello with a weight ratio of 1 : 2 was able to produce glucose yields of 97 and 93 % in the hydrolyses of filter paper and pretreated oil palm empty fruit bunch, respectively. This work presented the unprecedented high glucose yields from hydrolysis of lignocelluloses with immobilized enzymes. For the former reaction, 71 % of the initial activity was retained after 8 cycles. And for the latter reaction, 73 % of the initial activity was retained after 6 cycles. Such biocatalyst recyclabilities are good enough compared to the reported strong recyclabilities of immobilized cellulases and cellobiase for hydrolysis of lignocelluloses (20, 25, 26).

 

Yang et al. disclosed their work on chemo- and biocatalytic applications of nano-sized chemically crosslinked PNAGA hydrogels (15,27). The chemically crosslinked PNAGA hydrogels and the β-D-glucosidase (β-D-GS)-PNAGA hydrogels were synthesized using N, N’-methylenebis(acrylamide) (BIS) as the crosslinker by free radical polymerization in water. The silver nanoparticle (AgNP)-containing hybrid hydrogels were prepared by reduction of AgNO3 to AgNPs using NaBH4 inside the PNAGA hydrogels and the BG-PNAGA hydrogels, respectively. The dynamic light scattering study indicated that both the chemically crosslinked PNAGA hydrogels and the hybrid chemically crosslinked PNAGA hydrogels continuously swelled upon heating up to 70 oC in water. The particle size of the PNAGA-3.5 % BIS hydrogels in phosphate buffer increased by 1.9 times in volume upon heating from 10 to 52 oC (15). It approached the maximum above 52 oC. The broad temperature range from 10 to 52 oC was deemed to correspond to the phase transition temperature interval, analogous to the cases of other UCST-type polymers (12,17,28,29). The changes in the concentrations of BIS and β-D-GS added in the chemically crosslinked PNAGA hydrogels apparently did not change the phase transition of the PNAGA hydrogels which followed the same trend of particle size variation with temperature (15,20), differing from what happens to linear homologous PNAGA hydrogels (30-34). In the latter cases, the phase transition is sensitive to the polymer molecular weight, the polymer structure and the environmental factors, so that the Tcp values are susceptible to vary (30-34). Based on the comparative observation that the particle size of free β-D-GS dramatically rose around 50 oC due to aggregation, it was assumed that the aggregation of β-D-GS encapsulated inside the PNAGA hydrogels is suppressed at elevated temperatures (15). The suppression of β-D-GS aggregation may result from the hydrogen bonding between β-D-GS and the PNAGA hydrogels which renders β-D-GS dispersed on the PNAGA hydrogels. Likewise, the hybrid Ag-PNAGA-BIS hydrogels retained the phase transition of the PNAGA hydrogels, displaying an increased dispersion or swelling with increasing temperature up to at least 70 oC in water (20). Towards the enzymatic cleavage of p-nitrophenyl-β-D-glucopyranoside (pNGP), the β-D-GS -PNAGA hydrogels displayed a relative activity varying from 22 to 2200 % at pH 3-11 and 40 oC (15). The optimal activity was found at pH 7.0 with an apparent kinetic constant of 5.0 × 103 s-1 mmol-1.

 

Sun et al. designed nano-sized triblock copolymers using acrylamide (AAm) and N-acryloxysuccinimide (NAS) as comonomers that enabled both covalent binding and hydrogen bonding with proteins such as BSA and ovalbumin (OVA) in favour of facile formation of protein-copolymer hybrids for applications in biocatalysis and biomedicine (16). From the turbidity measurements of 0.1 % P(AAm-co-NAS-co-acrylic acid (AAc)) solutions, the Tcp increased with increasing NAS fraction, being tunable from 12 to 81 oC over an NAS fraction range from 1.5 to 7.3 mol%. The Förster resonance energy transfer study suggested that both the covalent bonds and the hydrogen bonds contribute to the enzyme-copolymer hybridization in the enzyme-P(AAm-co-NAS-co-AAc): the covalent bonding makes a strong chemical linkage between the enzyme and the copolymer, while the hydrogen bonding produces a loose enzyme-copolymer complex.

The circular dichroism study demonstrated that such a BSA-copolymer coassembly process does not alter the thermal stability of BSA. Towards the enzymatic hydrolysis of p-nitrophenyl acetate, BSA-P(AAm-co-NAS-co-AAc) could retain 95 % or above of the activity of free BSA, assuming the occurrence of less mass transfer limitations between BSA-P(AAm-co-NAS-co-AAc) and the substrate.

 

To sum up, Table 1 compiles all the reported cases of nano-sized UCST-type polymer-immobilized enzymes in biocatalytic applications.

CONCLUSIONS AND OUTLOOK
Almost all of the reported nano-sized UCST-type polymer-conjugated enzymes display superior or close activity to that of their free counterparts as well as superior stability to that of their free counterparts (11,12,14-17). Some of them even produce an impressive activity which is (59-670)-fold that of their free counterparts (12). The striking advantage of nano-sized UCST-type polymers in terms of relative activity consists in higher specific surface area which enables high dispersion of enzymes and less mass transfer limitations between the conjugates and the substrates. However, the disadvantage of nano-sized UCST-type polymers in terms of separation and recovery ought not to be ignored. In designing and developing nano-sized UCST-type polymer-engineered enzyme biocatalysts, the biocatalytic performance should be preferably compared to that of their micro-sized counterparts. In making an optimal choice between nano-sized and micro-sized UCST-type polymer counterparts, a trade-off is required between activity and separation and recovery.

 

UCST-type polymer-immobilized biocatalysis is of important significance, since UCST-type polymer-immobilized biocatalysts use a lower temperature for biocatalyst separation than for homogeneously biocatalytic reactions, which ensures prevention of the immobilized biocatalysts from deactivation (17, 30, 35, 36).
What’s more, the phase transition properties of thermoresponsive polymers can be tuned by taking advantage of copolymerization. By using different comonomers and adjusting related synthesis parameters adapted to the variation of hydrogen bonding with temperature, the properties of UCST-type copolymers can be expectedly optimized (13,14,16,17,20).

As such, there is bigger room for the application of UCST-type polymers in nano-enzyme biocatalysis. It can be envisioned that various types of polymers would be developed to meet the needs of rationally designing and fabricating versatile immobilized nano-enzyme biocatalysts.

 

Table 1. Reported nano-sized UCST-type polymer-immobilized enzymes for applications in biocatalytic reaction. a

 

ACKNOWLEDGEMENT
This work was supported by GlaxoSmithKline, Economic Development Board (Singapore) and Agency for Science, Technology and Research (Singapore).

 

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