Lectin Agglutinated Multienzyme Catalyst with Enhanced Substrate

May 6, 2016 - ... was conducted on a Zeiss LSM-710 NLO laser scanning confocal system equipped with Plan-Apochromat 100x/1.40 Oil DIC M27 objectives...
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Research Article pubs.acs.org/acscatalysis

Lectin Agglutinated Multienzyme Catalyst with Enhanced Substrate Affinity and Activity Yifei Zhang,† You Yong,† Jun Ge, and Zheng Liu* Key Lab for Industrial Biocatalysis, Ministry of Education, Department of Chemical Engineering, Tsinghua University, Beijing 100084, China S Supporting Information *

ABSTRACT: A method based on biological recognition was proposed to prepare cross-linked multiple glycoenzyme aggregates. With Concanavalin A (ConA) as a molecular glue, horseradish peroxidase (HRP) and glucose oxidase (GOx) were agglutinated and then cross-linked by glutaraldehyde, forming a GOx-ConA-HRP catalyst. The affinity of ConA to glucose enhanced the uptake of the substrate, reducing the Km of cross-linked GOx-ConA-HRP aggregates for glucose from 51 mM to 8.8 mM. The colocalization and clustering of cascade enzymes at nanoscale facilitated the intermediate consumption. These effects significantly improved the catalytic performance of the GOx-HRP cascade with a 1.5-fold increased specificity constant. The use of ConA as a molecular glue provides a facile way to construct a multienzyme catalyst with enhanced stability and activity. KEYWORDS: multienzyme systems, Concanavalin A, glycoenzyme aggregates, substrate affinity, enhanced activity

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INTRODUCTION Artificial multienzyme systems which perform multiple steps of cascade or coupled reactions by a combination of enzymes in vitro bring new opportunities in biosynthesis and biodiagnostics.1 Numerous potential benefits such as the protection of unstable intermediates and suppression of the undesired pathways and short-distance transport of intermediates are all conducive to high catalytic performances.2 Great efforts have been directed into fabricating a high-efficiency multienzyme catalyst, including assembling enzymes on DNA or RNA scaffolds,3,4 encapsulating enzymes in nanocontainers,5 and coimmobilizing enzymes on carriers.6 However, the former two methods are too elaborate to scale up, and the latter one usually reduces the activities of enzymes. Moreover, all enzymes are usually coimmobilized by the same chemical strategy, and the overall stability is determined by the less stable one.7 There is still a need for an economical and effective solution to construct high-efficiency multienzyme catalysts in a mild and biocompatible condition. As a scaffold-free method, cross-linking the aggregates of multiple enzymes together is an alternative for multienzyme system construction. The cross-linked enzyme aggregates (CLEA) proposed by Sheldon and colleagues has been applied in many practical industrial processes and has proven advantages in terms of enhanced stability, high recovery of activity, and ease of operation.8−10 This technique is also applicable to the coimmobilization of two or more enzymes in “combi-CLEAs” used for multiple-step catalysis in one-pot,11 as exemplified in the cascade synthesis of (S)-mandelic acid with a high yield and high enantiomeric purity.12 However, the use of © XXXX American Chemical Society

high concentration of ammonium sulfate (up to 5 M) and/or organic solvents (such as alcohols) to precipitate enzymes may lead to enzyme denaturing. The addition of necessary additives such as polyethylene glycol and sometimes sugars as stabilizer might introduce undesired impurities into the catalytic system, limiting their ranges of applications.10−14 However, the substrate uptake of the CLEAs may be hindered due to the cross-linked structures, leading to a reduced apparent activity. Concanavalin A, a type of lectin which can specifically bind to carbohydrates containing nonreducing terminal α-Dmannosyl or α-D-glucosyl moieties15 has been widely used in antitumor therapy,16 diagnostics,17,18 affinity chromatography,19 and enzyme embedding.20 In nature, glyco-proteins are widely distributed in eukaryotic cells and have also been found in some prokaryotic organisms.21 More than half of all proteins are estimated to be glycosylated.22 Most commonly used enzymes such as glucose oxidase from Aspergillus niger (GOx), horseradish peroxidase (HRP), bromelain from pineapple stem, catalase from bovine liver, and ribonucleic acid from calf liver are all glycosylated enzymes; thus, all of them can be recognized and agglutinated by ConA. It thus inspired us to use ConA as a natural glue to prepare multienzyme catalysts. Here, we report a new method of preparing multienzyme catalysts by using ConA to agglutinate enzymes. GOx and HRP were employed as a model system to demonstrate the feasibility of constructing lectin-mediated enzyme aggregates and to Received: April 12, 2016 Revised: May 2, 2016

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DOI: 10.1021/acscatal.6b01047 ACS Catal. 2016, 6, 3789−3795

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ACS Catalysis

ABTS. One unit of specific activity of the bienzyme system is defined as 1 mg of total enzymes consumed by 1.0 μmole of H2O2 per minute. High Performance Liquid Chromatography (HPLC). To determine the aggregation yield of HRP and GOx, the supernatant of the aggregate solution obtained by centrifugation was subject to HPLC on a TSK-GEL G2000SWXL column (5 μm, 7.8 × 300 mm) with a SHIMADZU SPD10AVP UV−vis detector. Typically, 20 μL of sample was injected and eluted by phosphate buffer solution (pH 6.7, 50 mM, containing 100 mM Na2SO4 and 0.5‰ NaN3) at a flow rate of 1 mL/min. Dynamic Light Scattering. Dynamic light scattering was carried out on a Malvin Zetasizer ZS-90 at a protein weight ratio around 1.0 mg/mL at room temperature. Synthesis of FITC-Labeled HRP and Rhodamine Blabeled GOx. The procedures of fluorescent labeling of HRP and GOx were described elsewhere.23 Laser Confocal Microscopy. Laser confocal microscopy was conducted on a Zeiss LSM-710 NLO laser scanning confocal system equipped with Plan-Apochromat 100x/1.40 Oil DIC M27 objectives. The excitation wavelengths for FITCGOx and Rhodamine B-HRP were 488 and 550 nm, respectively. The emission wavelengths were 560 and 590 nm, respectively. Fluorescent Spectra. Fluorescent spectra of various protein solutions were carried out on a SHIMADZU RF5301 PC fluorescence spectrophotometer. For unlabeled protein, the excitation wavelength was fixed at 280 nm. For FITC and Rhodamine B labeled protein, the excitation wavelengths were 488 and 560 nm, respectively. Transmission Electron Microscopy (TEM). The size and shape of enzyme aggregates were characterized by a highresolution transmission electron microscope (Hitachi JEOL 7401). Protein was stained by 1%, pH 7.0 sodium phosphotungstate before TEM observation, as detailed elsewhere.24

studying the catalytic process mechanism. Besides the effectiveness of assembling enzymes, the high substrate affinity of ConA for glucose facilitates the uptake of substrates, and the colocalization of cascade enzymes with nanoscale proximity enables a faster consumption of the intermediate. These effects would extend the applications of enzymatic catalysis, particularly in catalyzing reactions with ultralow substrate concentrations.

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EXPERIMENTAL SECTION Materials. Glucose oxidase from Aspergillus niger, horseradish peroxidase, Concanavalin A from Jack bean (type IV), Dglucose, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulponic acid)diammonium salt (ABTS), glutaraldehyde (25 wt % in water), fluorescein isothiocyanate (FITC), and rhodamine B isothiocyanate were purchased from Sigma-Aldrich Co., LLC. Hydrogen peroxide (30 wt % in water) and sodium borohydride (NaBH4) were purchased from Alfa Aesar, China. Synthesis of Enzyme-ConA Aggregates. The enzymeConA aggregates were spontaneously agglutinated by mixing the enzyme solution and ConA solution. Typically, for the synthesis of GOx-ConA aggregates, a certain volume (e.g., 20 μL) of 1.0 mg/mL GOx solution was added into 1 mL of 1.0 mg/mL ConA solution. The mixture was then stirred at room temperature for 1 h to allow aggregate formation, followed by cross-linking with glutaraldehyde (0.2% w/v in the reaction mixture) at room temperature for 2 h. NaBH4 (5% weight of protein) was added into the mixture to reduce the Schiff base for 4 h. With the similar method, the cross-linked HRP-ConA and GOx-ConA-HRP aggregates were synthesized. The enzyme aggregates can be purified by centrifugation. The supernatant was sampled and subjected to size exclusion chromatography to determine the content of free enzyme. The above procedure was also applied to determine the yield of HRP-ConA aggregates and GOx-ConA-HRP aggregates. Enzymatic Assays. All of the enzymatic assays were taken on the SHIMADZU UV-2450 spectrophotometer at room temperature. The increase in absorbance at 415 nm was measured for 1 min. The enzyme activity was calculated from the slope of absorbance versus time curve. For the HRP activity assay, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonicacid)-diammonium salt (ABTS) was used as the substrate. In a typical run, 20 μL of 10 μg/mL HRP solution and 50 μL of 0.3% H2O2 were added into 930 μL of phosphate buffer solution (pH 7.0, 50 mM) containing 0.5 mM ABTS. One unit activity of HRP is defined as the amount of HRP that catalyzes 1.0 μmole of H2O2 per minute at pH 7.0 at 25 °C. The specific activity of HRP is defined as 1 mg of HRP that catalyzes 1.0 μmole of H2O2 per minute. For the GOx activity assay, 20 μL of 20 μg/mL GOx solution (or suspension of ConA-GOx aggregates) and 100 μL of 100 μg/mL HRP solution were added to 890 μL of the substrate solution (50 mM phosphate buffer at pH 7.0, containing 100 mM glucose and 0.5 mM ABTS) to initiate the reaction. One unit activity of GOx was defined as the amount of GOx that generates 1.0 μmole of H2O2 per minute at pH 7.0 at 25 °C. The specific activity of GOx is defined as 1 mg of GOx that generates 1.0 μmole of H2O2 per minute. For the GOx-HRP system, 20 μL of GOx-HRP solution (in most experiments, the concentration of GOx and HRP were 20 μg/mL and 80 μg/mL) or suspension of GOx-ConA-HRP aggregates was added into 980 μL of phosphate buffer solution (pH 7.0, 50 mM) containing 100 mM glucose and 0.5 mM

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RESULTS AND DISCUSSION Preparation and Characterization of ConA-Agglutinated GOx Aggregates. The preparation of protein aggregates using ConA as agglutinant is shown in Scheme 1. Scheme 1. Preparation of Protein Aggregates Using ConA as Agglutinant and Glutaraldehyde as Cross-Linker

Once ConA is added into the aqueous solution of GOx or HRP, protein aggregation occurs spontaneously. The aggregation yields of GOx and HRP highly depended on the concentration of ConA (Figure 1a), as determined by size exclusion chromatography (Figure S1). We first prepared the GOx-ConA aggregates and studied the catalytic properties. The optimal mass ratio of GOx to ConA is set as 1:10 to obtain a high aggregation yield, from which about 98% of GOx and 80% of ConA were precipitated, suggesting that GOx was 3790

DOI: 10.1021/acscatal.6b01047 ACS Catal. 2016, 6, 3789−3795

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Figure 1. (a) ConA agglutinated GOx (labeled with FITC, yellow) and HRP (labeled with Rho-B, magenta) aggregates and their aggregation yields at various concentrations of ConA; (b) DLS of free GOx (blue) and cross-linked GOx-ConA aggregates (red).

Figure 2. (a) Relative activities of free GOx, GOx-ConA, and cosslinked GOx-ConA aggregates. (b) The Lineweaver−Burke plots of free GOx (black), ConA-GOx aggregates (red), and cross-linked GOx-ConA aggregates (blue); the inset shows the intercepts.

52 mM for free GOx). The cross-linking by glutaraldehyde led to a slight increase in Km of cross-linked GOx-ConA, which might be attributed to the additional steric hindrance and partial loss of enzyme conformation flexibility. We also crosslinked BSA (bovine serum albumin) with GOx and found that the Km of BSA-GOx slightly increased to 55 mM (Figure S4). These results suggested that the decrease in Km of the GOxConA aggregates can be attributed to the affinity of ConA toward glucose. To further confirm this conclusion, we determined the association constant of ConA for glucose using the method proposed by Kim et al. (see details in Figure S5).29 The binding constant Ka is 1970 M−1, which agrees well with the reported results.29,30 Such an affinity adsorption enriched glucose in the vicinity of GOx-ConA aggregates and the active sites, leading to a lower apparent Km. Rational tailoring of the substrate−carrier interaction is an emerging strategy for tuning enzyme kinetics. In earlier research, the electrostatic interaction was well introduced to modulate enzymatic activity for charged substrates. For example, You et al.31 prepared enzyme−nanoparticle complexes composed of α-chymotrypsin and amino-acid-functionalized gold nanoparticles. The substrate specificity (kcat/Km) was improved by 3-fold for the cationic substrate but decreased by 95% for the anionic substrate. Murata et al.32 grafted a positively charged polymer on the surface of α-chymotrypsin. The conjugate had an increased affinity for negatively charged substrate and a reduced inhibition effect against positively charged inhibitor. Wheeldon and colleagues33,34 presented that the nonspecific attraction can also be used to improve the

surrounded by Con A in the agglutinated aggregates with a molecular ratio of 1:12.8. Even at a very low concentration of GOx (10 μg/mL), over 90% of GOx can be agglutinated from the solution containing 1.0 mg/mL of ConA (Figure S2). To strengthen the enzyme aggregates, glutaraldehyde was applied to covalently cross-link the enzymes, yielding the more stable protein aggregates with an average diameter around 1−2 μm, as shown by dynamic light scattering (DLS) (Figure 1b). Glutaraldehyde is one of the most widely used reagents in enzyme immobilization and can react with amino groups from lysines with several different mechanisms, which has been well reviewed by Barbosa et al.25 The cross-linked enzyme aggregates showed an improved stability against the high concentration of glucose (Figure S3). The effect of ConA on GOx catalytic performance was examined. GOx activity assay was conducted in the presence of 50-fold excess of HRP to ensure a rapid and complete consumption of H2O2 generated by GOx. As shown in Figure 2a, the ConA agglutinated GOx aggregates retained 91% activity of the free GOx. It indicated that ConA is a mild agglutinant for preparing enzyme aggregates and is conducive to stabilize the conformation and the multimeric form of GOx.26 The further treatment with glutaraldehyde reduced the apparent activity to 57%. Such a significant reduction of GOx activity may be caused by the irreversible conformation change of the enzyme during cross-linking, as observed by other researchers.27,28 As shown in Figure 2b, a remarkable decrease in Km for glucose was observed (21 mM for GOx-ConA and 25 mM for cross-linked GOx-ConA aggregates, as compared with 3791

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Figure 3. (a) Aggregation yields of HRP and GOx in GOx-ConA-HRP aggregates at various ConA concentrations; (b) DLS of GOx-ConA-HRP and glutaraldehyde cross-linked GOx-ConA-HRP; (c) TEM of cross-linked GOx-ConA-HRP aggregates; (d) laser confocal microscopy of cross-linked GOx-ConA-HRP aggreagtes, in which GOx and HRP were labeled, respectively, with fluorescein (FITC) and rhodamine-B (RhoB) (scale bar presents 10 μm). The insets are zoomed-in images.

Figure 4. (a) Overall and specific activity of the free GOx with free HRP, GOx-ConA-HRP aggregates, and cross-linked GOx-ConA-HRP aggregates. (b) Activities of free HRP, HRP-ConA aggregates, and cross-linked HRP-ConA aggregates. (c) Overall activities of cross-linked GOx-ConA-HRP, cross-linked GOx-ConA with cross-linked HRP-ConA, and cross-linked GOx-ConA with free HRP (compared with free GOx and free HRP at the same concentrations). (d) Lineweaver−Burke plots of free HRP&GOx, HRP-ConA, aggregates, and cross-linked HRP-ConA aggregates.

catalytic efficiency due to the increase in effective molarity of the substrate. In enzyme−DNA complexes, they varied substrate−DNA interactions and confirmed that only the moderate interaction between substrate and scaffold is suitable for activity enhancement. A reduced Km and increased kcat/Km

were found both in HRP−DNA and aldo-keto reductase−DNA nanostructures.34 Here, we demonstrate that the biospecific affinity helps to increase the effective substrate concentration. The moderate attraction with the association constant at 1.97 × 103 M−1 agrees well with Wheeldon’s observation, where the 3792

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ACS Catalysis optimal dissociate constant Kd of the DNA scaffold toward substrates is the order of magnitude of 102 to 103 μM.33 Preparation and Characterization of ConA-Agglutinated GOx-HRP Aggregates. We then proceeded to a multienzyme system using ConA as agglutinate. Here, we chose HRP, a type of glycoprotein in which the carbohydrate chain accounts for 18% of total weight, to assemble an enzyme cascade system with GOx. As shown in Scheme 1, ConA was added to the bienzyme mixture to form GOx-ConA-HRP aggregates, followed by cross-linking with glutaraldehyde. According to our previous study, a molar ratio of GOx to HRP at 2:8 gave an optimal overall activity.23 The present study also confirmed this optimal ratio of the two enzymes for the multiple enzyme aggregates (Figure S6); thus, we fixed the enzyme ratio of GOx to HRP at 2:8 in the following experiments. The increase in ConA amounts improved the agglutination efficiencies of HRP and GOx, and over 90% of HRP as well as GOx were agglutinated when the concentration of ConA reached 500 μg/mL (Figure 3a). Considering the overall activity recovery after glutaraldehyde treatment, ConA concentration for enzyme agglutination was set at 1.0 mg/mL for the following experiments. Incubating with 20 μg/mL GOx and 80 μg/mL HRP for 1 h, about 96% of GOx, 93% HRP, and 94% of ConA were agglutinated and precipitated, suggesting the GOx/HRP/ConA molar ratio of 1:14:72 in aggregates, approximately one enzyme being surrounded by 5 ConA molecules. As shown in Figure 3b, the average size of ConA agglutinated GOx-HRP aggregates was around 0.8 μm, and the cross-linked GOx-ConA-HRP aggregates were around 1−2 μm in diameter with a wide distribution, suggesting that several ConA-enzyme aggregates were cross-linked by glutaraldehyde. The size of cross-linked aggregates was also confirmed by TEM (Figure 3c). The laser scanning confocal microscopy in Figure 3d shows the colocalization of GOx and HRP in the aggregates with a pseudohomogeneous configuration at the molecular level. For the GOx-ConA-HRP aggregates, H2O2 can be in situ generated by GOx. As shown in Figure 4a, the GOx-ConAHRP aggregates retained 93% of the overall activity compared with that of the free GOx and HRP mixture. The overall activity was further decreased to 78% after cross-linking. Note that the relative activity of cross-linked HRP-ConA aggregates was only 24% compared with that of free HRP (Figure 4b and Figure S7). As mentioned above, the activity of cross-linked GOxConA was just 57% compared with that of free GOx (Figure 2a). Therefore, it suggests a better throughput of cross-linked GOx-ConA-HRP aggregates than that of separately cross-linked enzyme aggregates. It is more explicit in Figure 4c that the cross-linked GOx-ConA-HRP aggregates showed an 87% increase in the overall activity compared with that of GOxConA aggregates with the same amount of free HRP. Meanwhile, the activity of cross-linked GOx-ConA showed no differences in the presence of cross-linked HRP-ConA or free HRP, indicating that in both cases, the overall activities were limited by the uptake of H2O2 by HRP. Similar to the GOx-ConA aggregates, the activity of bienzyme aggregates are also influenced by ConA due to the accumulation effect of glucose. We determined the enzymatic Michaelis−Menten kinetics of GOx in cross-linked bienzyme aggregates (Figure 4d and Table 1). The Km value for glucose of cross-linked GOx-ConA-HRP aggregates was 8.8 mM (24.2 mM for un-cross-linked aggregates), whereas the Km for free

Table 1. Enzymatic Kinetics of GOx in a Free Cascade Enzyme System, ConA Agglutinated Aggregates, and CrossLinked GOx-ConA-HRP Aggregates

free GOx and free HRP GOx-ConA-HRP aggregates cross-linked GOxConA-HRP aggregates

Vmax (μmol·L−1·s−1)

Km (mM)

kcat (s−1)

kcat/Km (s−1 mM−1)

1.9 0.81

52.3 24.2

759 322

14.5 13.4

0.47

8.8

186

21.3

GOx-HRP mixture was 52.3 mM. Moreover, the specificity constant (kcat/Km) for GOx in cross-linked GOx-ConA-HRP aggregates was higher than that of free GOx, indicating a more efficiency substrate uptake. According to the Michaelis− Menten equation, v = kcat[E][S]/(Km+[S]), when the substrate concentration is much less than Km, the cross-linked GOxConA-HRP aggregates will show an enhanced activity compared with that of their free counterparts. In addition, the cross-linked GOx-ConA-HRP aggregates showed an improved thermal stability than free enzymes (Figure S8). Another mechanism for activity enhancement is due to the colocalization of the cascade enzymes. The hydrogen peroxide produced by GOx can accumulate inside the cross-linked GOxConA-HRP particles so that the HRP can perform the reaction immediately, leading to a higher initial reaction rate. The same phenomena have been well revealed by Rodrigues et al.35 The proximity of GOx and HRP after agglutination by ConA was first confirmed by the quenching of intrinsic protein fluorescence, as shown in Figure 5a. Intrinsic protein fluorescence, which mainly originates from excitation of tryptophan (Trp) or tyrosine (Tyr) residues, is highly sensitive to the changes in the local environment of these residues. The remarkable decrease in fluorescence intensity after cross-linking indicates that the fluorophores are located in a more constrained environment.36 Furthermore, we determined the Förster resonance energy transfer (FRET) spectra of the crosslinked GOx-ConA-HRP by using the fluorescein isothiocyanate (FITC) labeled GOx and Rhodamine B (RhoB) labeled HRP. FRET occurs between two proximately located fluorophores via nonradiative dipole−dipole coupling and is an effective and sensitive technique to probe the distance between two fluorophores (typically on the 1−10 nm length scale). In fact, FRET has successfully been applied in the study of single biomolecular conformation,37 protein−protein interactions,38 molecular motors,39 etc. The FITC and RhoB can be excited by radiation at 490 and 560 nm. As shown in Figure 5b, a remarkable emission peak at 590 nm attributed to RhoB occurred in the GOx-ConA-HRP aggregates when excited at 488 nm. In contrast, such a peak was negligible in the aqueous mixture of free GOx-FITC and HRP-RhoB. This result confirmed that a significant proportion of GOx and HRP in the ConA-mediated aggregates colocalized within a distance of 10 nm. We further performed the FRET experiment on a Zeiss LSM-710 laser confocal microscope. As shown in Figure 5c, the average distance between donors (fluorescein) and acceptors (Rhodamine B) was interpreted as 6.9 nm, using the method described by Xia et al.40 (See Supporting Information for details.) In addition to the short distance, another advantage of ConA-mediated multiple enzyme aggregates is the crowding of the multiple copies of enzymes, enabling the fast consumption of the intermediate (H2O2) and preventing its escaping, which 3793

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Figure 5. (a) Fluorescent spectra of GOx-ConA-HRP aggregates and cross-linked aggregates. (b) Spectra of Förster resonance energy transfer and (c) laser confocal microscopies of (GOx-FITC)-ConA-(HRP-RhoB) in enzyme aggregates.

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is supposed to accelerate the overall enzymatic reaction permanently.41 Given the above advantages including affinitive uptake of glucose, facilitated consumption of intermediate, and improved thermal stability, this glutaraldehyde-cross-linked multienzyme catalyst promises a variety of applications, particularly for the sensitive detection of ultralow concentration glucose.

*E-mail: [email protected]. Author Contributions †

Y.Z. and Y.Y. contributed equally to this work.

Notes

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The authors declare no competing financial interest.

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CONCLUSIONS A novel multienzyme system using ConA as agglutinate was established. The affinity of ConA toward glucose resulted in an improved uptake of substrate by the GOx-ConA aggregates, as shown by a reduced Km compared to that of free GOx. The agglutination of cascade enzymes by ConA was confirmed by FRET and confocal microscopy, from which the average distance between GOx and HRP is 6.9 nm. The nanoscale colocalization of cascade enzymes enables the fast consumption of intermediate by the second enzyme, resulting in an enhanced overall activity. The specific affinity to glucose makes these enzyme aggregates capable of the sensitive detection of low concentration of glucose. Moreover, the general existence of glycoenzymes intrinsically promises the availability of this method for constructing various multiple enzyme systems.

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AUTHOR INFORMATION

Corresponding Author

ACKNOWLEDGMENTS This work was supported by National Natural Science Foundation of China under grant no. 21036003. REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acscatal.6b01047. Details for the determination of average distance between the fluorophores from confocal FRET microscopy; determination of association constant of ConA for glucose; typical size exclusion chromatograms; activities of GOx-ConA-HRP aggregates at different ratios of HRP to GOx; the Lineweaver−Burke plots of HRP activities of free HRP, HRP-ConA aggregates, and cross-linked HRP-ConA aggregates; Km of cross-linked BSA-GOx for glucose; and thermal inactivation kinetics of cross-linked GOx-ConA-HRP and free GOx&HRP (PDF) 3794

DOI: 10.1021/acscatal.6b01047 ACS Catal. 2016, 6, 3789−3795

Research Article

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DOI: 10.1021/acscatal.6b01047 ACS Catal. 2016, 6, 3789−3795