Low Enzyme Concentrations Research Articles

Biosterilant effects of Bacillus thuringiensis kurstaki HD-73 extract on male Wistar albino rats

Chemosterilants have long been used in sterilization programs for managing pet, stray, and wild animals but adverse effects such as trauma, incomplete responses to treatment, and complete abolition of gonadal sources of testosterone often occurs. This study describes the biosterilant effects of administering three doses of Bacillus thuringiensis kurstaki HD-73 (Bt 5, 50, and 250 mg/kg; 20 rats/dose) extract in adult male Wistar albino rats on testicular parameters, function, histology, and a number of biochemical markers of overall health, free radical production, and cell proliferation. Intratesticular administration of Bt extract to rats induces testicular oxidative stress and damages and consequently, perturb spermatogenesis, degeneration of testis, reduction in testes size, and depletion of testosterone and antioxidant enzyme concentrations in a dose-dependent manner because of free radical–mediated lipid peroxidation. No morbidity or mortality adverse effects were observed in both the saline control and Bt extract–treated rats. Significant variation was noted in clinical manifestations, weight and volume of testes, and hormonal and biochemical profiles between Bt doses in comparison with the saline control. Aspermia/azoospermia (100%) resulted in Bt-treated rats without any adverse effects. Histopathological analysis showed degeneration, necrosis, vacuolation, fewer germ cells, formation of multinucleated giant cells, and a lack of elongated spermatids in atrophic seminiferous tubules in Bt extract–treated groups in the presence of low concentrations of testosterone, antioxidant enzymes, and suppression of germ cell proliferation. Dose-dependent effects were evident in most parameters that were measured. The vast array of tests that were undertaken also provides some important indicators of the physiological effects associated with the treatments that were applied. Intratesticular injection of Bt extract impairs spermatogenesis and induces permanent sterility in rats.

Influence of NOM and SS on the BPA removal via peroxidase catalyzed reactions: Kinetics and pathways

Abstract In the present study, horseradish peroxidase (HRP) was employed to removal bisphenol A (BPA) with the presence of natural organic matter (NOM) and suspended solids (SS). Humic acid (HA) and kaolin have been selected as the typical NOM and SS in the experiment. According to the results, HA has shown a very interesting performance, which has both inhibitory and facilitation effects on BPA removal. For the low enzyme concentration (20 u/L and 50 u/L), small amount of HA could facilitate the BPA removal, while large amount of HA could inhibit the BPA removal. For the higher enzyme concentration (100–300 u/L), the addition of HA reduced the removal transformation of BPA and the inhibition effect of HA raised with the increase of HA concentrations. The facilitation nature of HA on enzyme catalytic processes can be attributed to the mitigation of enzyme inactivation, which has been proven by HRP inactivation rate. The inhibitory effect could be caused by the inhibition effect of HA on the enzyme activity, which has been suggested by both kinetic study and enzyme inactivation analysis. Kaolin showed a negative effect on BPA removal by HRP catalyzed reactions due to the inhibition effect of kaolin on HRP activity. The results have important implications for the engineering design by employing HRP in water and waste water treatment.

Label-Free Electrical Detection of Enzymatic Reactions in Nanochannels.

We report label-free electrical detection of enzymatic reactions using 2-D nanofluidic channels and investigate reaction kinetics of enzymatic reactions on immobilized substrates in nanoscale-confined spaces. Trypsin proteolysis is chosen for demonstration of the detection scheme. When trypsin cleaves poly-l-lysine coated on the surface of silica nanochannels, the resulting change of surface charge density can be detected by monitoring the ionic conductance of the nanochannels. Our results show that detection of such surface enzymatic reactions is faster than detection of surface binding reactions in nanochannels for low-concentration analytes. Furthermore, the nanochannel sensor has a sensitivity down to 5 ng/mL, which statistically corresponds to a single enzyme per nanochannel. Our results also suggest that enzyme kinetics in nanochannels is fundamentally different from that in bulk solutions or plain surfaces. Such enzymatic reactions form two clear self-propagating reaction fronts inside the nanochannels, and the reaction fronts follow square-root time dependences at high enzyme concentrations due to significant nonspecific adsorption. However, at low enzyme concentrations when nonspecific adsorption is negligible, the reaction fronts propagate linearly with time, and the corresponding propagation speed is related to the channel geometry, enzyme concentration, catalytic reaction constant, diffusion coefficient, and substrate surface density. Optimization of this nanochannel sensor could lead to a quick-response, highly sensitive, and label-free sensor for enzyme assay and kinetic studies.

Structural and Catalytic Characterization of a Fungal Baeyer-Villiger Monooxygenase.

Baeyer-Villiger monooxygenases (BVMOs) are biocatalysts that convert ketones to esters. Due to their high regio-, stereo- and enantioselectivity and ability to catalyse these reactions under mild conditions, they have gained interest as alternatives to chemical Baeyer-Villiger catalysts. Despite their widespread occurrence within the fungal kingdom, most of the currently characterized BVMOs are from bacterial origin. Here we report the catalytic and structural characterization of BVMOAFL838 from Aspergillus flavus. BVMOAFL838 converts linear and aryl ketones with high regioselectivity. Steady-state kinetics revealed BVMOAFL838 to show significant substrate inhibition with phenylacetone, which was more pronounced at low pH, enzyme and buffer concentrations. Para substitutions on the phenyl group significantly improved substrate affinity and increased turnover frequencies. Steady-state kinetics revealed BVMOAFL838 to preferentially oxidize aliphatic ketones and aryl ketones when the phenyl group are separated by at least two carbons from the carbonyl group. The X-ray crystal structure, the first of a fungal BVMO, was determined at 1.9 Å and revealed the typical overall fold seen in type I bacterial BVMOs. The active site Arg and Asp are conserved, with the Arg found in the “in” position. Similar to phenylacetone monooxygenase (PAMO), a two residue insert relative to cyclohexanone monooxygenase (CHMO) forms a bulge within the active site. Approximately half of the “variable” loop is folded into a short α-helix and covers part of the active site entry channel in the non-NADPH bound structure. This study adds to the current efforts to rationalize the substrate scope of BVMOs through comparative catalytic and structural investigation of different BVMOs.

Spatial organization of heterologous metabolic system in vivo based on TALE.

For years, prokaryotic hosts have been widely applied in bio-engineering. However, the confined in vivo enzyme clustering of heterologous metabolic pathways in these organisms often results in low local concentrations of enzymes and substrates, leading to a low productive efficacy. We developed a new method to accelerate a heterologous metabolic system by integrating a transcription activator-like effector (TALE)-based scaffold system into an Escherichia coli chassis. The binding abilities of the TALEs to the artificial DNA scaffold were measured through ChIP-PCR. The effect of the system was determined through a split GFP study and validated through the heterologous production of indole-3-acetic acid (IAA) by incorporating TALE-fused IAA biosynthetic enzymes in E. coli. To the best of our knowledge, we are the first to use the TALE system as a scaffold for the spatial organization of bacterial metabolism. This technique might be used to establish multi-enzymatic reaction programs in a prokaryotic chassis for various applications.

Comparative Processing of Enzyme Kinetic Data by Henri and Michaelis-Menten Equations

The deduction of Michaelis-Menten equation is based on the preposition of quasi-steady state, i.e. the concentration of the activated complexes is kept constant in time. In the general case it is not true, particularly at low enzyme concentrations compared to the substrate one. In the present study own experimental data on the nitrate reduction by crude enzyme extract stimulated by constant electric field are handled by both kinetic models. It was demonstrated that the more general model (i.e. the one of Henri) leads to more consistent results and reasonable values of the estimated kinetic constants than the Michaelis-Menten ones in the considered case.

Atypical profiles and modulations of heme-enzymes catalyzed outcomes by low amounts of diverse additives suggest diffusible radicals' obligatory involvement in such redox reactions.

Peroxidations mediated by heme-enzymes have been traditionally studied under a single-site (heme distal pocket), non-sequential (ping-pong), two-substrates binding scheme of Michaelis-Menten paradigm. We had reported unusual modulations of peroxidase and P450 reaction outcomes and explained it invoking diffusible reactive species [Manoj, 2006; Manoj etal., 2010; Andrew etal., 2011, Parashar etal., 2014 & Venkatachalam etal., 2016]. A systematic investigation of specific product formation rates was undertaken to probe the hypothesis that involvement of diffusible reactive species could explain undefined substrate specificities and maverick modulations (sponsored by additives) of heme-enzymes. When the rate of specific product formation was studied as a function of reactants' concentration or environmental conditions, we noted marked deviations from normal profiles. We report that heme-enzyme mediated peroxidations of various substrates are inhibited (or activated) by sub-equivalent concentrations of diverse redox-active additives and this is owing to multiple redox equilibriums in the milieu. At low enzyme and peroxide concentrations, the enzyme is seen to recycle via a one-electron (oxidase) cycle, which does not require the substrate to access the heme centre. Schemes are provided that explain the complex mechanistic cycle, kinetics & stoichiometry. It is not obligatory for an inhibitor or substrate to interact with the heme centre for influencing overall catalysis. Roles of diffusible reactive species explain catalytic outcomes at low enzyme and reactant concentrations. The current work highlights the scope/importance of redox enzyme reactions that could occur "out of the active site" in biological or in situ systems.

Eco-Friendly Degumming Technology for Ramie Fiber

Ramie fiber represents almost 25–28% gum in its decorticated form. Unlike other common lignocellulosic bast fibers, e.g., jute, kenaf, etc., ramie is difficult to ret naturally. However, effective removal of gum from ramie determines its appropriate end-uses. In the present study, an eco-friendly degumming technology has been explained where pectinolytic and hemi cellulytic enzymes (pectinase, hemicellulase) in pure form have been mixed in a synergistic ratio to develop a unique ramie degumming formulation. Degumming of ramie with this new enzyme formulation has been investigated and the process is optimized considering loss in weight, tensile properties of degummed ramie fiber, and economy of processing. Residual gum content of 5.36% has been achieved at comparatively lower enzyme concentration (2.0% on wt. of fiber) under ambient temperatures (28–35°C) in a considerably less time (24 h). Tensile strength (17.85 g/tex) and corresponding fineness (0.74 tex) of degummed ramie fiber obtained under optimized degumming process substantiate its suitability in subsequent textile applications. Appearance and aesthetics of eco-degummed ramie fiber improved significantly (scanning electron microscopic pictures), which are beneficial for its commercial exploitation on industrial scale.

Waste Frying Oil Hydrolysis in a Reverse Micellar System

In this work we explore the feasibility of using reverse micelles to carry out the enzymatic hydrolysis of waste frying oils (WFO). The effect of some relevant parameters, such as the water/surfactant molar ratio (W0), the oil volume fraction in the organic phase, and the enzyme concentration was studied. As a preliminary result, we observed that the extent of the reverse micellar system was slightly higher in mixtures containing WFO than when prepared with virgin oil. The hydrolysis extent was found to strongly depend on the amount of water inside the micelles, and an optimum value of 13.3 was found for W0. Although the hydrolysis took place for every volume fraction of oil in the organic phase, the reaction conversion was limited at high oil fractions. A maximum hydrolysis extent was found at high enzyme concentrations, but the specific activity of the enzyme was much higher at low enzyme concentrations. An analysis of the product distribution showed that free fatty acids are the main hydrolysis product...

Saccharification Protocol for Small-scale Lignocellulosic Biomass Samples to Test Processing of Cellulose into Glucose

Second generation biofuels are derived from inedible lignocellulosic biomass of food and non-food crops. Lignocellulosic biomass is mainly composed of cell walls that contain a large proportion of cellulosic and hemicellulosic polysaccharides. An interesting route to generate biofuels and bio-based materials is via enzymatic hydrolysis of cell wall polysaccharides into fermentable sugars, a process called saccharification. The released sugars can then be fermented to fuels, e.g. by use of yeast. To test the saccharification efficiency of lignocellulosic biomass on a lab-scale, a manual saccharification protocol was established that uses only small amounts of biomass and a low concentration of enzyme. This protocol can be used for different plant species like Arabidopsis thaliana, tobacco, maize and poplar. The low enzyme concentrations make it possible to detect subtle improvements in saccharification yield and to analyze the speed of hydrolysis. Although a specific acid and alkali pretreatment were included, the saccharification step can be preceded by any other pretreatment. Because no advanced equipment is necessary, this protocol can be carried out in many laboratories to analyze saccharification yield. The protocol was initially described in Van Acker et al. (2013).

Size product modulation by enzyme concentration reveals two distinct levan elongation mechanisms in Bacillus subtilis levansucrase.

Two levan distributions are produced typically by Bacillus subtilis levansucrase (SacB): a high-molecular weight (HMW) levan with an average molecular weight of 2300 kDa, and a low-molecular weight (LMW) levan with 7.2 kDa. Previous results have demonstrated how reaction conditions modulate levan molecular weight distribution. Here we demonstrate that the SacB enzyme is able to perform two mechanisms: a processive mechanism for the synthesis of HMW levan and a non-processive mechanism for the synthesis of LMW levan. Furthermore, the effect of enzyme and substrate concentration on the elongation mechanism was studied. While a negligible effect of substrate concentration was observed, we found that SacB elongation mechanism is determined by enzyme concentration. A high concentration of enzyme is required to synthesize LMW levan, involving the sequential formation of a wide variety of intermediate size levan oligosaccharides with a degree of polymerization (DP) up to ∼70. In contrast, an HMW levan distribution is synthesized through a processive mechanism producing oligosaccharides with DP <20, in reactions occurring at low enzyme concentration. Additionally, reactions where levansucrase concentration was varied while the total enzyme activity was kept constant (using a combination of active SacB and an inactive SacB E342A/D86A) allowed us to demonstrate that enzyme concentration and not enzyme activity affects the final levan molecular weight distribution. The effect of enzyme concentration on the elongation mechanism is discussed in detail, finding that protein-product interactions are responsible for the mechanism shift.

Homogeneous single-label tyrosine kinase activity assay for high throughput screening

Protein post-translational modifications (PTMs) are regulatory mechanisms carried out by different enzymes in a cell. Kinase catalyzed phosphorylation is one of the most important PTM affecting the protein activity and function. We have developed a single-label quenching resonance energy transfer (QRET) assay to monitor tyrosine phosphorylation in a homogeneous high throughput compatible format. Epidermal growth factor receptor (EGFR) induced phosphorylation was monitored using Eu3+-chelate labeled peptide and label-free phosphotyrosine specific antibody in presence of a soluble quencher molecule. In the QRET kinase assay, antibody binding to phosphorylated Eu3+-peptide protects the Eu3+-chelate from luminescence quenching, monitoring high time-resolved luminescence (TRL) signals. In the presence of specific kinase inhibitor, antibody recognition and Eu3+-chelate protection is prevented, allowing an efficient luminescence quenching. The assay functionality was demonstrated with a panel of EGFR inhibitors (AG-1478, compound 56, erlotinib, PD174265, and staurosporine). The monitored IC50 values ranged from 0.08 to 155.3 nM and were comparable to those found in the literature. EGFR activity and inhibition assays were performed using low nanomolar enzyme and antibody concentration in a 384-well plate format, demonstrating its compatibility for high throughput screening (HTS).

A Raman spectroscopic approach to investigate the production of biodiesel from soybean oil using 1-alkyl-3-methylimidazolium ionic liquids with intermediate chain length

We present a Raman spectroscopic study of the conversion of soybean oil into biodiesel by methanol using the lipase B enzyme pseudomonas cepacia as the bio-catalyst and 1-alkyl-3-methylimidazolium ionic liquids (CnC1Im) as co-solvents, where the alkyl chain length is varied from ethyl (n = 2) to decyl (n = 10). We have limited this study to a low reaction temperature, 40 °C, and a low enzyme concentration to investigate the possibility of producing biodiesel with low energy and cost demands. We also demonstrate that Raman spectroscopy is a powerful and straightforward method to estimate the yield of the transesterification reaction, by analysis of the characteristic CO stretching mode found in the range ∼1730–1750 cm−1. Our results indicate that both reaction yield and reaction rate increase with the chain length, but also that for chains longer than decyl further improvements are marginal. We discuss these results from the viewpoint of local interactions and local structure of the investigated ionic liquids.

Effect of enzymatic hydrolysis on surface activity and surface rheology of type I collagen

We describe the adsorption behaviour and rheological properties of a calf skin type I collagen, and of its hydrolysates obtained using a Clostridium histolyticum collagenase (CHC) under moderate conditions (pH 7, 37°C). The effect of CHC concentration (2×10−9–2×10−6M) and incubation time (35–85min) was studied and optimised to achieve the highest decrease of surface tension and the highest dilational surface viscoelasticity of the adsorbed layers. SDS-PAGE electrophoresis and reverse-phase high performance liquid chromatography (RP-HPLC) were used to characterise the hydrolysis products. The results show that even simple modifications (heat treatment, pH change, partial hydrolysis) of collagen enhances its surface properties, especially in terms of surface dilational elasticity modulus. The use of low enzyme concentration (CHC-to-collagen molar ratio of 4×10−3) and short incubation time (<45min) results in moderately hydrolysed products with the highest ability to lower surface tension (γ=53.9mNm−1) forming highly elastic adsorbed layers (surface dilational elasticity, E′=74.5mNm−1).

Effect of a double‐structured microporous polymer support on the catalytic activity, stability and aggregation behavior of immobilized enzymes

AbstractA double‐structured microporous polymer composite support consisting of a microporous skeleton for protection of immobilized enzymes and a gel‐like polymer bearing different functionalities has been synthesized for immobilization of various enzymes. Among three approaches based on two types of interactions (physical and chemical), we found that under our experimental conditions the covalent bonding between amine moieties of enzymes and epoxide groups of the support was the best, with enzyme leaching being reduced to as low as zero for nine enzymes and catalytic activities increased by factors of 1–4 for two lipases. The improvement in catalytic activity could be attributed to the amphiphilic soft gel which might create a kind of lipid–water interface or microenvironment in aqueous solution, favoring the interfacial activation of certain lipases. Furthermore, the thermal and operational stability, and reusability of all the enzymes could be enhanced to varying degrees. We also found that enzyme aggregates of different sizes could be formed on the spherical support surface at quite low enzyme concentrations, which might also influence the final catalytic activities of the immobilized enzymes. © 2015 Society of Chemical Industry

Effective reaction rates in diffusion-limited phosphorylation-dephosphorylation cycles.

We investigate the kinetics of the ubiquitous phosphorylation-dephosphorylation cycle on biological membranes by means of kinetic Monte Carlo simulations on the triangular lattice. We establish the dependence of effective macroscopic reaction rate coefficients as well as the steady-state phosphorylated substrate fraction on the diffusion coefficient and concentrations of opposing enzymes: kinases and phosphatases. In the limits of zero and infinite diffusion, the numerical results agree with analytical predictions; these two limits give the lower and the upper bound for the macroscopic rate coefficients, respectively. In the zero-diffusion limit, which is important in the analysis of dense systems, phosphorylation and dephosphorylation reactions can convert only these substrates which remain in contact with opposing enzymes. In the most studied regime of nonzero but small diffusion, a contribution linearly proportional to the diffusion coefficient appears in the reaction rate. In this regime, the presence of opposing enzymes creates inhomogeneities in the (de)phosphorylated substrate distributions: The spatial correlation function shows that enzymes are surrounded by clouds of converted substrates. This effect becomes important at low enzyme concentrations, substantially lowering effective reaction rates. Effective reaction rates decrease with decreasing diffusion and this dependence is more pronounced for the less-abundant enzyme. Consequently, the steady-state fraction of phosphorylated substrates can increase or decrease with diffusion, depending on relative concentrations of both enzymes. Additionally, steady states are controlled by molecular crowders which, mostly by lowering the effective diffusion of reactants, favor the more abundant enzyme.

Study of wettability of calcite surfaces using oil–brine–enzyme systems for enhanced oil recovery applications

Enzymes have recently been considered as possible agents for enhanced oil recovery (EOR) acting at the liquid–solid interface. One way to assess this is via measuring the wettability of calcite surfaces, important for EOR methods in carbonaceous reservoirs. In the present work, we have experimentally investigated the effect of enzymes on the wettability of calcite mineral surfaces with oil–brine systems. The action of various enzymes, including esterases/lipases, carbohydrases, proteases and oxidoreductases (along with two commercial mixtures) was studied by contact angle measurements and adhesion behaviour tests. Comparative studies with a surfactant, protein, purified enzyme, enzyme stabiliser using n-decane (as a model for the oil) have also been carried out in order to verify experimental results. The enzymes that have the highest effect on the wettability have been identified. Those enzymes, which were found the most promising from a practical perspective, have shown the ability to fully detach oil from the surface, even at very low enzyme concentrations. For example, esterases/lipases were found to strongly affect the wettability and to remove adhesion at concentrations as low as 0.1% of the enzyme product (corresponding to 0.002–0.005% protein). Likewise, proteases could also improve wettability, although the effect was not consistent and was dependent on impurities. Other enzymes had no effect on the wettability of calcite at the concentration studied. The main mechanism of enzymatic action has been found to be replacement of oil at the solid surface by the enzyme. Other mechanisms (modification of the surface tension or catalytic modification of hydrocarbons resulting in reducing the oil viscosity) have shown to be much less pronounced from the measurements reported here.

Use of 2-Photon Fluorescence Correlation Spectroscopy and Electron Microscopy to Ellucidate the Dependence of RLPFK Self Association on Ligand Concentration

Phosphofructokinase (PFK) catalyzes the first committed step of glycolysis. Allosteric regulation of PFK makes its activity, and thus glycolysis, sensitive to intercellular metabolic conditions. Regulation of PFK from liver is complex because of the liver's responsibility to maintain glucose homeostasis, which means that glycolysis and gluconeogenesis must be regulated reciprocally. Allosteric regulation of PFK activity is achieved by modifying the binding affinity of its substrate, fructose 6-phosphate (Fru-6-P). However, in vitro studies performed on rat liver PFK (RLPFK) suggest that at physiological concentrations of Fru-6-P RLPFK activity is negligible even in the presence of known allosteric activators. Additional mechanisms must exist to account for the activity required to support flux through glycolysis. The smallest active oligomer of RLPFK is a tetramer, however fluorescence polarization studies have previously demonstrated that at a physiological enzyme concentration both Fru-6-P and other activators stabilize species much larger than a tetramer. A Weber linkage argument predicts that the highly associated species would demonstrate a higher affinity for Fru-6-P resulting in activation. RLPFK exists as a tetramer in the dilute enzyme concentrations necessary for in vitro activity assays whereas, at physiological concentrations, RLPFK can be highly associated. 2-photon fluorescence correlation spectroscopy (FCS) and electron microscopy have been used to quantify the oligomeric state of RLPFK at high and low enzyme concentrations. FCS performed on alexa-488 labeled RLPFK suggests a complex self-association behavior in the presence of Fru-6-P. Electron microscopy indicates that, in the presence of Fru-6-P, rat liver PFK can form long fibrils that consist of up to 36 PFK tetramers extending over 200 nm in length. These effects are counteracted by MgATP, a known allosteric inhibitor of the enzyme. Funding: NIH-GM33216, NIH-CBI and Welch-A1543

Hysteresis and positive cooperativity as possible regulatory mechanisms of Trypanosoma cruzi hexokinase activity

In Trypanosoma cruzi, the causal agent of Chagas disease, the first six or seven steps of glycolysis are compartmentalized in glycosomes, which are authentic but specialized peroxisomes. Hexokinase (HK), the first enzyme in the glycolytic pathway, has been an important research object, particularly as a potential drug target. Here we present the results of a specific kinetics study of the native HK from T. cruzi epimastigotes; a sigmoidal behavior was apparent when the velocity of the reaction was determined as a function of the concentration of its substrates, glucose and ATP. This behavior was only observed at low enzyme concentration, while at high concentration classical Michaelis–Menten kinetics was displayed. The progress curve of the enzyme's activity displays a lag phase of which the length is dependent on the protein concentration, suggesting that HK is a hysteretic enzyme. The hysteretic behavior may be attributed to slow changes in the conformation of T. cruzi HK as a response to variations of glucose and ATP concentrations in the glycosomal matrix. Variations in HK's substrate concentrations within the glycosomes may be due to variations in the trypanosome's environment. The hysteretic and cooperative behavior of the enzyme may be a form of regulation by which the parasite can more readily adapt to these environmental changes, occurring within each of its hosts, or during the early phase of transition to a new host.

Preparation of cross-linked enzyme aggregates of trehalose synthase via co-aggregation with polyethyleneimine.

Trehalose synthase (TreS) from Meiothermus ruber was co-aggregated with polyethyleneimine (PEI) and precipitated with polyethylene glycol (PEG), followed by cross-linking with glutaraldehyde to obtain TreS-polyethyleneimine cross-linked enzyme aggregates (termed as CLEAs-PEI-PEG). The TreS solution at 0.5 mg mL(-1) protein concentration, with PEI at a mass ratio of 1:0.8 (enzyme/PEI, w/w) and 25 % (w/v) PEG concentration were found to be most adequate for the co-aggregation of TreS. CLEAs-PEI-PEG was most active with glutaraldehyde at a mass ratio of 1:0.5 (enzyme/glutaraldehyde, w/w) to cross-link the co-aggregates. The CLEAs-PEI-PEG prepared in this work had an optimum pH of 6.5 and optimum temperature of 60 °C. For lower concentrations of enzyme, using PEI could enhance the cross-linking efficiency of TreS. The thermal stability and pH tolerance of CLEAs-PEI-PEG were significantly improved. Scanning electron microscopy revealed that the main structure of CLEAs-PEI-PEG showed scaffolding morphology which was constituted by structured ball-like particles with a size of 1-2.5 μm in diameter.