Enzyme Aggregates Research Articles

Improved development in magnetic Xyl-CLEAs technology for biotransformation of agro-industrial by-products through the use of a novel macromolecular cross-linker

Cross-Linked Enzyme Aggregates (CLEAs) technologies for enzyme immobilization are influenced by mass transference problems as the degree of molecular crosslinking achieved strongly affects the enzyme exposure to the substrates. Therefore, this work seeks to improve the accessibility of high molecular weight substrates by using macromolecular cross-linkers to the synthesis of a xylanolytic biocatalyst. After confirming that commercial polymers used as macromolecular cross-linkers significantly upgraded the xylanase activity from a crude preparation, a novel biopolymer/amyloid protein complex (BPAP) extracted from a microbial biofilm was used producing a remarkable recovery (83%) of the enzyme activity.A response surface methodology was applied to contrast the features of a previously developed biocatalyst with glutaraldehyde (GA@Xyl-CLEAs) and a novel one synthesized with BPAP combined with functionalized magnetic nanoparticles: mBPAP@Xyl-CLEAs. It was observed that the crosslinking agent used was the factor that most affected the enzyme activity. Also, the mBPAP system showed a similar and higher hydrolytic activity than those synthesized with GA, which was not affected by the mNPs/protein ratio. Finally, the mBPAP@Xyl-CLEAs were successfully tested for xylooligosaccharides production from agroindustrial-derived substrates, making this technology a promising practice to obtain green and suitable biocatalysts.

A novel polyphenol oxidase immobilized polyglycine/reduced graphene oxide composite electrode for sensitive determination of catechol

In this study, we report a new type of flexible and free standing paper electrochemical sensor fabricated by integrating reduced graphene oxide (rGO), poly(glycine) and polyphenol oxidase (PPO) enzyme. For the sensor development, firstly, electrochemical polymerization of glycine at the rGO paper electrode was carried out by the cyclic voltammetric method, and then the poly(glycine)/rGO paper electrode was further utilized as a matrix to immobilize the PPO enzyme purified from potato. The developed PPO-poly(glycine)/rGO immobilized paper sensor was characterized by scanning electron microscopy, X-ray photoelectron spectroscopy and Raman spectroscopy. Enzyme aggregation and distribution within the poly(glycine)/rGO paper electrode were studied using a fluorescence microscope. The electrochemical behavior of catechole (CC) on PPO-poly(glycine)/rGO immobilized paper electrode was investigated by cyclic voltammetry and amperometry. The sensor performed well in the concentration range of 0.1–800 µM, with a detection limit of 0.07 µM and a sensitivity of 0.0512 mA µM cm−2.

Crosslinked Enzyme Aggregates (CLEAs) of Laccases from Pleurotus citrinopileatus Induced in Olive Oil Mill Wastewater (OOMW).

The enzymatic factory of ligninolytic fungi has proven to be a powerful tool in applications regarding the degradation of various types of pollutants. The degradative potential of fungi is mainly due to the production of different types of oxidases, of which laccases is one of the most prominent enzymatic activities. In the present work, crude laccases from the supernatant of Pleurotus citrinopileatus cultures grown in olive oil mill wastewater (OOMW) were immobilized in crosslinked enzyme aggregates (CLEAs), aiming at the development of biocatalysts suitable for the enzymatic treatment of OOMW. The preparation of laccase CLEAs was optimized, resulting in a maximum of 72% residual activity. The resulting CLEAs were shown to be more stable in the presence of solvents and at elevated temperatures compared to the soluble laccase preparation. The removal of the phenolic component of OOMW catalyzed by laccase-CLEAs exceeded 35%, while they were found to retain their activity for at least three cycles of repetitive use. The described CLEAs can be applied for the pretreatment of OOMW, prior to its use for valorization processes, and thus, facilitate its complete biodegradation towards a consolidated process in the context of circular economy.

Novel cross-linked enzyme aggregates of levanase from Bacillus lehensis G1 for short-chain fructooligosaccharides synthesis: Developmental, physicochemical, kinetic and thermodynamic properties

Short-chain fructooligosaccharides (scFOSs) can be produced from the levan hydrolysis using levanase. Levanase from Bacillus lehensis G1 (rlevblg1) is an enzyme that specifically converts levan to scFOSs. However, the use of free levanase presents a lack of stability and reusability, thus hindering the synthesis of scFOSs for continuous reactions. Here, CLEAs for rlevblg1 were prepared and characterized. Cross-linked levanase aggregates using glutaraldehyde (CLLAs-ga) and bovine albumin serum (CLLAs-ga-bsa) showed the best activity recovery of 92.8% and 121.2%, respectively. The optimum temperature of CLLAs-ga and CLLAs-ga-bsa was increased to 35 °C and 40 °C, respectively, from its free rlevblg1 (30 °C). At high temperature (50 °C), the half-life of CLLAs-ga-bsa was higher than that of free rlevblg1 and CLLAs-ga. Both CLLAs exhibited higher stability at pH 9 and pH 10. Hyperactivation of CLLAs-ga-bsa was achieved with an effectiveness factor of more than 1 and with improved catalytic efficiency. After 3 h reaction, CLLAs-ga-bsa produced the highest total scFOSs yield of 35.4% and total sugar of 60.4% per gram levan. Finally, the reusability of CLLAs for 8 cycles with more than 50% activity retained makes them as a potential synthetic catalyst to be explored for scFOSs synthesis.

Synthesis of dehydroepiandrosterone by co‐immobilization of keto reductase and glucose dehydrogenase

AbstractBACKGROUNDDehydroepiandrosterone (DHEA), a steroid hormone drug and a precursor of other steroid hormone drugs, is industrially important. We aimed to establish the co‐immobilization of keto reductase and glucose dehydrogenase by a crosslinked enzyme aggregate (co‐CLEA) and to determine whether co‐CLEAs can improve DHEA yield compared with crude enzymes.RESULTSHeterologous expression plasmids containing genes coding for keto reductase or glucose dehydrogenase were constructed. The size of each of these proteins was determined using SDS‐PAGE and purification on a nickel column. After optimizing the expression conditions for these two enzymes, the co‐CLEAs were prepared with 80% acetone as a precipitant and 0.005% glutaraldehyde as a crosslinking agent. Compared with the crude enzyme solutions, the co‐CLEAs showed better thermal stability and pH stability. After six cycles of use, the co‐CLEAs still showed 70% (keto reductase) and 46% (glucose dehydrogenase) of their initial activities. After four reaction cycles, the accumulated production of DHEA using the co‐CLEAs reached 183 g L–1.CONCLUSIONSThis research provides a possible strategy for the production of clinically useful DHEA through co‐immobilization. © 2020 Society of Chemical Industry

Pectin methylesterase inactivation by pulsed light

Pectinmethylesterase (PME) is a degradative enzyme of the cloudiness of fruit juices. Pulsed light (PL) is a non-thermal technology able to inactivate enzymes. This work aimed to assess the potential inactivation of PME by PL and to explain how it occurs. To this, a suspension of the enzyme was treated with PL up to 128 J/cm2. The inactivation curve was built and structural changes were determined by far-UV circular dichroism, intrinsic and extrinsic fluorescence and spectrophotometry. Furthermore, the presence/absence of unfolding intermediaries was assessed by phase-diagram analysis. PL was able to inactivate PME with a first-order kinetic rate of 0.034 cm2/J. The inactive protein seemed to have a molten-globule structure, due to the conserved secondary structure and modified tertiary structure. Phase diagram data fitted a single line, which is consistent with absence of unfolding intermediaries. A significant increase in absorbance at 420 nm (p < 0.05) showed that PL promoted enzyme aggregation.

The role of cations in regulating reaction pathways driven by Bacillus circulans β-galactosidase

A β-galactosidase (EC 3.2.1.23) from Bacillus circulans (Biolacta FN5) can catalyse transgalactosylation reactions with lactose as a donor. In addition to their function as cofactors and structural stabilisers in biocatalytic reactions, cations can play a role in salt-bridge interactions and electrostatic charge screening of proteins. In this work, we investigated the impact of calcium, magnesium, sodium and potassium, commonly found in dairy whey systems, on the transgalactosylation kinetics of the β-galactosidase from Bacillus circulans. Both molecular modeling and quantitative experimental methods were used to assess enzyme aggregation and resulting loss in enzyme activity that is initiated by high concentrations of these cations. The effect of this loss in activity with time was studied during the transgalactosylation of N-acetylglucosamine (GlcNAc) to N-acetyllactosamine (LacNAc) using lactose as the donor. No significant change in hydrolysis or transgalactosylation reaction kinetics was observed at low concentrations of divalent cations (Ca2+ or Mg2+) or up to 100 mM of monovalent cations (Na+ or K+). The enzymatic yield and selectivity, however, were significantly affected at concentrations of 100 mM of Ca2+ or Mg2+. These changes were the result of both the loss in enzyme activity and a reduction in the reaction rate constant for hydrolysis and formation of the undesired isomer, Allo-LacNAc. In particular, addition of magnesium enhanced the selectivity for LacNAc over Allo-LacNAc, with no significant reduction in the LacNAc yield. These findings suggest that cations can be employed to regulate the action of β-galactosidase during transgalactosylation through the formation of protein aggregates.

Preparation of CLEAs and magnetic CLEAs of a recombinant l-arabinose isomerase for d-tagatose synthesis

The insolubilization of a recombinant l-arabinose isomerase (l-AI) from Enterococcus faecium by cross-linked enzyme aggregates (CLEA) was investigated, aiming the biochemical production of d-tagatose from d-galactose. d-tagatose is a functional sweetener that has many health benefits, sweetening properties and lower calorific value. Different precipitants (ammonium sulfate, ethanol, acetone, polyethylene glycol 4000) were used in the first step of the protocol, in order to establish the precipitation conditions, and the best results of yield and activity were achieved with ammonium sulfate. In order to facilitate the recovery of the biocatalyst, a new strategy for immobilization of the multimeric enzyme l-arabinose isomerase was proposed. Magnetic cross-linked enzyme aggregates (m-CLEA) were obtained using ammonium sulfate as precipitant and magnetic nanoparticles (MNP) functionalized with APTES (3- Aminopropyltriethoxysilane). Another immobilization strategy was to immobilize the enzyme onto MNP-APTES, as a control. The best results were achieved when the m-CLEA was produced with 20 mg of MNP, 7.69 U. g−1 of enzymatic activity, 7.61 % of recovered activity, 99 % of yield of immobilization. On the other hand, the enzyme immobilized onto MNP-APTES, presented only 2.12 U. g−1 of enzymatic activity, 32.3 % of recovered activity, and 15 % of yield of immobilization.

Biotechnological relevance of the lipase A from Candida antarctica

This review intends to present some of the latest studies on the lipase A from Candida antarctica (CALA). This lipase is among the most stable ones and has some capability to attack the sn-2 position of triglycerides. This makes it a very interesting lipase, especially considering that it is commercially available. The cloning and production of the enzyme together with some structural facts and applications will be discussed in this review. Special focus will be put on the immobilization of the enzyme. The use of the commercially available crosslinked enzyme aggregates of this enzyme will be explained, together with the use of the enzyme in some new trends in enzyme immobilization, such as bio-imprinting of the open form of CALA by detergents and the fixation of the open structure, the design of heterofunctional supports able to take full advantage of the immobilization via interfacial activation but preventing enzyme release, or the design of strategies for the preparation of multiple layers of lipase enzymes (using just CALA or combining CALA with other lipases).

Catalytic properties of variously immobilized mushroom tyrosinase: A kinetic study for future development of biomimetic amperometric biosensors

Mushroom tyrosinase was immobilized by direct embedding into electrode material (modified carbon paste electrode), incorporation of cross-linked enzyme aggregates into a polymer membrane (glassy carbon electrode covered by thin layer of Nafion®), and covalent attachment using self-assembled monolayers (gold electrode with the chemically bound enzyme). Both, standard UV–Vis spectrophotometry and amperometry in a batch configuration are presented as complementary methods to study the tyrosinase enzyme kinetics, whose catecholase activity results in electroactive products (ortho-quinones). Due to higher sensitivity of amperometric detection, evident advantage in the enzyme consumption was obtained. Prepared amperometric tyrosinase biosensors were characterized using cyclic voltammetry and atomic force microscopy. The Michaelis constant values of immobilized and unbound tyrosinase (free enzyme solution) towards dopamine and catechol were compared. The apparent Michaelis constant values for immobilized tyrosinase are significantly lower than the declared value of 0.840 mmol L−1 dopamine for the unbound enzyme. The enzyme tyrosinase arranged in self-assembled monolayer serves as an efficient sensor due to low apparent Michaelis constant of 0.061 mmol L−1 dopamine and high maximum reaction velocity of 0.458 μA s−1. This fact reflects the ideal arrangement of enzyme molecules causing high availability of the binding site. Tris-glycine sodium dodecyl sulphate polyacrylamide gel electrophoresis and atomic force microscopy clarified that the protein of molecular weight 25 kDa is bound preferably on chemically modified gold electrode. A sensor prepared by the immobilization of tyrosinase on gold electrode results in higher catecholase activity towards dopamine than in case of CPE and GC electrodes, where enzyme is immobilized physically.

Removal of bisphenol A in aqueous solution using magnetic cross-linked laccase aggregates from Trametes hirsuta

Enzymatic removal of Bisphenol A (BPA), acknowledged as an environmentally friendly approach, is a promising method to deal with hard degradable contaminants. However, the application of “enzymatic treatment” has been limited due to lower operational stability and practical difficulties associated with recovery and recycling. Enzyme immobilization is an innovative approach which circumvents these drawbacks. In this study, laccase from Trametes hirsuta was used for BPA removal. Amino-functionalized magnetic Fe3O4 nanoparticles were synthesized via the co-precipitation method followed by surface modification with (3-aminopropyl)trimethoxysilane (APTMS). The as-prepared nanoparticles were utilized for the immobilization of laccase with the magnetic cross-linked enzyme aggregates method (MCLEAs). Activity recovery of 27% was achieved, while no immobilized laccase was observed in the cross-linked enzyme aggregates method. The performance of immobilized laccase was measured by analyzing the degradation of BPA pollutant. The maximum removal efficiency of 87.3% was attained with an initial concentration of 60 ppm throughout 11 h.

Entrapment of enzyme aggregates in chitosan beads for aroma release in white wines

Glycosidases are enzymes involved in the cascade reactions leading to the release of aromatic compounds in white wines. However, the use of commercial soluble glycosidases is facing difficulties due to their fast inactivation, poor reaction control, low efficiency of enzyme use, and the presence of catalyst residues in the product. Co-immobilization as cross-linked enzyme aggregates (combi-CLEAs) is a sound alternative allowing the immobilization of enzymes in their own protein matrix, yielding highly stable and active biocatalysts. Notwithstanding, their micrometer sized particles limit their application in industrial processes. To overcome this, combi-CLEAs of β-D-glucosidase (βG) and α-L-arabinofuranosidase (ARA) were entrapped in polymeric chitosan beads. The effect of crosslinking reagents and crosslinking time on the specific activity and stability of combi-CLEAs was studied, and the best conditions for the entrapment of the combi-CLEAs in polymeric chitosan beads were determined varying the concentration of the chitosan solution and the pH of the gelation agent solution. The resulting biocatalyst beads (average diameter 1.24 mm), retained full activity after 91 days of incubation under winemaking conditions, having specific activities of 0.91 and 0.88 international units of activity per gram for βG and ARA, respectively. Such characteristics make them suitable for aroma enhancement in wines.

Greener production of low methoxyl pectin via recyclable enzymatic de-esterification using pectin methylesterase cross-linked enzyme aggregates captured from citrus peels

We propose more green and sustainable enzymatic production of low methoxyl (LM) pectin with desired degree of esterification (DE) using a reusable and practically cost-free biocatalyst captured from renewable waste. The pectin methyl esterase (PME) was directly captured as recyclable biocatalyst from the waste citrus peels through cross-linked enzyme aggregates (CLEAs). The optimization of biocatalyst preparation specifically recovered 85% of PME activity available in the waste citrus peels by ammonium sulphate (50%, v/v) precipitation followed by cross-linking for 5 h with 70 mM glutaraldehyde. The PME-CLEAs were characterized by FTIR and SEM and used for batch de-esterification of high methoxyl (HM) pectin obtained from citrus, mango, and pomegranate to LM-pectin. The PME-CLEAs exhibited higher stability in acidic pH (most desired for HM-pectin de-esterification), thermostability, and similar kinetics of de-esterification compared to the native PME. The PME-CLEAs were extremely versatile as they achieved maximum reduction in DE by 75%, 68%, and 66% for pectin derived from citrus, mango, and pomegranate, respectively without changing their molecular weight (MW) and GalA content under the same optimal batch reaction conditions (pectin loading 1% w/v, PME loading 40 U/g of pectin, 35 °C, and pH 6.5, 4 h). De-esterified pectin's strong gelling in presence of Ca2+ and decreased methyl ester peak density in FTIR further confirmed production of LM-pectin by PME-CLEAs. Finally, the PME-CLEAs were recycled for 7 batches of LM-pectin production with consistent DE, MW and GalA content of LM-pectin produced in each batch and PME activity which makes this process promising to pectin industry.

Tyrosinase-Catalyzed Phenol-Mediated Immobilization of β-Agarase on l-Lysine-Coated Magnetic Particles for the Production of Neoagarooligosaccharides from Gelidium amansii

Cross-linked enzyme aggregates of β-agarase (Aga2) were immobilized on l-lysine-coated magnetic nanoparticles (Lys@Fe₃O₄) for the production of neoagarooligosaccharides (NAOSs) from Gelidium amansii. The support Lys@Fe₃O₄ was prepared in a one-pot aqueous medium, whereas Aga2 was engineered to carry five tyrosine residues at its N-terminus (Aga2Y5N). Cross-linked immobilization of Aga2Y5N on Lys@Fe₃O₄ was effectively catalyzed by tyrosinase but its efficiency was further improved with phenol addition, affording the best biocatalyst, Aga2Y5N-TP@Lys@Fe₃O₄. Although its kinetic properties were slightly reduced, the overall results show that Aga2Y5N-TP@Lys@Fe₃O₄ performed remarkably better than free Aga2Y5N, as reflected by its excellent stability at wider pH and temperature ranges. Moreover, Aga2Y5N-TP@Lys@Fe₃O₄ can be effectively collected and recycled through magnetization, unlike the free Aga2Y5N, which cannot be easily retrieved from the reaction mixture after use. Hydrolysis results demonstrate the capability of Aga2Y5N-TP@Lys@Fe₃O₄ to effectively and selectively produce NAOS as a mixture of neoagarotetraose and -hexaose. The proposed route could be useful for the immobilization of other enzymes in various biotechnological applications.

Entrapment of porous cross-linked enzyme aggregates of maltogenic amylase from Bacillus lehensis G1 into calcium alginate for maltooligosaccharides synthesis

Maltooligosaccharides (MOSs) are emerging oligosaccharides in food-based applications and can be synthesized through the enzymatic synthesis of maltogenic amylase from Bacillus lehensis G1 (Mag1). However, the lack of enzyme stability makes this approach unrealistic for industrial applications. The formation of cross-linked enzyme aggregates (CLEAs) is a promising tool for improving enzyme stability, and the substrate accessibility problem of CLEA formation was overcome by the addition of porous agents to generate porous CLEAs (p-CLEAs). However, p-CLEAs exhibited high enzyme leaching and low solvent tolerance. To address these problems, p-CLEAs of Mag1 (Mag1-p-CLEAs) were entrapped in calcium alginate beads (CA). Mag1-p-CLEAs-CA prepared with 2.5% (w/v) sodium alginate and 0.6% (w/v) calcium chloride yielded 53.16% (17.0 U/mg) activity and showed a lower deactivation rate and longer half-life than those of entrapped free Mag1 (Mag1-CA) and entrapped non-porous Mag1-CLEAs (Mag1-CLEAs-CA). Moreover, Mag1-p-CLEAs-CA exhibited low enzyme leaching and high tolerance in various solvents compared to Mag1-p-CLEAs. A kinetic study revealed that Mag1-p-CLEAs-CA exhibited relatively high affinity towards beta-cyclodextrin (β-CD) (Km = 0.62 mM). MOSs (300 mg/g) were synthesized by Mag1-p-CLEAs-CA at 50 °C. Finally, the reusability of Mag1-p-CLEAs-CA makes them as a potential biocatalyst for the continuous synthesis of MOSs.

Laccase immobilisation on Poly(ethylene) terephthalate grafted with maleic anhydride (PET-g-MAH) nanofiber mat

A comparative study was carried out on three different enzyme immobilisation methods which were physical adsorptions (PA), covalent bonding (CV) and covalent bonding of cross-linked enzyme aggregates (CL) for laccase immobilisation on Poly(ethylene) Terephthalate (PET) grafted with Maleic Anhydride (MAH) nanofiber mats. The chemically inert PET was successfully grafted with MAH at temperature between 40 – 45 °C and used as the carrier to immobilised laccase in the form of electrospun nanofiber mats. The peaks from carbonyl (C=O) and alkene (C=C) groups appeared on the spectral subtraction between PET and PET-g-MAH nanofiber mats. These groups might be the potential group to form covalent bond between the amine groups of laccase enzyme. Laccase immobilised on the PET-g-MAH nanofiber mats using CL methods using glutaraldehyde as crosslinker gave the best performance with the highest enzyme loading and immobilisation yield which were 40.88 μg/mg and 48.37 %. On top of that, the immobilised laccase on PET-g-MAH nanofiber mats also managed to retain 69.01 % of its initial activity after 10 repeated cycles of 2, 2-azino-bis 3-ethylbenzothiazoline-6-sulfonic acid (ABTS) oxidations. These results demonstrate that PET-g-MAH nanofiber is a good material to be considered as laccase carrier.

Immobilization of Lipozyme TL 100L for methyl esterification of soybean oil deodorizer distillate.

An immobilization method for binding cross-linked enzyme aggregates of Lipozyme TL 100L on macroporous resin NKA (CLEA-TLL@NKA) was developed in this study. The esterification activity of CLEA-TLL@NKA reached 6.4 U/mg. The surface structure of immobilized lipase was characterized by scanning electron microscopy. Methyl esterification reaction of soybean oil deodorizer distillate (SODD) was catalyzed by CLEA-TLL@NKA, which the conversion rate reached 98% and its activity retained over 90% after 20 batches of reaction. Compared with the commercial enzyme Lipozyme TLIM, half-life (t 1/2) of CLEA-TLL@NKA increased by 25 times and the catalytic activity increased by approximate 10 times. Thus, CLEA-TLL@NKA had high catalytic activity, good operational stability, and potential industrial application in the field of oil processing.

A Millifluidic Device with Embedded Cross-Linked Enzyme Aggregates for Degradation of H2O2.

Millifluidic devices decorated with enzymes have been used for enzymatic reactions in continuous processes, but low enzymatic activity and enzyme leaching remain as challenges. Herein, we develop a strategy to embed cross-linked enzyme aggregates (CLEAs) on the surfaces of millifluidic devices to achieve higher enzymatic activity and better stability. Catalase was chosen as a model enzyme to degrade H2O2 in wastewater samples. First, CLEA of catalase (153 ± 10 nm) was formed by simultaneous precipitation and cross-linking with 25.0 wt % acetonitrile containing 0.025 wt % glutaraldehyde in a millifluidic device. To immobilize CLEA, we first swell a piece of plastic tubing by using 5.0 wt % acetonitrile and then immerse it in an aqueous solution with 5.0 wt % (3-aminopropyl)triethoxysilane (APTES) and 5.0 wt % dextran polyaldehyde (DPA) subsequently. After CLEA is absorbed inside the expanded polymer network of the tubing, the tubing is tightened by using a vacuum to secure the immobilized CLEA. The millifluidic device decorated with CLEA of catalase has total activity of 660 U for degradation of H2O2, and it shows good stability under a flow rate of 200 μL/min. The tubing can be used to degrade 0.1 wt % H2O2 solution continuously for 3 h or remove 2 wt % residual H2O2 in wastewater for 2 h. The technique is general enough and can be applied to other types of enzymes for continuous enzymatic reactions.

Biochemical and Structural Characterization of Cross-Linked Enzyme Aggregates (CLEAs) of Organic Solvent Tolerant Protease

Cross-linked enzyme aggregates (CLEAs) is an immobilization technique that can be used to customize enzymes under an optimized condition. Structural analysis on any enzyme treated with a CLEA remains elusive and has been less explored. In the present work, a method for preparing an organic solvent tolerant protease using a CLEA is disclosed and optimized for better biochemical properties, followed by an analysis of the structure of this CLEA-treated protease. The said organic solvent tolerant protease is a metalloprotease known as elastase strain K in which activity of the metalloprotease is measured by a biochemical interaction with azocasein. Results showed that when a glutaraldehyde of 0.02% (v/v) was used under a 2 h treatment, the amount of recovered activity in CLEA-elastase was highest. The recovered activity of CLEA-elastase and CLEA-elastase-SB (which was a CLEA co-aggregated with starch and bovine serum albumin (BSA)) were at an approximate 60% and 80%, respectively. The CLEA immobilization of elastase strain K allowed the stability of the enzyme to be enhanced at high temperature and at a broader pH. Both CLEA-elastase and CLEA-elastase-SB end-products were able to maintain up to 67% enzyme activity at 60 °C and exhibiting an enhanced stability within pH 5–9 with up to 90% recovering activity. By implementing a CLEA on the organic solvent tolerant protease, the characteristics of the organic solvent tolerant were preserved and enhanced with the presence of 25% (v/v) acetonitrile, ethanol, and benzene at 165%, 173%, and 153% relative activity. Structural analysis through SEM and dynamic light scattering (DLS) showed that CLEA-elastase had a random aggregate morphology with an average diameter of 1497 nm.

Immobilization of Enzymes as Cross-Linked Enzyme Aggregates: General Strategy to Obtain Robust Biocatalysts.

Among carrier-free immobilization techniques, cross-linked enzyme aggregates (CLEA) have been extensively described for a great number of diverse enzymes. During the last two decades, numerous efforts have been devoted to identify and understand the main variables involved in CLEA's preparation process leading to robust immobilized biocatalysts. Since every enzyme immobilized as CLEA requires specific conditions and protocols, herein we provide a general preparation strategy where main parameters are highlighted and correlated with a possible desired improved enzyme feature.