Preparation Of Cross-linked Enzyme Aggregates Research Articles

Silanized maghemite for cross-linked enzyme aggregates of recombinant xylanase from Trichoderma reesei

Numerous state-of-the-art technologies and new types of carriers have been developed recently to improve enzyme immobilization. Cross-linked enzyme aggregate (CLEA) technology is a lucrative prospect, as several robust biocatalysts have been generated using this simple method of carrier-free immobilization with the possibility of using semipurified enzyme. However, the low lysine content in the enzyme remains a challenge for effective crosslinking. In this work, maghemite (γ- Fe2O3), a recently sought after nanoparticle, was silanized with (3-aminopropyl) triethoxysilane (APTES) for use in the preparation of cross-linked enzyme aggregates of recombinant xylanase from Trichoderma reesei (Xyl-CLEA-silanized maghemite). Prior screening revealed ethanol to be the best precipitant, and a 0.2: 1 (v:v) glutaraldehyde to enzyme ratio was essential to form active CLEAs. The Xyl-CLEA-silanized maghemite succeeded in increasing the activity recovery 1.66- and 1.5-fold compared to conventional Xyl-CLEAs and Xyl-CLEA-BSA, respectively. The silanization of maghemite with APTES was proven feasible when a 0.0075:1 (v:v) maghemite to enzyme ratio was able to achieve a 78% activity recovery of the xylanase aggregates, whereas the non-silanized maghemite only achieved a 47% activity recovery. At an elevated temperature of 60°C, Xyl-CLEA-silanized maghemite retained approximately 50% of its initial activity, compared to the free enzyme, for which the activity recovery had plummeted to 20%. Conversely, Xyl-CLEA suffered a total loss of activity at this temperature, whereas Xyl-CLEA-BSA retained only a 6% activity. Xyl-CLEA-silanized maghemite also successfully retained more than 50% of its activity after up to 6 cycles, whereas Xyl-CLEA retained approximately 10% of its initial activity after only 5 cycles. The surface morphology, particle size and loading of silanized maghemite were confirmed by field emission scanning electron microscopy (FESEM) and Fourier transform infrared (FTIR) spectroscopy. The combination of the effective surface modification, high enzyme activity recovery, improved stability and enhanced reusability of Xyl-CLEA-silanized maghemite presents an attractive process for xylanase immobilization and provides a promising catalyst for the biomass industry.

Magnetic macromolecular cross linked enzyme aggregates (CLEAs) of glucoamylase

This work illustrates the preparation of magnetic macromolecular glucoamylase CLEAs using dialdehydic pectin, as a cross linker instead of traditional glutaraldehyde. The effect of precipitators type and amount, cross linker concentration, cross linking time and amount of amino functionalized magnetic nanoparticles (AFMNs) on glucoamylase activity was studied. Glucoamylase magnetic macromolecular CLEAs prepared by precipitation in presence of AFMNs by ammonium sulfate were subsequently cross linked by dialdehydic pectin. After cross-linked by pectin, 95.4% activity recovery was achieved in magnetic macromolecular CLEAs, whereas in case of glutaraldehyde cross linker, 85.3% activity recovery was achieved. Magnetic macromolecular CLEAs showed 2.91 and 1.27 folds higher thermal stability as compared to free and magnetic glutaraldehyde CLEAs. In kinetics study, magnetic macromolecular CLEAs retained same Km values, whereas magnetic glutaraldehyde CLEAs showed higher Km value than free enzyme. The porous structure of magnetic macromolecular CLEAs was not only enhanced mass transfer toward macromolecular substrates, but also showed compression resistance for 5 consecutive cycles which was checked in terms of effectiveness factor. At the end, in reusability study; magnetic macromolecular CLEAs were retained 84% activity after 10th cycle without leaching of enzyme which is 22% higher than traditional magnetic CLEAs.

Preparation of cross-linked enzyme aggregates with crude-pored microspherical silica core

Preparation of cross-linked enzyme aggregates with crude-pored microspherical silica core

Cross-linked enzyme aggregates (CLEAs) of PepX and PepN – production, partial characterization and application of combi-CLEAs for milk protein hydrolysis

Abstract The proline-specific X-prolyl-dipeptidyl aminopeptidase (PepX; EC 3.4.14.11) and the general aminopeptidase N (PepN; EC 3.4.11.2) from Lactobacillus helveticus ATCC 12046 were individually immobilized as cross-linked enzyme aggregates (CLEA). The influence of the precipitant, precipitant concentration, cross-linking time, cross-linking agent concentration, the proteic feeder casein, and sodium borohydride was investigated to find the optimal conditions for CLEA preparation. Ammonium sulfate was selected as the most efficient precipitation agent for both enzymes. After optimization of the CLEA preparation conditions (PepX: 3 M (NH 4 ) 2 SO 4 , 2 h cross-linking time, 100 mM glutaraldehyde, 0.087 mg/mL casein, no addition of sodium borohydride; PepN: 4 M (NH 4 ) 2 SO 4 , 4 h cross-linking time, 50 mM glutaraldehyde, no addition of casein and sodium borohydride), the highest activity yield CLEA was about 16% and 8% for PepX-CLEAs and PepN-CLEAs, respectively. The temperature stability, for example, of the PepX-CLEAs (66% residual activity at 50 °C after 4 d compared to 50% for the free enzyme) and the optimum temperature of PepN-CLEAs (40 °C compared to 30 °C for the free enzyme) increased. Additionally, combi-CLEAs of PepX/PepN were prepared under optimal conditions evaluated for the single CLEAs. Here, the highest activity yield CLEA was about 18% and 9% for PepX and PepN activity, respectively. These combi-CLEAs were applied for hydrolysis into an Alcalase® pre-hydrolyzed casein solution. As proof of principle, it was shown that the combi-CLEAs are suitable for application in protein hydrolysis. The relative degree of hydrolysis was increased by approximately 52% compared to the Alcalase® pre-hydrolyzed casein solution.

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.

ChemInform Abstract: Glutaraldehyde in Bio‐Catalysts Design: A Useful Crosslinker and a Versatile Tool in Enzyme Immobilization

AbstractReview: 173 refs.

Co-aggregation of Laccase and Nature Egg White: a Simple Method to Prepare Stable and Recyclable Biocatalyst

Cross-linked enzyme aggregates (CLEAs) are a versatile and effective method for enzyme immobilization, which is exquisitely simple and amenable to rapid optimization. In this study, nature egg white, which is low cost, easily available, and nontoxic, was used as protein feeder to replace traditional protein feeder (bovine serum albumin, etc.) in the preparation of laccase CLEAs (CLEAs-egg). The effects of the various parameters--nature of the precipitant, temperature, glutaraldehyde concentration, and cross-linking time--on the activity recovery of the resulting CLEAs were studied. The laccase CLEAs-egg exhibited increased stability compared to the free and the laccase CLEAs without protein feeder. The thermal stability of CLEAs-egg was improved and showed 1.3- and 1.8-fold increase in activity at 40 and 60 °C after 5 h incubation, respectively. The stability of CLEAs-egg against denaturants (urea and GndHCl) and protease (trypsin) was also improved. Laccase CLEAs-egg was also demonstrated to be an active and stable biocatalyst in the removal of chlorophenol. After 30 h, 83.6 and 91.5% of 4-chlorophenol and 2,4-dichlorophenol can be removed.

Glutaraldehyde in bio-catalysts design: a useful crosslinker and a versatile tool in enzyme immobilization

Glutaraldehyde is one of the most widely used reagents in the design of biocatalysts. It is a powerful crosslinker, able to react with itself, with the advantages that this may bring forth. In this review, we intend to give a general vision of its potential and the precautions that must be taken when using this effective reagent. First, the chemistry of the glutaraldehyde/amino reaction will be commented upon. This reaction is still not fully clarified, but it seems to be based on the formation of 6-membered heterocycles formed by 5 C and one O. Then, we will discuss the production of intra- and inter-molecular enzyme crosslinks (increasing enzyme rigidity or preventing subunit dissociation in multimeric enzymes). Special emphasis will be placed on the preparation of cross-linked enzyme aggregates (CLEAs), mainly in enzymes that have low density of surface reactive groups and, therefore, may be problematic to obtain a final solid catalyst. Next, we will comment on the uses of glutaraldehyde in enzymes previously immobilized on supports. First, the treatment of enzymes immobilized on supports that cannot react with glutaraldehyde (only inter and intramolecular cross-linkings will be possible) to prevent enzyme leakage and obtain some enzyme stabilization via cross-linking. Second, the cross-linking of enzymes adsorbed on aminated supports, where together with other reactions enzyme/support crosslinking is also possible; the enzyme is incorporated into the support. Finally, we will present the use of aminated supports preactivated with glutaraldehyde. Optimal glutaraldehyde modifications will be discussed in each specific case (one or two glutaraldehyde molecules for amino group in the support and/or the protein). Using preactivated supports, the heterofunctional nature of the supports will be highlighted, with the drawbacks and advantages that the heterofunctionality may have. Particular attention will be paid to the control of the first event that causes the immobilization depending on the experimental conditions to alter the enzyme orientation regarding the support surface. Thus, glutaraldehyde, an apparently old fashioned reactive, remains the most widely used and with broadest application possibilities among the compounds used for the design of biocatalyst.

Preparation of cross-linked enzyme aggregates in water-in-oil emulsion: Application to trehalose synthase

Abstract In this work, a novel method to prepare cross-linked enzyme aggregates (CLEA) in a water-in-oil emulsion was reported. Spherical CLEA of recombinant trehalose synthase (TreS) from Meiothermus rubber was obtained through the emulsion based process. By varying the experimental parameters, the optimum conditions for the preparation of spherical CLEA were determined. The optimum catalytic temperature and thermal stability of spherical CLEA were enhanced significantly compared with the free enzyme. Spherical CLEA was also more tolerant to metal ions and chemical denaturants. Optical and scanning electron microscopy analysis showed that instead of the amorphous clumping shape of conventionally prepared CLEA, spherical CLEA has a more structured ball-like appearance with a size of 20–60 μm in diameter that could be easily separated.

Preparation and Thermal Kinetic Deactivation of Cross-linked Enzyme Aggregates of Penicillin Acylase†

Cross-linked enzyme aggregates( CLEAs) is an efficient approach to obtain immobilized enzymes without the use of any pre-existing carriers. The preparation of CLEAs usually consisted of two simple steps: precipitation and cross-linking. In this work,CLEAs of penicillin acylase was preparaed using 50% ammonium sulphate solution as precipitant and 0. 35% glutaraldehyde as cross-linking agent. The resulting CLEAs obtained an activity yield of 30. 1% and higher optimal temperature( 57 ℃) than free penicillin acylase( 47 ℃) as well as an obvious shift of optimal pH from 8. 3 to 10. 0. Moreover,compared with free enzyme, the thermal stability of CLEAs was largely enhanced which facilitated the application of penicillin acylase in high-temperature reaction systems. Based on the thermal deactivation kinetics study,it was found that the deactivation model of penicillin acylase changed from one-step model to serial model through immobilizing as CLEAs. The higher deactivation energy of CLEAs( 549. 2 kJ / mol) than free enzyme( 248. 8 kJ / mol) also explained the excellent stability of CLEAs under high temperature environment. In addition,CLEAs exhibited favorable reusability after using 7 times.

A Simple Technique of Preparing Stable CLEAs of Phenylalanine Ammonia Lyase Using Co-aggregation with Starch and Bovine Serum Albumin

Cross-linked enzyme aggregates (CLEAs) have been recently proposed as an alternative to conventional immobilization methods on solid carriers. However, the low cross-linking efficiency causes the major activity loss and instability in the conventional protocol for CLEA preparation. Herein, the effects of bovine serum albumin and starch addition on the cross-linking efficiency of CLEAs of phenylalanine ammonia lyase (PAL) from Rhodotorula glutinis were evaluated. A co-aggregation strategy was developed to improve cross-linking efficiency by adding starch and bovine serum albumin (BSA). CLEAs of PAL prepared in the presence of BSA and starch (PSB-CLEAs) retained 36 % activity, whereas CLEAs prepared without BSA and starch (PAL-CLEAs) retained only 8 % activity of the starting enzyme preparation. Compared with PAL-CLEAs, the thermal stability of PSB-CLEAs has improved considerably, maintaining 30 % residual activity after 4 h of incubation at 70 °C, whereas the PAL-CLEAs have only 13 % residual activity. PSB-CLEAs also exhibited the expected increased stability of PAL against hydrophilic organic solvents, superior operability, and higher storage stability. The proposed technique of preparing CLEAs using co-aggregation with starch and BSA would rank among the potential strategies for efficiently preparing robust and highly stable enzyme aggregates.

Parameters in preparation and characterization of cross linked enzyme aggregates (CLEAs)

In the past couple of decades, cross linked enzyme aggregates (CLEAs) have emerged as a novel and versatile carrier free immobilization technique. The immobilization of enzymes as cross linked enzyme aggregates (CLEAs) involves precipitation of an enzyme from aqueous solution followed by cross linking with a bi-functional reagent. It is worth noting that many parameters alter the enzyme precipitation and the aggregate cross linking and hence affect the activity and stability of CLEAs. Therefore to endorse CLEAs for industrial application, each newly synthesised CLEA is characterized. This review intends to investigate the effects of various parameters, such as the nature and purity of the enzyme, the nature and amount of precipitant, the nature and amount of cross linker, the cross linking time, the pH and temperature during CLEA preparation and washing and separation techniques on the activity and stability of CLEAs. The major parameters such as catalytic properties, particle size and morphology, stability and reusability required for approval of industrial applicability of newly synthesized CLEAs are critically reviewed. Furthermore the scope of CLEAs in non-aqueous solvent, the development of one pot cascade processes and the design of different types of enzyme reactors is also discussed.

Catalytic performance of cross-linked enzyme aggregates of Penicillium expansum lipase and their use as catalyst for biodiesel production

Cross-linked enzyme aggregates (CLEAs) of lipase from Penicillium expansum (PEL) were prepared directly from fermentation broth, a more practical and economically viable procedure than the generally used methods that require purified or partially purified enzymes for CLEA preparation. A systematic study of the activity and stability of PEL-CLEAs was undertaken in aqueous solution, organic solvents, and ionic liquids (ILs). Immobilization of the enzyme resulted in a significantly enhanced stability in aqueous solution with regard to pH and temperature. PEL-CLEAs showed an improved activity in the IL [BMIm][PF6] relative to that observed in hexane, both keeping increased with temperature (up to 90°C in the IL and 60°C in hexane). The effect of water content and water activity in these two nonaqueous media showed similar patterns as for the uncrosslinked enzyme. The half life of the CLEAs was higher in hydrophobic organic solvents (hexane and chloroform) than in aqueous solution, and presented a sigmoid relationship with the logP of the organic solvent tested. PEL-CLEAs catalyzed biodiesel production from microalgal oil in the IL [BMIm][PF6] with a conversion of 85.7%, demonstrating that they can be taken as a promising catalyst for this application.

Highly efficient biosynthesis of sucrose-6-acetate with cross-linked aggregates of Lipozyme TL 100 L

As a short chain monoester, sucrose-6-acetate (S-6-a) is a key intermediate in the preparation of an eminent sweetener (sucralose). To replace the traditional multi-step chemical route for sucralose biosynthesis, enzymatic synthesis of S-6-a was investigated, using cross-linked enzyme aggregates (CLEAs) of Lipozyme TL 100 L. The optimal CLEA preparation conditions was obtained as follows: using 33.3% (v/v) PEG600 co-precipitated with additive of D-sorbierite, then cross-linking with 1.5% (v/v) glutaraldehyde at 0 °C for 4 h. As a result, the immobilized Lipozyme had high specific bioactivity (34.64 U/g) of transesterification in non-aqueous media. With these immobilized enzymes, the optimum transesterification conditions were investigated systematically, including CLEA loading, the mole ratio of vinyl acetate versus sucrose, temperature and reaction time, etc. The results showed that the highest concentration and yield of S-6-a was 49.8 g/L and 87.46%, respectively. Further experiments showed that the resulting CLEAs also had much higher operational stability than the commercial Lipozyme TLIM. The present work has paved a new path for the large-scale bioproduction of S-6-a with immobilized lipase in the future.

Preparation of cross-linked enzyme aggregates of l-aminoacylase via co-aggregation with polyethyleneimine

Abstract l -Aminoacylase from Aspergillus melleus was co-aggregated with polyethyleneimine and subsequently cross-linked with glutaraldehyde to obtain aminoacylase–polyethyleneimine cross-linked enzyme aggregates (termed as AP-CLEA). Under the optimum conditions, AP-CLEA expressed 74.9% activity recovery and 81.2% aggregation yield. The said method of co-aggregation and cross-linking significantly improved the catalytic stability of l -aminoacylase with respect to temperature and storage. AP-CLEA were employed for enantioselective synthesis of three unnatural amino acids (namely: phenylglycine, homophenylalanine and 2-naphthylalanine) via chiral resolution of their ester-, amide- and N-acetyl derivatives. The enantioselectivity of AP-CLEA was the highest for hydrolysis of amino acid amides; was moderate for hydrolysis of N-acetyl amino acids and was the least for hydrolysis of amino acid esters. Furthermore, AP-CLEA were found to retain more than 92% of the initial activity after five consecutive batches of (RS)-homophenylalanine hydrolysis suggesting an adequate operational stability of the biocatalyst.

Influence of the pH of glutaraldehyde and the use of dextran aldehyde on the preparation of cross-linked enzyme aggregates (CLEAs) of lipase from Burkholderia cepacia

The preparation of cross-linked enzyme aggregates (CLEAs) of lipase has been a challenge due the low amount of lysine residues that lipases have on their surface. The results show that CLEAs prepared using dextran aldehyde (100-200KDa) have a higher hydrolysis activity and particle size (activities between 3186 ± 21 U/g of CLEA and 4800 ± 30 U/g of CLEA and particle sizes between 52.6 ± 18.7 µm and 126.2 ± 53.5 µm) than CLEAs prepared with glutaraldehyde (0.1 KDa) (activities between 894 ± 16 U/g of CLEA and 2874 ± 20 U/g of CLEA and particle sizes between 21.2 ± 5.1 µm and 83.4 ± 24.9 µm); Thermal stability assays of bioctalysts at 60oC at pH 7.0 using phosphate buffer 25 mM showed that CLEAs prepared with dextran aldehyde have lower residual activity after 50 hrs (maximum residual activity of 46.8% in the CLEA) than CLEAs prepared with glutaraldehyde (maximum residual activity of 70.2% in CLEA). When considering hydrolysis activity, thermal stability and residual activity of CLEAs as a criteria for selecting the best preparation conditions, it has been found that the best condition for CLEAs preparation are to use glutaraldehyde as cross-linking reagent at pH 9.5, at a concentration of 3.5 g/l, and an enzyme/albumin ratio of 15.

Cross-Linked Enzyme Aggregates as Industrial Biocatalysts

The immobilization of enzymes as cross-linked enzyme aggregates (CLEAs) and their applications in industrially relevant biotransformations are reviewed. The preparation of CLEAs involves precipitation from aqueous buffer followed by cross-linking with a bifunctional reagent, usually a dialdehyde such as glutaraldehyde or dextran polyaldehyde. The technique is exquisitely simple and broadly applicable. CLEAs have several benefits in the context of industrial biocatalysis. They have a significantly enhanced shelf life and operational stability, are easy to recover and reuse, and are completely stable towards leaching in aqueous media. They circumvent the use of, often expensive, carriers and have high volumetric and catalyst productivities as a result of the absence of noncatalytic ballast. The applications of CLEAs for a wide variety of enzymes, including various types of hydrolases, oxidoreductases, and lyases in industrially relevant biotransformations, are discussed. Co-aggregation and cross-linking of two or more enzymes affords combi-CLEAs that are ideally suited for catalyzing one-pot, enzymatic cascade processes, for example, the successful application of a triple-decker combi-CLEA comprising a hydroxynitrile lyase, a nitrilase, and an amidase in the one-pot conversion of benzaldehyde to S-mandelic acid in >99% ee at 96% conversion. Finally, reactor concepts are discussed in the context of industrial applications.

Preparation of multipurpose cross-linked enzyme aggregates and their application to production of alkyl ferulates

Commercial multicomponent enzyme preparations, Ultraflo L, Depol 740L and Depol 670L, with feruloyl esterase activity, were tested for the transesterification of methyl ferulate to 1-butyl ferulate in their free and immobilized form using as a reaction system a ternary water–organic mixture consisting of n-hexane, 1-butanol and water. A number of factors affecting enzymes precipitation and cross-linking into cross-linked enzyme aggregates (CLEAs) have been investigated. Consecutive optimization of the precipitant type and cross-linker concentration resulted in CLEAs showing higher operational stability and synthetic activity compared to the free enzymes’ forms. Under certain optimization conditions, conversion yields of 97%, 87% and 5%, were obtained by CLEAs prepared from Ultraflo L, Depol 740L and Depol 670L, respectively. The activities initially present in the three commercial preparations were completely retained after cross-linking resulting in multipurpose biocatalysts which have the potential to carry out different and independent reactions. This work is consistent to the novel CLEA concept called combi-CLEA.

Cross-Linked Amorphous Nitrilase Aggregates for Enantioselective Nitrile Hydrolysis

A recombinant Escherichia coli strain was constructed which efficiently expressed the enantioselective nitrilase from Alcaligenes faecalis DSMZ 30030 as a hisitidine-tagged enzyme variant under the control of a rhamnose inducible promoter. The enzyme was purified from cell extracts and used for the preparation of cross-linked enzyme aggregates (CLEAs). The conditions for the preparation of the CLEAs were optimized using various organic solvents and cross-linking agents and a procedure was developed which combined a precipitation with 85 % (v/v) isopropyl alcohol and a cross-linking with 30 mM glutaraldehyde. Thus, about 80 % of the initial nitrilase activity could be incorporated into the CLEAs. The hydrolysis of racemic mandelonitrile to (R)-mandelic acid was compared between the soluble nitrilase preparations and their CLEAs (nit-CLEAs). The nitrilase activity in the CLEAs was at 30 °C and 60 °C about 5 times more stable than in the soluble preparations. The CLEAs could be reused 5 times with only about 10 % reduction in activity. The enantioselectivity of the nitrilase for the formation of (R)-mandelic acid from racemic mandelonitrile decreased for both preparations with increasing temperatures (10 °C to 50 °C), but this effect was significantly less pronounced for the CLEAs. A detailed analysis of solvent effects on nitrilase enantioselectivity allowed thermodynamic insights into contributions from free energy component (activation enthalpy and entropy) to chiral preference of nitrilase in such non conventional media.

Preparation of cross-linked enzyme aggregates by using bovine serum albumin as a proteic feeder

Addition of bovine serum albumin (BSA) as a proteic feeder facilitates obtaining cross-linked enzyme aggregates (CLEAs) in cases where the protein concentration in the enzyme preparation is low and/or the enzyme activity is vulnerable to the high concentration of glutaraldehyde required to obtain aggregates. CLEAs of Pseudomonas cepacia lipase and penicillin acylase were prepared. CLEA of lipase prepared in the presence of BSA retained 100% activity whereas CLEA prepared without BSA retained only 0.4% activity of the starting enzyme preparation. Lipase CLEA showed 12-fold increase in activity over free enzyme powder when the CLEA was used in transesterification of tributyrin. For the transesterification of Jatropha oil, while free enzyme powder required 8 h and 50 mg lipase to obtain 77% conversion, CLEA required only 6 h and 6.25 mg lipase to obtain 90% conversion. In the case of penicillin acylase, 86% activity could be retained in CLEA prepared with BSA whereas CLEA made without BSA retained only 50% activity. CLEA prepared without BSA lost 20% activity after 8 h at 45 °C whereas CLEA with BSA retained full activity. CLEA prepared with BSA showed V max/ K m of 36.3 min −1 whereas CLEA prepared without BSA had V max/ K m of 17.4 min −1 only. Scanning electron microscopy analysis showed that CLEAs prepared in the presence of BSA were less amorphous and closer in morphology to CLEAs of other enzymes described in the literature.