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The preparation of cross-linked enzyme aggregates (CLEAs) with albumin as an additive is demonstrated successfully with lipase Thermomyces lanuginosus (TL) and lipase B of Candida antarctica (CalB). Investigations regarding the albumin admixture and the temperature optimum are employed. Compared to non-cross-linked lipase, CalB delivers the best results at a mass ratio of 1:16 lipase/albumin, which leads to a 24-fold increase of activity; TL at a mass ratio of 1:12, which leads to a eightfold increased activity. A transesterification yield of 61% m/m ethyl ester can be reached with 2.5% m/m CLEAs of lipase TL applied on rapeseed oil within 1.5 h reaction time. A catalyst mass of 10% m/m yields 94% m/m ethyl ester in the same time. Within six cycles of reuse, the catalyst remains stable. CLEAs of CalB and Novozym 435 (a commercial immobilized lipase B of C. antarctica) show comparable rates in ethanolysis and glycerolysis reaction. Practical applications: CLEAs have become of increasing interest in terms of synthesis of organic compounds via bio-catalytic (green) approaches. The findings presented here are targeting at transesterification of fats and oils into corresponding alkyl esters applicable, e.g., as alternative fuel substitutes. High conversion rates coupled with hyperactivation effects as well as highly appealing reusability behavior indicate the practical benefits even for technical applications. Conversion of lipids into derivatives by CLEAs as demonstrated here, might extend utilization and conversion possibilities of biomass. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Highlights: [•] Highly active and stable CLEAs of Mung bean EHs were successfully prepared. [•] CLEAs were superior to free enzyme in terms of activity, stability and catalytic efficiency. [•] CLEAs of Mung bean EHs were more efficient in catalyzing asymmetric hydrolysis of SO to (R)-PED. [•] The efficient biocatalytic process with CLEAs was feasible on a 250-mL preparative scale. [Copyright &y& Elsevier]

Abstract: Chitosan (CS) nanofibers with a diameter of 150–200nm were fabricated from a mixed chitosan/poly (vinyl alcohol) (PVA) solution by the electrospinning method. The nascent CS/PVA nanofibers were treated with 0.5M NaOH solution to make stable CS nanofibers by removing PVA under aqueous conditions. Hen egg-white lysozyme was immobilized on electrospun CS nanofibers via cross-linked enzyme aggregates (CLEAs) and used for effective and continuous antibacterial applications. The maximum amount of lysozyme immobilized on the CS nanofibers was determined to be 62.3mg/g of nanofibers under the optimum conditions. The immobilized lysozyme-CLEA retained more than 75.4% of its initial activity after 80 days of storage at room temperature, while the free lysozyme lost all of its activity under the same conditions. In addition, the immobilized lysozyme-CLEA retained more than 76% of its activity after 100 consecutive uses. Finally, the durability of the lysozyme-CLEA immobilized CS nanofibers showed bacteriostasis ratios of 82.4%, 79.8%, 83.4%, and 84.1% after 10 cycles against 4 pathogenic bacteria, viz. Staphylococcus aureus, Bacillus subtilis, Shigella flexneri, and Psedomonas aeruginosa, respectively. These results demonstrated that lysozyme-CLEA immobilized CS nanofibers could be used as a promising material for enhanced and continuous antibacterial applications. [Copyright &y& Elsevier]

Abstract: Laccase and laccase-based cross-linked enzyme aggregates (CLEAs) were stabilized through the formation of a surrounding polymeric network made of chitosan and 3-aminopropyltriethoxysilane. The thermoresistance of the resulting enzyme polymer engineered structures of laccase (EPES-lac) and CLEAs (EPES-CLEA) were more than 30times higher than that of free laccase and CLEAs at pH 3 and 40°C. The EPES showed higher residual activity than the unmodified biocatalysts against chaotropic salts (up to 10times), EDTA (up to 5times), methanol (up to 15times) and acetone (up to 20times). The Michaelis–Menten kinetic parameters revealed that the affinity for 2,2’-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) has doubled for the EPES-lac and EPES CLEA compared to their unmodified forms. The EPES-lac structures acted optimally at pH 4 and their activity was nearly temperature-independent, while the laccase activity of EPES-CLEA was optimal at pH 4 and 60°C. Globally, the EPES have shown significantly improved properties which make them attractive candidate for the development of laccase-based applications. [Copyright &y& Elsevier]

Abstract: The production of porous cross-linked enzyme aggregates (p-CLEAs) is a simple and effective methodology for laccase immobilization. A three-phase partitioning technique was applied to co-precipitate laccase and starch, followed by cross-linking with glutaraldehyde and removal of starch by α-amylase to create pores in the CLEAs. Scanning electron microscopy revealed a very smooth spherical structure with numerous large pores. The half-life of free laccase at 55°C was calculated to be 1.3h, while p-CLEAs did not lose any activity even after 14h. p-CLEAs also exhibited improved storage stability, catalytic efficiency and could be recycled 15 times with 60% loss of activity. The catalysts decolorized triphenylmethane and reactive dyes by 60–70% at initial dye concentrations of 2 and 0.5gL−1, respectively, without any mediators. These results suggest the potential of CLEA technology in waste-water treatment. [Copyright &y& Elsevier]

Cross-linked enzyme aggregates (CLEAs) of β-glucosidase were prepared and characterized. Under the optimum conditions, the activity recovery of CLEAs reached 84 %. The reduction by NaBH resulted in slightly lower activities of CLEAs, while their thermostability was enhanced. CLEAs were more thermally stable than free enzyme (half lives, 973 vs. 518 min at 50 °C), while less stable than seed meal (half life, 1,090 min). In 90 % (v/v) t-butanol, the half lives of CLEAs and free enzyme were 53 and 6.7 h, respectively. Besides, the catalytic efficiency ( V/ K) of CLEAs was comparable to free enzyme (0.42 vs. 0.47 min mg). This carrier-free immobilized enzyme had a network structure with multiple layers. The productivity of salidroside using CLEAs reached 150 g/l g catalyst, while being 6.3 g/l g with seed meal. [ABSTRACT FROM AUTHOR]

Abstract: Cross-linked enzyme aggregates of poly-3-hydroxybutyrate (PHB) depolymerase from Streptomyces exfoliatus (PhaZ Sex–CLEAs) have been prepared. Acetone was used as the precipitating agent, while addition of bovine serum albumin (BSA) facilitated CLEAs formation. Conditions for enzyme precipitation and cross-linking have been optimized, and confocal scanning microscopy showed a homogeneous enzyme distribution in the biocatalyst. Obtained PhaZ Sex –CLEAs presented an average size of 50–300μm, showing a high PHB depolymerase activity of 255U/g wet biocatalyst at 40°C and pH 7.0. Temperature-activity profile of PhaZ Sex –CLEAs at pH 8.0 showed that the highest activity for pNPB hydrolysis was achieved at 60°C, whereas pH-activity profile at 40°C indicated that highest activity for PHB hydrolysis was achieved at pH 7.0. Additionally, immobilized biocatalyst could be recycled at least for 20 consecutive batch reactions without loss of catalytic activity, and showed higher pH and temperature stability, and better tolerance to several organic solvents than its soluble counterpart. [Copyright &y& Elsevier]

Cross-linked enzyme aggregates of phenylalanine ammonia lyase (PAL -CLEAs) from Rhodotorula glutinis were prepared. The effects of the type of aggregating agent, its concentration, and that of cross-linking agent were studied. PAL-CLEAs production was most effective using ammonium sulfate (40 % saturation), followed by cross-linking for 1 h with 0.2 % ( v/ v) glutaraldehyde. Moreover, the storage and operational stability of the resulting PAL-CLEAs were also investigated. Compared to the free enzyme, the PAL-CLEAs exhibited the expected increased stability of the enzyme against various deactivating conditions such as pH, temperature, denaturants, and organic solvents and showed higher storage stability than its soluble counterpart. Additionally, the reusability of PAL-CLEAs with respect to the biotransformation of l-phenylalanine was evaluated. PAL-CLEAs could be recycled at least for 12 consecutive batch reactions without dramatic activity loss, which should dramatically increase the commercial potential of PAL for synthesis of l-phenylalanine. To the best of our knowledge, this is the first report of immobilization of PAL as cross-linked enzyme aggregates. [ABSTRACT FROM AUTHOR]

Aim: The purpose of the work is the use of L-Lysine amino acid cross-linker in the achievement of alternative and compatible enzyme aggregate. Cross-linked enzyme aggregates (CLEAs) were prepared from several enzymes (glucose oxidase, peroxidase and urease) by precipitation and subsequent cross-linking using glutaraldehyde, 1,8-octanediamine and L-Lysine. Material and Methods: The effects of cross-linking agents on CLEAs activity were investigated and immobilized enzymes were characterized. The initial enzyme concentration was constant as 4x10-3 mg/ml. BSA were used as precipitant in CLEA's method as described in our previously study. Results: The concentration of cross-linkers were 2% for glutaraldehyde and 1,8-octanediamine and 4% for L-Lysine. Activities of both free and immobilised enzymes were obtained by measuring the amount of substrate conversion, spectrophotometrically. Kinetic parameters of native and immobilised enzyme were calculated by using Lineweaver-Burk plots. Conclusion: L-Lysine was applied successfully as a cross-linker for the formation of CLEA's. [ABSTRACT FROM AUTHOR]

Abstract: Cross-linked enzyme aggregates (CLEAs), protein coated microcrystals (PCMCs), cross-linked protein coated microcrystals (CLPCMCs) of Candida antarctica lipase B (CALB) were used for esterification of glycerol with palmitic acid in acetone under low water condition. With CLEAs, 81% monoglyceride (MG) along with 4.5% diglyceride (DG) were produced at 1% (v/v) water content in 24h. The water content in the medium was managed by stepwise addition of the molecular sieves at appropriate time intervals. With PCMCs (potassium sulfate as a core material), 82% monoglyceride along with 4.0% diglyceride were obtained, with 0.5% water (v/v) added initially to anhydrous acetone with molecular sieves present in the reaction medium. With CLPCMC (prepared by cross-linking with 200mM glutaraldehyde), 87% monoglyceride and 3.3% diglyceride were produced in 24h in presence of 1% (v/v) water (added initially) and with appropriate amount of molecular sieves added in the reaction medium. The results offer a comparative study on the performance of three high activity preparations of CALB for preparation of monopalmitin with ≤10% of the diglyceride content. [Copyright &y& Elsevier]

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. [Copyright &y& Elsevier]

Abstract: The precipitation of enzyme causes the major activity loss in the conventional protocol for CLEAs preparation. Herein, a sugar-assisted strategy was developed to minimize the activity loss in the step of enzyme precipitation by adding sugar as the stabilizer, which contributed to improve the activity yield of resulting CLEAs. Penicillin G acylase (PGA) was employed as a model enzyme. The effects of glucose, sucrose and trehalose on the activity yields of CLEAs were investigated. The highest activity was obtained in the case of adding trehalose. Confocal laser scanning microscopy and Fourier transform infrared spectroscopy showed that the polar microenvironment and the secondary structure of native enzyme were preserved to some extent when PGA was prepared as sugar-assisted CLEAs, resulting in PGA''s higher activity than sugar-free CLEAs. Scanning electron microscope revealed the different inner morphologies, and the kinetic studies showed the higher affinity and resist-inhibition capacity of sugar-assisted CLEAs. Furthermore, stability experiments demonstrated that CLEAs prepared in sugar-assisted strategy remained higher thermal stability when it was incubated at high temperature. [Copyright &y& Elsevier]

Abstract: Cross-linked enzyme aggregates (CLEA®s) were prepared from Candida rugosa lipase (CrL) using glutaraldehyde as the cross-linker. The optimum conditions of the immobilization process were determined (precipitant: ethanol, crosslinker concentration: 25mM, enzyme concentration: 50mg/ml, crosslinking time: 45min.). CLEAs were shown to have several advantages compared to the free enzyme. They were more stable at 50°C and 60°C and had good reusability; retaining 40% of their initial activity after 15 recycles in aqueous media and remaining constant at that level thereafter, suggesting some initial leaching in water. The CLEAs catalyzed esterification reactions in cyclohexane, affording higher conversions than with the free enzyme, especially when longer fatty acids and alcohols were used as substrates. [Copyright &y& Elsevier]

Abstract: A recombinant esterase (EC 3.1.1.1) cloned from Bacillus subtilis 0554 (BSE) was carrier-freely immobilized with the method of cross-linked enzyme aggregates. The conditions for preparing the cross-linked aggregates of BSE (CLA-BSE) were optimized, including the type and concentration of precipitants, and the concentration of cross-linker, and a simple and efficient procedure for preparing CLA-BSE was developed, consisting of a precipitation step with 0.5gmL−1 (NH4)2SO4 and a cross-linking step with 60mM glutaraldehyde for a period of 3h as the cross-linking time. As a result, about 70% of the initial free BSE activity was incorporated into the CLA-BSE. The thermal stabilities of the immobilized enzyme at 30°C and 50°C were >360 and 14 times those of free BSE, respectively. More importantly, the operational stability of CLA-BSE was also considerably improved. In the kinetic resolution of dl-menthyl acetate to produce l-menthol with CLA-BSE gave ee p >94% at conversion of >40% and the CLA-BSE could be reused for 10 times with only about 8% reduction in activity. Therefore, the new biocatalyst immobilized through the methodology of CLEAs could significantly decrease the manufacturing cost of l-menthol and would be more beneficial for its practical applications. [Copyright &y& Elsevier]

To obtain robust and thermo-stable enzyme aggregates, p-benzoquinone was used as cross-linker and bovine serum albumin (BSA) as crowding macromolecules to prepare cross-linked enzyme aggregates (CLEAs) of lipase. Effects of cross-linking time and cross-linker content on the activity, thermal stability and characteristics of enzyme aggregates were examined carefully. It was observed that when the content of p-benzoquinone was 5 mM and amount of BSA was 125% of that of lipase (w/w), the specific activity of cross-linked co-aggregates of lipase and BSA was 79.8 U mg, 2.44-fold of that of cross-linked enzyme aggregates of lipase without BSA. Moreover, after heat treatment for 96 h at 50 °C, the CLEAs prepared with this facile routine kept 75.18% of their initial activity, 5.01-fold more than that of the just CLEAs using glutaraldehyde. Furthermore, BSA macromolecules in lipase CLEAs enhanced the catalytic efficiency of free and just lipase CLEAs without BSA by 1.45 and 2.83 times, respectively. The proposed crosslinking technique would rank among the potential strategies for efficiently preparing robust and thermo-stable enzyme aggregates. [ABSTRACT FROM AUTHOR]

The article presents a study on the immobilization of candida rugosa lipase on amino-functionalized magnetic beads using cross-linked enzyme aggregates (CLEA). It states that batch operation mode was used to examine immobilized lipase usability while the immobilized and free lipases were stored in a solution of sodium phosphate buffer. Results show that 80% of the immobilized lipase's initial activity was maintained.

Abstract: The para-nitrobenzyl esterase (PNBE), which was encoded by pnbA gene from Bacillus subtilis, was immobilized on amino-functionalized magnetic supports as cross-linked enzyme aggregates (CLEA). The maximum amount of PNBE-CLEA immobilized on the magnetic beads using glutaraldehyde as a coupling agent was 31.4mg/g of beads with a 78% activity recovery after the immobilization. The performance of immobilized PNBE-CLEA was evaluated under various conditions. As compared to its free form, the optimal pH and temperature of PNBE-CLEA were 1unit (pH 8.0) and 5°C higher (45°C), respectively. Under different temperature settings, the residual enzyme activity was highest for the PNBE-CLEA, followed by covalently fixed PNBE without further cross-linking and the free PNBE. During 40 days of storage pried, the PNBE-CLEA maintained more than 90% of its initial activity while the free PNBE maintained about 60% under the same condition. PNBE-CLEA also retained more than 80% activity after 30 reuses with 30min of each reaction time, indicating stable reusability under aqueous medium. [Copyright &y& Elsevier]

The operational stabilities of nitrilases from Aspergillus niger K10 and Fusarium solani O1 were examined with 4-cyanopyridine as the substrate in continuous-stirred membrane reactors (CSMRs). The former enzyme was fairly stable at 30 °C with a deactivation constant ( kd) and enzyme half-life of 0.014 h−1 and 50 h, respectively, but the latter exhibited an even higher stability characterized by kd = 0.008 h−1 and half-life of 87 h at 40 °C. Another advantage of this enzyme was its high chemoselectivity, i.e., selective transformation of nitriles into carboxylic acids, while the amide formed a high ratio of A. niger K10 nitrilase product. High conversion rates (>90%) were maintained for about 52 h using the nitrilase from F. solani O1 immobilized in cross-linked enzyme aggregates (CLEAs). The purity of isonicotinic acid was increased from 98% to >99.9% by using two CSMRs connected in series, the first one containing the F. solani O1 nitrilase and the second the amidase from Rhodococcus erythropolis A4 (both enzymes as CLEAs), the amidase hydrolyzing the by-product isonicotinamide. [ABSTRACT FROM AUTHOR]

Abstract: The microstructure and the catalytic properties of cross-linked enzyme aggregates (CLEA) of penicillin acylase (PA) obtained under different conditions were investigated. The period of time left between the enzyme precipitation and the cross-linking step was found to influence the structural organization of the resulting enzyme preparation. Confocal fluorescent microscopy of the so-called “fresh” and “mature” CLEAs PA allowed to estimate the “characteristic” diameter of CLEA PA particles, which appeared to be about 1.6μm, and revealed that the “mature” type was composed of relatively big particles as compared to the “fresh” type. Complementary kinetic studies showed that the “mature” CLEA PA were more effective in both hydrolytic and synthetic reactions. It was suggested that the aggregate size might regulate the extent of covalent modification of PA and thereby influence the catalytic properties of CLEA. [Copyright &y& Elsevier]

Abstract: Pseudomonas sp. Lipase (PSL) immobilized with the method of cross-linked enzyme aggregates (CLEAs) using acetone as the optimal precipitant is investigated. The immobilization efficiency (%) and activity retention (%) of the immobilized lipase (CLEA-PSL) are 70.6 and 45.1%, respectively. In the kinetic resolution of N-(2-ethyl-6-methylphenyl) alanine, CLEA-PSL not only keeps excellent enantioselectivity (E-value>100) as the free PSL, but also shows higher catalytic activity and thermal stability. CLEA-PSL requires only 12h to obtain 50% conversion whereas the free PSL needs 48h; the residual activity of CLEA-PSL and free PSL are respectively 72.2 and 23.3% after incubated 24h at 60°C. Furthermore, CLEA-PSL can be re-used through ten cycles and the efficiency loss in activity is found to be only 19.1%. [Copyright &y& Elsevier]

Cross-linked enzyme aggregates (CLEAs) are prepared by precipitation of an enzyme and then chemical cross-linking the precipitate. Three CLEAs of lipase with glutaraldehyde concentrations of 10 mM (CLEA A), 40 mM (CLEA B) and 60 mM (CLEA C) were prepared. Studies show that there is a trade-off between thermal stability vs transesterification/hydrolysis rate vs enantioselectivity. The initial rates for transesterification of β-citronellol for the uncross-linked enzyme and CLEAs A, B and C were 243, 167, 102 and 40 µmol mg-1 h-1, respectively. Their thermal stabilities in aqueous media, as reflected by their half-life values at 55°C, were 6, 9, 13 and 16 h, respectively. The enantioselectivity, E values (for kinetic resolution of β-citronellol by transesterification) were 19, 74, 11 and 6, respectively. These results show that CLEA C was the most thermostable; the uncross-linked enzyme was best at obtaining the highest transesterification rate; and CLEA A was best suited for the enantioselective synthesis. Scanning electron microscopy (SEM) showed that the morphology of CLEA was dependent upon the extent of cross-linking. [ABSTRACT FROM AUTHOR]

Abstract: The potential of the Aspergillus niger type A feruloyl esterase (AnFaeA) for the synthesis of various phenolic acid esters was examined using a ternary-organic reaction system consisting of a mixture of n-hexane, 1- or 2-butanol and water. Reaction parameters including the type of methyl hydroxycinnamate, the composition of the reaction media, the temperature, and the substrate concentration were investigated to evaluate their effect on initial rate and conversion to butyl esters of sinapic acids. Optimisation of the reaction parameters lead to 78% and 9% yield for the synthesis of 1-butyl and 2-butyl sinapate, respectively. For the first time, a feruloyl esterase was introduced in the reaction system as cross-linked enzyme aggregates (CLEAs), after optimisation of the immobilisation procedure, allowing the recycling and reuse of the biocatalyst. The inhibition of copper-induced LDL oxidation by hydroxycinnamic acids and their corresponding butyl esters was investigated in vitro. Kinetic analysis of the antioxidation process demonstrates that sinapate derivatives are effective antioxidants indicating that esterification increases the free acid''s antioxidant activity especially on dimethoxylated compounds such as sinapic acid compared to methoxy-hydroxy-compounds such as ferulic acid. [Copyright &y& Elsevier]

Abstract: A semi-purified nitrile hydratase from Rhodococcus erythropolis A4 was applied to biotransformations of 3-oxonitriles 1a–4a, 3-hydroxy-2-methylenenitriles 5a–7a, 4-hydroxy-2-methylenenitriles 8a–9a, 3-hydroxynitriles 10a–12a and 3-acyloxynitrile 13a into amides 1b–13b. Cross-linked enzyme aggregates (CLEAs) with nitrile hydratase and amidase activities (88% and 77% of the initial activities, respectively) were prepared from cell-free extract of this microorganism and used for nitrile hydration in presence of ammonium sulfate, which selectively inhibited amidase activity. The genes nha1 and nha2 coding for α and β subunits of nitrile hydratase were cloned and sequenced. [Copyright &y& Elsevier]

Abstract: Tyrosinase from mushroom was immobilized as a cross-linked enzyme aggregate (CLEA) via precipitation with ammonium sulfate and cross-linking with glutaraldehyde. The effects of precipitation and cross-linking on CLEA activity were investigated and the immobilized tyrosinase was characterized. Sixty percent ammonium sulfate saturation and 2% glutaraldehyde were used; a 3-h cross-linking reaction at room temperature, at pH 7.0 was performed; particle sizes of the aggregates were reduced; consequently, 100% activity recovery was achieved in CLEAs with enhanced thermal and storage stabilities. Slight changes in optimum pH and temperature values of the enzyme were recorded after immobilization. Although immobilization did not affect V max, substrate affinity of the enzyme increased. Highly stable CLEAs were also prepared from crude mushroom tyrosinase with 100% activity recovery. [Copyright &y& Elsevier]

Abstract: Laccase from the white rot fungus Coriolopsis polyzona was immobilized for the first time through the formation of cross-linked enzyme aggregates (CLEAs). Laccase CLEAs were produced by using 1000g of polyethylene glycol per liter of enzyme solution as precipitant and 200μM of glutaraldehyde as a cross-linking agent. These CLEAs had a laccase activity of 148Ug−1 and an activity recovery of 60.2% when using 2,2′-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) as substrate. CLEAs formed by co-aggregation with bovine serum albumin (BSA) as a stabilizer showed lower laccase activity and affinity for ABTS than those without BSA. The CLEAs co-aggregated with BSA showed higher residual activity against a protease, NaN3, EDTA, methanol and acetone. The thermoresistance was higher for CLEAs than for free laccase and also higher for CLEAs co-aggregated with BSA than for simple CLEAs when tested at a pH of 3 and a temperature of 40°C. Finally, laccase CLEAs were tested for their capacity to eliminate the known or suspected endocrine disrupting chemicals (EDCs) nonylphenol, bisphenol A and triclosan in a fluidized bed reactor. A 100-ml reactor with 0.5mg of laccase CLEAs operated continuously at a hydraulic retention time of 150min at room temperature and pH 5 could remove all three EDCs from a 5mgl−1 solution. [Copyright &y& Elsevier]

Abstract: Lipases from two different sources Candida rugosa (CRL) and Burkholderia cepacia (BCL) were formulated as enzyme precipitated and rinsed with organic solvents, organic solvent rinsed enzyme preparation, cross-linked enzyme aggregates (CLEAs) and protein coated micro-crystals (PCMCs). These various enzyme formulates were evaluated for the kinetic resolution of (±)-1-phenylethanol in ionic liquid [Bmim][PF6] by transesterification with vinyl acetate. Of all the enzyme forms evaluated EPRP and PCMC in the case of CRL showed the best results with 26 % (E value=153) and 53% (E value=79) conversion, respectively, at 35°C in 24h. Carrying out this conversion with PCMC at lower temperature of 25°C further improved the E value to 453 (with 44% conversion in 12h). For BCL the acetone-rinsed enzyme preparation (AREP), CLEA and PCMC performed equally well with % conversion of 50 and 99 eep (%) (E value >1000) in just 2h, whereas, the free lipase gave only 8% conversion. [Copyright &y& Elsevier]

Aims: Optimization of 6-aminopenicillanic acid (6-APA) production using cross-linked enzyme aggregates (CLEA) of Bacillus badius penicillin G acylase (PAC). Methods and Results: CLEA–PAC was prepared using purified/partially purified PAC with phenylacetic acid as active-site blocking agent and glutaraldehyde as cross-linker. Conversion of penicillin G to 6-APA by CLEA–PAC was optimized using response surface methodology (RSM) (central composite rotatable design) consisting of a three-factor–two-level pattern with 20 experimental runs. Conclusion: Nearly, 80% of immobilization yield was obtained when partially purified enzyme was used for the preparation of CLEA–PAC. Quantitative conversion of penicillin G to 6-APA was observed within 60 min and the CLEA–PAC was reusable for 20 repeated cycles with 100% retention of enzyme activity. Significance and Impact of the Study: The faster conversion of penicillin G to 6-APA by CLEA–PAC and efficient reusability holds a strong potential for the industrial application. [ABSTRACT FROM AUTHOR]

Abstract: Aspergillus oryzae β-galactosidase was immobilized by three different techniques, namely adsorption on celite, covalent coupling to chitosan and aggregation by cross-linking. These techniques were compared in terms of the yield of immobilized preparation, enzymatic characteristics, stability and efficiency in oligosaccharide synthesis. Immobilization led to increase in K m in each case. Immobilization on chitosan gave maximum enzyme yield and oligosaccharide synthesis. At 60°C, the chitosan-immobilized enzyme was stabilized (by 1.6-fold) due to protection effect of the matrix. However, at 65°C, the t 1/2 of cross-linked enzyme aggregates (CLEA) of β-galactosidase was 1.07h as compared to 0.79h in the case of free enzyme. Both chitosan-immobilized enzyme and CLEA were used for oligosaccharide synthesis. Using 20% (w/v) lactose, the chitosan-immobilized enzyme gave maximum oligosaccharide yield (17.3% of the total sugar) as compared to free enzyme (10.0%) in 2h at 40°C. CLEA were instead found effective in lactose hydrolysis yielding 78% monosaccharide in 12h. [Copyright &y& Elsevier]

Abstract: 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 8h and 50mg lipase to obtain 77% conversion, CLEA required only 6h and 6.25mg 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 8h at 45°C whereas CLEA with BSA retained full activity. CLEA prepared with BSA showed V max/K m of 36.3min−1 whereas CLEA prepared without BSA had V max/K m of 17.4min−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. [Copyright &y& Elsevier]

A new method for monitoring reactions catalyzed by an immobilized enzyme, cross-linked penicillin acylase aggregates (PA CLEA), is suggested. Appropriate chromogenic substrates for spectrophotometric assay of catalytic activity of immobilized enzyme were chosen and their kinetic parameters determined. Active sites in PA CLEA preparations were titrated by the suggested method; it is shown that almost all active sites are retained during immobilization. This method is characterized as highly expressive, simple, and precise, and may be used for control of PA immobilization efficiency as well as for study of operational, thermal, and pH stability immobilized enzyme preparations. [ABSTRACT FROM AUTHOR]

[Display omitted] • Magnetite cross-linked inulinase aggregates were prepared for inulin hydrolysis. • The immobilization process was optimized using D-optimal Design. • 18.1 g/L fructose from inulin was obtained at 60℃ in 4 h. • The immobilized magnetic inulinase was recycled for 25 cycles. The utilization of advanced nanoengineered technologies opens up attractive horizons towards sustainable treatment of waste biomass to produce fine chemicals. Production of fructose syrup (FS) in a single step using inulinase is a major achievements of enzyme technology as it eliminates the need for a multi-enzyme system. Functionalization of particles was done by coating chitosan on the surface of the magnetite and further, the chitosan coated magnetic nanoparticle (cMNP) was added to the mixture of cross-linked inulinase aggregates, and the physio-chemical properties of the aggregates were characterized. The immobilized biocatalyst was further utilized for the production of FS. Upon inulin hydrolysis for 4 h at 60 °C, 18.1 g/L of fructose was obtained with immobilized system. The immobilized inulinase was recycled up to 25 cycles and could be separated from the solution in 20 s by an external magnetic field. [ABSTRACT FROM AUTHOR]

α-Galacto-oligosaccharides (α-GOSs) have great functions as prebiotics and therapeutics. This work established the method of batch synthesis of α-GOSs by immobilized α-galactosidase for the first time, laying a foundation for industrial applications in the future. The α-galactosidase from Aspergillus niger L63 was immobilized as cross-linked enzyme aggregates (CLEAs) nano-biocatalyst through enzyme precipitating and cross-linking steps without using carriers. Among the tested agents, the ammonium sulfate showed high precipitation efficacy and induced regular structures of α-galactosidase CLEAs (Aga-CLEAs) that had been analyzed by scanning electron microscopy and Fourier-transform infrared spectroscopy. Through optimization by response surface methodology, the ammonium sulfate-induced Aga-CLEAs achieved a high activity recovery of around 90% at 0.55 U/mL of enzymes and 36.43 mM glutaraldehyde with cross-linking for 1.71 h. Aga-CLEAs showed increased thermal stability and organic solvent tolerance. The storage ability was also improved since it maintained 74.5% activity after storing at 4 °C for three months, significantly higher than that of the free enzyme (21.6%). Moreover, Aga-CLEAs exhibited excellent reusability in the α-GOSs synthesis from galactose, retaining above 66% of enzyme activity after 10 batch reactions, with product yields all above 30%. [ABSTRACT FROM AUTHOR]

Abstract: An optimally prepared Candida rugosa lipase aggregate cross-linked with bovine serum albumin, was found to overcome acetaldehyde deactivation during transacetylation of a series of benzyl alcohols with vinyl acetate. The formulation, under the same reaction conditions, exhibited 4–30× enhancement in the reaction rate as compared to the celite immobilized lyophilized formulation and 25–133× enhancement as compared to the free lyophilized enzyme depending upon the alcohol chosen. The racemic 1-phenylethanol, taken as one of the alcohols, underwent a more efficient enantioselective transacetylation giving 80% enantiomeric excess of the product, (R)-1-phenylethyl acetate, at 38% conversion (E = 15) within 24h while the enzyme immobilized on celite gave 83% enantiomeric excess at 18% conversion (E =13) during the same period of time. [Copyright &y& Elsevier]