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Phenolic contamination is one of the crucial concerns for the safety of drinking water. Enzymatic degradation is a green and efficient manner for phenolic compounds removal from water. However, enzymatic degradation of phenolic pollutants in water is limited as a result of the low activity, stability and reusability of the enzyme. Herein, we propose a novel strategy to degrade phenolic pollutants in water by using an enzyme‐metal hybrid catalyst constructed by in situ formation of ultrafine Cu nanoparticles on the cross‐linked Laccase aggregates. The designed Cu/Lac CLEAs showed excellent performance on phenolic pollutants removal due to the cooperative catalysis between Lac CLEA and Cu NPs and the enrichment of phenolic pollutants in hybrid catalyst. The degradation efficiency of 2,4,6‐trichlorophenol catalyzed by Cu/Lac CLEAs was improved by 33 % compared to the Lac CLEAs, while Cu NPs barely catalyzed the degradation process of phenolic pollutants. The Cu/Lac CLEAs hybrid catalyst exhibit high catalytic activity at room temperature in a wide pH range of 5–8, making the degradation of phenolic pollutants more practically operational. In other words, this study develops a novel hybrid catalyst for the efficient removal of pollutants from water. [ABSTRACT FROM AUTHOR]

Enzymatic degradation of biomass is preferred over chemical methods for environmental protection. However, in most cases, a chemical pretreatment is still required to help improve enzyme accessibility of the biomass. Therefore, it is necessary to develop highly efficient enzymes for those reasons and to save costs accordingly. Costs can be further reduced if the enzymes are recycled. XynR8(N58D) is an engineered and highly active xylanase originating from the rumen fungus Neocallimastix patriciarum. In this study, XynR8(N58D) was extracellularly over-expressed by Pichia pastoris. The resulting crude enzyme solution, with a purity of XynR8(N58D) of over 80%, was used to prepare the cross-linked enzyme aggregates (CLEA) of XynR8(N58D). Specific activity of the crude enzyme solution was 11,599.39 IU/mg, and that of CLEA was 6129.42 IU/mg. The immobilization yield and the immobilization efficiency were 62.68 ± 16.20% and 84.31 ± 3.11%, respectively. The thermostability, stress tolerance, and shelf life of CLEA were significantly better than those of the free enzyme. The free enzyme and CLEA both effectively decomposed insoluble xylan and autoclaved corncob powders. Therefore, XynR8(N58D) made into CLEA not only maintained high activity, but also improved its stability and permitted its reuse or recyclability. Both forms of the enzyme can effectively degrade lignocellulosic biomass without relying on chemical pretreatment to reduce recalcitrance of the biomass structure. The application of XynR8(N58D) and its CLEA to biomass degradation can help establish an eco-friendly and more cost-effective process. [ABSTRACT FROM AUTHOR]

Glioblastoma is one of the most lethal tumors, displaying striking cellular heterogeneity and drug resistance. The prognosis of patients suffering from glioblastoma after 5 years is only 5%. In the present work, capsaicin analogues bearing modifications on the acyl chain with long-chain fatty acids showed promising anti-tumoral activity by its cytotoxicity on U-87 and U-138 glioblastoma multiforme cells. The capsaicin analogues were enzymatically synthetized with cross-linked enzyme aggregates of lipase B from Candida antarctica (CALB). The catalytic performance of recombinant CALB-CLEAs was compared to their immobilized form on a hydrophobic support. After 72 h of reaction, the synthesis of capsaicin analogues from linoleic acid, docosahexaenoic acid, and punicic acid achieved a maximum conversion of 69.7, 8.3 and 30.3% with CALB-CLEAs, respectively. Similar values were obtained with commercial CALB, with conversion yields of 58.3, 24.2 and 22% for capsaicin analogues from linoleic acid, DHA and punicic acid, respectively. Olvanil and dohevanil had a significant cytotoxic effect on both U-87 and U-138 glioblastoma cells. Irrespective of the immobilization form, CALB is an efficient biocatalyst for the synthesis of anti-tumoral capsaicin derivatives. Key points: • This is the first report concerning the enzymatic synthesis of capsaicin analogues from docosahexaenoic acid and punicic acid with CALB-CLEAs. • The viability U-87 and U-138 glioblastoma cells was significantly affected after incubation with olvanil and dohevanil. • Capsaicin analogues from fatty acids obtained by CALB-CLEAs are promising candidates for therapeutic use as cytotoxic agents in glioblastoma cancer cells. [ABSTRACT FROM AUTHOR]

Glioblastoma is one of the most lethal tumors, displaying striking cellular heterogeneity and drug resistance. The prognosis of patients suffering from glioblastoma after 5 years is only 5%. In the present work, capsaicin analogues bearing modifications on the acyl chain with long-chain fatty acids showed promising anti-tumoral activity by its cytotoxicity on U-87 and U-138 glioblastoma multiforme cells. The capsaicin analogues were enzymatically synthetized with cross-linked enzyme aggregates of lipase B from Candida antarctica (CALB). The catalytic performance of recombinant CALB-CLEAs was compared to their immobilized form on a hydrophobic support. After 72 h of reaction, the synthesis of capsaicin analogues from linoleic acid, docosahexaenoic acid, and punicic acid achieved a maximum conversion of 69.7, 8.3 and 30.3% with CALB-CLEAs, respectively. Similar values were obtained with commercial CALB, with conversion yields of 58.3, 24.2 and 22% for capsaicin analogues from linoleic acid, DHA and punicic acid, respectively. Olvanil and dohevanil had a significant cytotoxic effect on both U-87 and U-138 glioblastoma cells. Irrespective of the immobilization form, CALB is an efficient biocatalyst for the synthesis of anti-tumoral capsaicin derivatives. Key points: • This is the first report concerning the enzymatic synthesis of capsaicin analogues from docosahexaenoic acid and punicic acid with CALB-CLEAs. • The viability U-87 and U-138 glioblastoma cells was significantly affected after incubation with olvanil and dohevanil. • Capsaicin analogues from fatty acids obtained by CALB-CLEAs are promising candidates for therapeutic use as cytotoxic agents in glioblastoma cancer cells. [ABSTRACT FROM AUTHOR]

Enzymes are commonly used as biocatalysts for various biological and chemical processes. However, some major drawbacks of free enzymes (e.g. poor reusability and instability) significantly restrict their industrial practices. How to overcome these weaknesses remain considerable challenges. Enzyme immobilization is one of the most effective ways to improve the reusability and stability of enzymes. Cross-linked enzyme aggregates (CLEAs) has been known as a novel and versatile carrier-free immobilization method. CLEAs is attractive due to its simplicity and robustness, without purification. It generally shows: high catalytic specificity and selectivity, good operational and storage stabilities, and good reusability. Moreover, co-immobilization of different kinds of enzymes can be acquired. These CLEAs advantages provide opportunities for further industrial applications. Herein, the preparation parameters of CLEAs were first summarized. Next, characterization of structural and catalytic properties, stability and reusability are also proposed. Finally, some important applications of this technique in: environmental protection, industrial chemistry, food industry, and pharmaceutical synthesis and delivery are introduced. Potential challenges and future research directions, such as improving cross-linking efficiency and internal mass transfer efficiency, are also presented. This implies that CLEAs provide an efficient and feasible technique to improve the properties of enzymes for use in the industry. [ABSTRACT FROM AUTHOR]

The invertase enzyme has been immobilized onto various support materials to enhance operational stability, shelf life, and reusability compared to free ones. Among the immobilization methods, carrier-free immobilization, such as cross-linked enzyme aggregates (CLEAs), attracts attention. However, recovery of the CLEAs from the reaction environment by centrifugation or filtration is a great challenge. The use of magnetic nanoparticles (Fe3O4) can overcome these separation limitations of the aggregates. For this aim, in this study, the surface of magnetite nanoparticles was functionalized with a significant number of free amino groups through silanization reaction. After precipitation of these aggregates and then cross-linking to the functionalized structure, the resulting magnetite cross-linked invertase aggregates (MCLIAs) were obtained, having different enzyme and glutaraldehyde concentrations. Magnetic nanoparticles were characterized by SEM, VSM, and FTIR analysis. The assay parameters, both constructional and experimental ones on the activity of MCLIAs, were practically determined by a personal glucose meter (PGM). The results indicate that magnetite nanoparticles with superparamagnetic behaviour were successfully functionalized with amino groups. The activity results demonstrated that experimental parameters were more effective than constructional parameters. MCLIAs also exhibited maximum activity at pH 5. Furthermore, 30-min incubation time and 35 °C were the best activity assay conditions. [ABSTRACT FROM AUTHOR]

Laccases are versatile oxidases that are capable of decolorizing various synthetic dyes. Recombinant Bacillus amyloliquefaciens laccase was immobilized as magnetic cross-linked enzyme aggregates (M-CLEAs) for application in dye decolorization. Several parameters influencing the activity recovery were evaluated during the synthesis of M-CLEAs. With ammonium sulfate as precipitant, maximum activity was recovered by cross-linking with 0.16% glutaraldehyde for 1 h. The prepared M-CLEAs exhibited improved activity under alkaline conditions. It remained 74% activity after incubation at 60 °C for 5 h. Enhanced tolerance towards NaCl was also observed for the M-CLEAs, with 68% activity remaining in the presence of 1 M NaCl. The immobilized laccase could rapidly decolorize more than 93% of reactive black 5 and indigo carmine in 1 h, while its catalytic efficiency towards reactive blue 19 was relatively low. After four cycles of consecutive reuse, the M-CLEAs could decolorize 92% of indigo carmine. The easy recovery and reusability of M-CLEAs facilitate the potential application of bacterial laccase in dye decolorization. [ABSTRACT FROM AUTHOR]

α-Galactosidases hold immense potential due to their biotechnological applications in various industrial and functional food sectors. In the present study, soluble and covalently cross-linked aggregated forms of a low molecular weight, thermo-labile α-galactosidase from Vigna mungo (VM-αGal) seeds were immobilized onto chitosan-coated magnetic nanoparticles for improved stability and repeated usage by magnetic separation. Parameters like precipitants (type, amount, and ratio), glutaraldehyde concentration, and enzyme load were optimized for the preparation of chitosan-coated magnetic nanocomposites of cross-linked VM-αGal (VM-αGal-MC) and VM-αGal (VM-αGal-M) resulted in 100% immobilization efficiency. Size and morphology of VM-αGal-M were studied through dynamic light scattering (DLS) and scanning electron microscopy (SEM), while Fourier transform infrared spectroscopy (FTIR) was used to study the chemical composition of VM-αGal-MC and VM-αGal-M. VM-αGal-MC and VM-αGal-M were found more active in a broad range of pH (3–8) and displayed optimal temperatures up to 25 °C higher than VM-αGal. Addition of non-ionic detergents (except Tween-40) improved VM-αGal-MC activity by up to 44% but negatively affected VM-αGal-M activity. Both VM-αGal-MC (15% residual activity after 21 min at 85 °C, Ed 92.42 kcal/mol) and VM-αGal-M (69.0% residual activity after 10 min at 75 °C, Ed 39.87 kcal/mol) showed remarkable thermal stability and repeatedly hydrolyzed the substrate for 10 cycles. [ABSTRACT FROM AUTHOR]

A new design of cross-linked enzyme aggregates (CLEAs) of Burkholderia cepacia lipase (BCL) based mainly on the use of lignocellulosic residue of palm fiber as an additive was proposed. Different parameters for the preparation of active CLEAs in the hydrolysis of olive oil, such as precipitation agents, crosslinking agent concentration, additives, and coating agents were investigated. The highest activity yield (121.1 ± 0.1%) and volumetric activity (1578.1 ± 2.5 U/mL) were achieved for CLEAs prepared using the combination of a coating step with Triton® X-100 and polyethyleneimine plus the use of palm fiber as an additive. The variations of the secondary structures of BCL-CLEAs were analyzed by second-derivative infrared spectra, mainly indicating a reduction of the α-helix structure, which was responsible for the lipase activation in the supramolecular structure of the CLEAs. Thus, these results provided evidence of an innovative design of BCL-CLEAs as a sustainable and biocompatible opportunity for biotechnology applications. [ABSTRACT FROM AUTHOR]

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With proper design, immobilization can be useful tool to improve the stability of enzymes, and in certain cases even their activity, selectivity, productivity and economic viability. An immobilized β-glucosidase (BGL, EC 3.2.1.21) through matrix adsorption and cross-linked enzyme aggregate (ad-CLEA) technology is presented in this work. After adsorption and precipitation, BGL was immobilized to poly(glycidyl methacrylate-co-ethylenedimethacrylate) (PGMA/EDMA) microparticles using glutaraldehyde as the cross-linker. Immobilized BGL exhibits lower apparent Km but much higher Vmax than that of the soluble enzyme, suggesting greater enzyme–substrate affinity and rapid velocity. Besides, ad-CLEA-BGL presents better thermostability retaining activity nearly 70% for 3 h and approximately 50% for 5 h at 70 °C, high operational reusability remaining more than 90% activity after nine uses and excellent storage stability holding about 95% activity after 45 days. Furthermore, the cellobiose is completely hydrolyzed within 1 h with ad-CLEA-BGL, which is significantly more efficient than soluble enzyme (about 4 h). Therefore, BGL was successfully immobilized on PGMA/EDMA microparticles with an ad-CLEA technology and the immobilization greatly enhances the biochemical characteristics. This work indicates promising application for ad-CLEA-BGL in utilizing agricultural remnants, bio-converting cellobiose to fermentable reducing sugar and ethanol production. [ABSTRACT FROM AUTHOR]

This study focuses on well-known but commonly overlooked or unreported factors in the preparation of cross-linked enzyme nano-aggregates (nano-CLEAs). The parameters including the ionic strength of the protein solution, protein, precipitant and cross-linker concentrations, pH and addition order of the reagents were fine-tuned for nanoaggregate preparation without the need of non-protein support material, special equipment or sophisticated procedures. For this purpose, precipitation as nano-aggregates and then cross-linking while maintaining submicron size distribution were studied independently. Moreover, nano-aggregate formation from reverse micellar solutions was also investigated to improve the scope of the method to membrane-bound enzymes. Five different precipitation agents together with three different cross-linkers were investigated for immobilization of the Trametes versicolor laccase as cross-linked nano-aggregates. Although complete activity recovery was possible for micro-aggregates, the best activity results for nano-aggregates were 40.5%±5.0. The Km values of the immobilized enzymes were slightly lower than the Km values of the free counterparts which indicate little or no mass transfer limitation due to the nano-immobilization process. However, Vmax values were also lower. The activity loss and Vmax reduction upon immobilization were found to mainly result from the modification of the amine groups instead of excess crosslinking. The thermal stabilities of the crosslinked laccase nano-aggregates were significantly higher (∼10–30 fold at 60 °C) compared to free laccase and the nano-CLEAs retained up to 30% of their initial activities upon 7 consequent usages. The sizes of the obtained immobilized enzyme products were found to be greatly variable depending on the cross-linker type. The smaller particles (∼200 nm radius) were obtained when EDAC was used as the cross-linker. The larger products (∼600 nm radius) were prepared when the cross-linker was dextran poly-aldehyde. The addition order of the reagents was found to be effective on particle size and thermal stability values. [ABSTRACT FROM AUTHOR]

BACKGROUND: Cross‐linked enzyme aggregates (CLEAs) of α‐galactosidase, partially purified from maize (Zea mays) flour, were prepared. The impact of various parameters on enzyme activity was examined to optimize the immobilization procedure. Biochemical characterization of the free and immobilized enzyme was carried out. Stability (thermal, pH, storage and operational stability) and reusability tests were performed. The potential use of the free enzyme and the CLEAs in hydrolysis processes of raffinose‐type oligosaccharides present in soymilk was investigated. RESULTS: α‐galactosidase CLEAs were prepared with 47% activity recovery under optimum conditions [1:5 (v/v) enzyme solution:saturated ammonium sulfate solution ratio; 7.5 mg protein and 0.1% (v/v) glutaraldehyde, 6 h, 4 °C, 150 rpm]. α‐galactosidase CLEAs exhibited increased stability in comparison to the free enzyme. The CLEAs and the free enzyme showed a maximum activity at 40°C and their optimal pH values were5.5 and 6.0, respectively. Kinetic constants (KM, Vmax and kcat) were calculated for the free enzyme and the CLEAs in the presence of p‐nitrophenyl‐α‐d‐galactopyranoside, stachyose, melibiose and raffinose. The effect of various chemicals and sugars on enzyme activity showed that both enzyme forms were significantly inhibited by HgCl2 and galactose. The CLEAs hydrolyzed 85% of raffinose and 96% of stachyose. CONCLUSION: The α‐galactosidase CLEAs, with their satisfactory enzymatic characteristics, have much potential for use in the food and feed industry. © 2019 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

The current industrial revolution signifies the high-value of protein engineering. The development of multipurpose biocatalysts is significantly expanding as a result of increased access and enzyme tailoring ability to satisfy the ever-increasing industrial demands. Enzyme-catalyzed processes offers multi-benefits at a time, e.g., low catalyst loading, high specificity, selectivity, mild processing for a complex and chemically unstable compounds, capability to reduce or eliminate reaction by-products, overall reusability and cost-effective ratio via immobilization, and potential to carry out conventional multi-stage processes via one-pot reaction. In this review, we critically elaborated recent achievements in applying new and/or state-of-the-art sophisticated protein engineering approaches to tailor the catalytic properties of enzymes or design enzymes with new and improved activities to catalyze desired biochemical transformations by orders of magnitude. We focused on different protein engineering approaches such as substrate engineering, medium engineering, and post-translational enzyme modification, structure-assisted protein tailoring, advanced computational modeling, and the exploration of inimitable synthetic scaffolds to develop multipurpose biocatalyst and improve the performance of multi-enzyme systems. In short, this study demonstrates an array of molecular biology insights and computational designs speeding up the tailored design of new and industrial biocatalysts. Continuous key developments in this direction together with protein engineering in unique ways might offer the ever-increasing opportunities for impending biocatalysis research for industrial bioprocesses. [ABSTRACT FROM AUTHOR]

In the present study, Candida antarctica lipase B was immobilized on amine‐functionalized silica microspheres as cross‐linked enzyme aggregates (CLEA) and utilized for the biomanufacturing of rhamnolipids (RL). Lipase CLEA synthesized under optimized conditions of 2.0:1.0 by volume of silica microsphere/enzyme concentration, a 1.0:2.5 (v/v) ratio of enzyme/2‐propanol, 7 mM glutaraldehyde concentration, when incubated at pH 9.0 and 40 °C, for a cross‐linking time of 30 min were observed to exhibit superior biocatalytic properties and a maximum enzyme load of 770 U g−1. Lipase CLEA exhibited enhanced pH stability in acidic and alkaline media and increased temperature resistance as compared to free lipase. Both free and CLEA lipases were used to synthesize RL in different solvent systems. After 12 h, from initiation of the esterification, the degree of esterification (molar conversion yield) reached 46% and 71% in the batch mode. 1H and 13C nuclear magnetic resonance (NMR) and high‐performance liquid chromatographic (HPLC) analysis confirm RL production by CLEA lipase. The CLEA showed greater confrontation to enzyme‐mediated bioprocess approach as compared to its soluble counterpart and exhibited excellent RL production and catalytic activity even after its tenth successive reuse. [ABSTRACT FROM AUTHOR]

BACKGROUND: High‐purity α‐, β‐, or γ‐cyclodextrin (CD) production using cyclodextrin glycosyltransferase (CGTase) as biocatalyst to cyclize starch is advantageous owing to its low cost of purification and product specificity. Several approaches have been investigated to enhance product specificity of CGTase, such as new CGTase isolation, CGTase gene (cgt) mutation, and chemical addition. RESULTS: Recombinant CGTase was cross‐linked by glutaraldehyde to obtain cross‐linked enzyme aggregates of recombinant CGTase (CLEA‐CGTase) at 85 °C because free CGTase showed high product specificity at 85 °C. The CLEA‐CGTase was prepared under the conditions 75 U mL−1 CGTase with 0.1% (v/v) glutaraldehyde for 10 min at 85 °C after optimization first, and produced a high proportion of β‐CD from soluble starch at 50 °C in the conversion system. The conversion process was carried out with 10 to 200 U mL−1 CLEA‐CGTase, resulting in 100% β‐CD from soluble starch after 420 min conversion of the potato starch solution at 50 °C. And the proportion of β‐CD was still higher than 90% when 8000 U mL−1 CLEA‐CGTase was added. CONCLUSIONS: CLEA technology maintained CGTase conformation of 85 °C, and produced a high proportion of β‐CD at 50 °C. This research showed the CLEA technology kept the conformation of enzyme for specific product production. © 2018 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

The present study focusses on the enhancement of the catalytic activity and stability of an acetylesterase enzyme isolated from Staphylococcus spp. as Cross-Linked Enzyme Aggregates (CLEAs). The various parameters governing the activity of CLEAs were optimized. The magnetite and graphene oxide nanoparticles were successfully prepared via the chemical co-precipitation and Hummer's method, respectively. These nanoparticles supported the preparation as magnetite nanoparticle-supported cross-Linked Enzyme Aggregates (MGNP-CLEAs) and graphene oxide-supported Cross-Linked Enzyme Aggregates (GO-CLEAs). The activity and stability of these immobilized CLEAs were compared with the free enzyme at various temperature, pH, and organic solvents along with its storage stability and reusability. The immobilized preparations were analyzed by Scanning Electron Microscopy (SEM) and Fourier Transform Infrared spectroscopy (FT-IR) techniques. Acetylesterase precipitated with 60% saturated ammonium sulfate salt (SAS) solution and cross-linked with 100 mM glutaraldehyde for 4 h at 30 °C was found to be optimal to produce CLEAs with highest activity recovery of 99.8%. The optimal pH at 8.0 and temperature at 30 °C remained the same for both the free and immobilized enzyme, respectively. Storage stability significantly improved for the immobilized enzyme as compared to free enzyme. SEM showed type-I aggregate and FT-IR revealed the successful immobilization of the enzyme. MGNP-CLEAs were found to have better activity and stability in comparison to other immobilized preparations. [ABSTRACT FROM AUTHOR]

Feruloyl esterases (FAEs, E.C. 3.1.1.73) are biotechnologically important enzymes with several applications in ferulic acid production from biomass, but also in synthesis of hydroxycinnamic acid derivatives. The use of such biocatalysts in commercial processes can become feasible by their immobilization, providing the advantages of isolation and recycling. In this work, eight feruloyl esterases, immobilized in cross-linked enzyme aggregates (CLEAs) were tested in regard to their transesterification performance, towards the production of prenyl ferulate (PFA) and arabinose ferulate (AFA). After solvent screening, comparison with the activity of respective soluble enzymes, and operational stability tests, FAE125 was selected as the most promising biocatalyst. A central composite design revealed the optimum conditions for each transesterification product, in terms of water content, time, and substrate ratio for both products, and temperature and enzyme load additionally for prenyl ferulate. The optimum product yields obtained were 83.7% for PFA and 58.1% for AFA. FAE125 CLEAs are stable in the optimum conditions of transesterification reactions, maintaining 70% residual activity after five consecutive reactions. Overall, FAE125 CLEAs seem to be able to perform as a robust biocatalyst, offering satisfactory yields and stability, and thus showing significant potential for industrial applications. [ABSTRACT FROM AUTHOR]

Lipases are the most widely employed enzymes in commercial industries. The catalytic mechanism of most lipases involves a step called "interfacial activation". As interfacial activation can lead to a significant increase in catalytic activity, it is of profound importance in developing lipase immobilization methods. To obtain a potential biocatalyst for industrial biodiesel production, an effective strategy for enhancement of catalytic activity and stability of immobilized lipase was developed. This was performed through the combination of interfacial activation with hybrid magnetic cross-linked lipase aggregates. This biocatalyst was investigated for the immobilization of lipase from Rhizomucor miehei (RML). Under the optimal conditions, the activity recovery of the surfactant-activated magnetic RML cross-linked enzyme aggregates (CLEAs) was as high as 2058%, with a 20-fold improvement over the free RML. Moreover, the immobilized RML showed excellent catalytic performance for the biodiesel reaction at a yield of 93%, and more importantly, could be easily separated from the reaction mixture by simple magnetic decantation, and retained more than 84% of its initial activities after five instances of reuse. This study provides a new and versatile approach for designing and fabricating immobilized lipase with high activation and stability. [ABSTRACT FROM AUTHOR]

The use of general and specific exopeptidases is of great interest for the hydrolysis of food proteins. Protein hydrolysates with a high degree of hydrolysis and, therefore, a reduced bitterness and improved antioxidative capacity can be produced due to the synergistic specificities of the general aminopeptidase PepN and the proline-specific peptidase PepX. These two activities were previously combined in a fusion protein and the latter showed both specific activities. However, due to its solubility an application of the fusion protein in continuous processes will be complicated in the future. Therefore, the aim of this study was the production, characterization and use of cross-linked enzyme aggregates (CLEAs) from the fusion protein (FUS-PepN_PepX CLEAs). The FUS-PepN_PepX CLEAs produced had activity for both specific enzymes. The biochemical characteristics determined (e.g., pH and temperature optima, environmental conditions) showed that the CLEAs are suitable for application in a complex matrix, such as food protein hydrolysates. The relative degree of hydrolysis of a prehydrolyzed casein solution was increased by 100% and the hydrolysate obtained showed a strong antioxidative capacity (ABTS-IC value: 7.85 µg mL). The stability against NaCl and the possibility of using ethanol as a microbial hurdle as well as the size of the FUS-PepN_PepX CLEAs seem promising for an application in an enzyme membrane reactor in the future. In summary, using these CLEAs, casein hydrolysates with a high degree of hydrolysis, a potentially reduced bitterness and high antioxidative capacity can be produced. [ABSTRACT FROM AUTHOR]

BACKGROUND Crosslinked enzyme aggregates ( CLEA) technology is a rapid and versatile method to produce immobilized enzymes via precipitation and cross-linking. A direct relationship between CLEA final activity and process parameters is however yet to be clarified. To address the issue, we have used a factorial design to test the formation and optimization of CLEA made from technical grade cellulase ( EC 3.2.1.4). Three types of precipitant (ammonium sulfate, polyethylene glycol, tert-butyl alcohol) were used at varied levels of cross-linker concentration, cross-linking time, and temperature. RESULTS It was found that the CLEAs produced with tert-butyl alcohol were inactive, whereas the polyethylene glycol- and ammonium sulfate- CLEA, recovered 29 and 17% of the free enzyme activity, respectively. Testing for re-usability, we observed that the polyethylene glycol- CLEA maintained 40% of the initial activity after four cycles, in contrast the ammonium sulfate- CLEA only maintained 10% of its activity after one cycle. CONCLUSION Our study highlights the importance of evaluating the effect of the precipitant on final CLEA activity rather than re-solubilized enzyme activity. It was demonstrated that polyethylene glycol, despite not being able to precipitate the enzymes as readily as ammonium sulfate, resulted in better performing CLEA. © 2016 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

In this study, manganese peroxidase (MnP) from an indigenous white-rot fungus Ganoderma lucidum IBL-05 was insolubilized in the form of cross-linked enzyme aggregates (CLEAs) using various aggregating agents, i.e., acetone, ammonium sulfate, ethanol, 2-propanol, and tert-butanol, followed by glutaraldehyde (GA) cross-linking. The precipitant type, MnP, and GA concentrations affected the CLEAs activity recovery and aggregation yield. Among precipitants used, acetone appeared to be the most efficient aggregation agent, providing the highest activity recovery and aggregation yield of 31.26 and 73.46%, respectively. Optimal cross-linking was noticed using 2.0% ( v/ v) GA and 8:1 ( v/ v) MnP to GA ratio at 3.0 h cross-linking time under continuous agitation at 4 °C. The highest recovered activity and aggregation yield were determined to be 47.57 and 81.26%, respectively. The MnP-CLEAs, thus synthesized, were tested to investigate their bio-catalytic capacity for removing two known endocrine-disrupting chemicals (EDCs), e.g., nonylphenol and triclosan in a packed bed reactor system. The insolubilized MnP efficiently catalyzed the biodegradation of both EDCs, transforming over 80% in the presence of MnP-based system. A maximal of 100% decolorization was recorded for Sitara textile (SIT-based) effluent, followed by 95.5% for Crescent textile (CRT-based) effluent, 88.0% for K&N textile (KIT-based) effluent, and 84.2% for Nishat textile (NIT-based) effluent. [ABSTRACT FROM AUTHOR]

The utilization of renewable biomass resources to produce high-value chemicals by enzymatic processes is beneficial for alternative energy production, due to the accelerating depletion of fossil fuels. As immobilization techniques can improve enzyme stability and reusability, a novel magnetic cross-linked cellulase aggregate has been developed and applied for biomass bioconversion. The cross-linked aggregates could purify and immobilize enzymes in a single operation, and could then be combined with magnetic nanoparticles (MNPs), which provides easy separation of the materials. The immobilized cellulase showed a better activity at a wider temperature range and pH values than that of the free cellulase. After six cycles of consecutive reuse, the immobilized cellulase performed successful magnetic separation and retained 74% of its initial activity when carboxylmethyl cellulose (CMC) was used as the model substrate. Furthermore, the structure and morphology of the immobilized cellulase were studied by Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). Moreover, the immobilized cellulase was shown to hydrolyze bamboo biomass with a yield of 21%, and was re-used in biomass conversion up to four cycles with 38% activity retention, which indicated that the immobilized enzyme has good potential for biomass applications. [ABSTRACT FROM AUTHOR]

Interfacial activation via surfactant (Tween 80, Triton X-100) treatment was conducted to improve the esterification activity of Rhizopus oryzae lipase that had undergone immobilization through cross-linked enzyme aggregates (CLEA®) technique. Surfactant pretreated immobilized enzymes exhibited better esterification activity compared to free and non-pretreated immobilized enzyme (Control CLEAs) since higher conversion rates were obtained within shorter times. The superiority of surfactant pretreated CLEAs, especially Tween 80 pretreated CLEAs (T 80 PT CLEAs), were clearly pronounced when longer alcohols were used as substrates. Conversion values exceeded 90% for octyl octanoate, oleyl octanoate and oleyl oleate synthesis with T 80 PT CLEAs whereas Control CLEAs and free enzyme showed no activity. Maximum conversions were achieved in the case equal molars of the substrates or in the case excess of the alcohol to acid in cyclohexane. In solvent free medium containing equal molars of substrates the conversion rates were 85% and 87% with T 80 PT CLEAs respectively for octyl octanoate and oleyl oleate within 2 hours. T 80 PT CLEAs showed 59% of its original activity after 7 consecutive usage for oleyl oleate synthesis. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:899-904, 2016 [ABSTRACT FROM AUTHOR]

Nanofibers prepared from cellulose derivatives are a suitable support for enzyme immobilization. We selected a methyl cellulose (MC) nanofibrous mat for enzyme immobilization. First, we established the electrospinning conditions for the preparation of the MC nanofibrous mat. A mixture of ethanol and water at a volume ratio of 1:1 was selected as the solvent of MC for electrospinning. An MC concentration of 5 wt% and a flow rate of 0.5 mL/h were employed to ensure stable electrospinning without bead formation. By varying the applied voltage, the sizes of the MC nanofibers could be controlled in the range of 50 to 80 nm, and an applied voltage of 18 kV was selected for further experimentation. The MC nanofibrous mats were cross-linked with glutaraldehyde under acidic conditions for 12 h in order to increase the stability of the mats in water. Lipase was directly immobilized onto cross-linked MC nanofibrous mats without a further activation step, and 34.82 µg of lipase was immobilized per mg of support. The reusability test showed an unexpected loss of activity after second reuse due to a loss of lipase during the washing procedure promoted by the surface erosion of the MC nanofiber. This problem was solved by introducing crosslinked enzyme aggregates (CLEA) to the surfaces of MC nanofibers. CLEA formation enhanced the activity of lipase per mg of support by almost 5-fold compared with the original lipase-immobilized MC nanofibrous mat. The reusability was also enhanced: more than 90% of the initial activity was retained after seven reuses. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Cross-linked enzyme aggregates from bovine pancreatic lipase were prepared by co-aggregation of lipase and BSA (Lipase-BSA-CLEAs). The main factors in the preparation of lipase-BSA-CLEAs were optimized. The highest activity recovery was around 75% under the condition of using 1% (v/v) glutaraldehyde as cross-linker and 0.05 g/L bovine serum albumin as feeder for 2 h cross linking. The optimum temperature for both lipase-CLEAs and lipase- BSA-CLEAs was measured as 60 °C, which is 10 °C higher than that of free lipase. Moreover, the lipase-BSA-CLEAs evidenced higher thermal stability and excellent reusability in comparison with the lipase-CLEAs. Lipase-BSA-CLEAs retained more than 75% of the initial activity after eight cycles of reuse, while lipase-CLEAs only retained 20% of its initial activity. Additionally, lipase-BSA-CLEAs showed more storage stability than free lipase and lipase-CLEAs. The high stability and recyclability of lipase-BSA-CLEAs make it efficient for different industrial applications. [ABSTRACT FROM AUTHOR]

BACKGROUND Cellulose as a renewable biomaterial has forced attention on the use of cellulose-hydrolyzing enzymes for industrial bioconversion of lignocellulosic materials to glucose. This paper reports immobilization of cross-linked cellulase aggregates ( CLEA) on the amine-functionalized Fe3O4@silica core-shell magnetic nanoparticles ( MNPs). RESULTS The optimum pH of the cellulase cocktail upon immobilization (cellulase CLEA-MNP) shifted a little to the acidic side whereas the optimum temperature did not change significantly. The behavior of CMCase activity in the cellulase CLEA-MNP at pH and temperature values higher than the optimum was significantly different compared with free cellulase. Cellulase CLEA-MNP retained about 45% of its maximum activity at pH values higher than 4.8, while free cellulase lost its activity sharply. Immobilized cellulase in contrast to the free form retained about 65% of its maximum activity at 80°C. Cellulase CLEA-MNP had improved thermal stability at 65°C. Operational stability of the immobilized cellulase was also noticeable. After a sharp decrease during two cycles of CMC hydrolysis, cellulase CLEA-MNP retained 30% of its initial activity through six cycles of reuse. CONCLUSION Simple separation of MNPs from reaction medium and durability of CLEA-MNP composite during repeated use may overcome the major bottleneck against comprehensive applications of cellulase enzymes in industry. © 2014 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Cross-linked enzyme aggregates (CLEAs) were prepared from several precipitant agents using glutaraldehyde as a cross-linking agent with and without BSA, finally choosing a 40% saturation of ammonium sulfate and 25 mM of glutaraldehyde. The CLEAs obtained under optimum conditions were biochemically characterized. The immobilized enzyme showed higher thermal activity and a broader range of pH and organic solvent tolerance than the free enzyme. Arylesterase from Gluconobacter oxydans showed activity toward cephalosporin C and 7-aminocephalosporanic acid. The CLEAs had a Kcat/KM of 0.9 M−1/S−1 for 7-ACA (7-aminocephalosporanic acid) and 0.1 M−1/S−1 for CPC (cephalosporin c), whereas free enzyme did not show a typical Michaelis-Menten kinetics. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:36-42, 2016 [ABSTRACT FROM AUTHOR]

Copyright of Journal of Hebei University of Science & Technology is the property of Hebei University of Science & Technology, Journal of Hebei University of Science & Technology and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

BACKGROUND Gamma(γ)-aminobutyric acid ( GABA) is produced through an α-decarboxylation reaction of L-monosodium glutamate ( MSG) using glutamate decarboxylase ( GAD). The pH rise caused by the reaction inactivates the enzyme catalyst, which is active only under acidic conditions, and consequently leads to low reaction conversions. Employment of conventional acids and buffers inevitably forms salts, which result in serious problems in separation and purification of GABA. It is essential to render GAD active even at neutral and alkaline pHs. In the present study, we first apply a cross-linked aggregation method in order to extend the active range of GAD toward alkaline pH. RESULTS GAD from Escherichia coli was prepared as cross-linked enzyme aggregate ( CLEA) in which the enzyme was precipitated using ammonium sulfate (60% saturation) and then cross-linked with glutaraldehyde (2%) in sodium acetate buffer (0.2 mol L−1, pH 4.6). The cross-linked aggregation extended an active pH range of GAD from 5.5 up to 8.0; as a result, the reaction conversion of 1 mol L−1 MSG into GABA was improved from 13% to 22%. Moreover, the CLEA of GAD was easily recovered after the reaction and reused retaining >95% of its initial activity during the first four cycles and >60% activity at the 10th cycle. CONCLUSION Cross-linked aggregation could make GAD active even at neutral and alkaline pHs. It is shown to be a useful method capable of facilitating recovery and reuse of the enzyme as well as increasing the reaction conversion by extending the active pH range of GAD. © 2015 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Copyright of Journal of Hebei University of Science & Technology is the property of Hebei University of Science & Technology, Journal of Hebei University of Science & Technology and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

The amidase of Rhodococcus rhodochrous 4-1 was immobilized by covalent attachment to activated chitosan by physical sorption on carbon adsorbents and by the formation of cross-linked aggregates in the absence of carrier. Comparative analysis of particular catalytic properties of the free and chitosan-immobilized amidase was performed. It was shown that the enzyme retained 50-60% of its initial activity after covalent immobilization on chitosan and was characterized by increased temperature stability as compared to soluble amidase. Moreover, the immobilized enzyme retained more than 20% of its activity after five 24-h cycles of acrylamide transformation. The effects of different types of immobilization on amidase stereoselective properties were studied by the model reaction of racemic lactamide hydrolysis to D-lactic acid and L-lactic acid. It was shown that cross-linked amidase aggregates possessed high D-stereoselectivity (up to 77-94%). The immobilized enzyme showed the highest enantioselectivity at 60°C. [ABSTRACT FROM AUTHOR]

Over the past decade, disaccharide phosphorylases have been successfully applied for the synthesis of numerous α-glucosides. In contrast, much less research has been done with respect to the production of β-glucosides. Although cellobiose phosphorylase was already successfully used for the synthesis of various disaccharides and branched trisaccharides, its glycosylation potential towards small organic compounds has not been explored to date. Unfortunately, disaccharide phosphorylases typically have a very low affinity for non-carbohydrate acceptors, which urges the addition of solvents. The ionic liquid AMMOENGTM 101 and ethyl acetate were identified as the most promising solvents, allowing the synthesis of various β-glucosides. Next to hexyl, heptyl, octyl, nonyl, decyl and undecyl β- D-glucopyranosides, also the formation of vanillyl 4- O-β- D-glucopyranoside, 2-phenylethyl β- D-glucopyranoside, β-citronellyl β- D-glucopyranoside and 1- O-β- D-glucopyranosyl hydroquinone was confirmed by nuclear magnetic resonance spectroscopy and mass spectrometry. Moreover, the stability of cellobiose phosphorylase could be drastically improved by creating cross-linked enzyme aggregates, while the efficiency of the biocatalyst for the synthesis of octyl β- D-glucopyranoside was doubled by imprinting with octanol. The usefulness of the latter system was illustrated by performing three consecutive batch conversions with octanol imprinted cross-linked enzyme aggregates, yielding roughly 2 g of octyl β- D-glucopyranoside. [ABSTRACT FROM AUTHOR]

Pancreatic trypsin was immobilized by cross-linked enzyme aggregates (CLEA) which is a carrier free immobilization method. Ammonium sulfate was chosen for enzyme precipitation which was followed by cross linking of formed aggregates via glutaraldehyde. Concentrations of precipitant and cross linker were respectively optimized as 60% ammonium sulfate and 1% glutaraldehyde. Optimum pH and temperature for CLEA was increased compared to free enzyme. Furthermore, pH, thermal and storage stability were improved. Presence of additives had no effects on enzyme activity. Prepared cross-linked trypsin aggregates are convenient for in situ protein fragmentation and can be used for protein identification. [ABSTRACT FROM AUTHOR]

Cross-linked enzyme aggregate (CLEA) technology has been regarded as an effective carrier-free immobilization method. This method is very attractive due to its simplicity and robustness, as well as for the possibility of using the crude enzyme extract and the opportunity to co-immobilize multiple different enzymes. The resulting CLEAs generally exhibit high catalyst productivities, improved storage and operational stability and are easy to recycle. Nowadays, although the technology has been applied to various enzymes, some undesirable properties have limited its further application. To overcome these limitations, novel strategies have been developing in recent years. This mini-review focuses on process optimization, new improved strategies and the latest advances on CLEAs technology. [ABSTRACT FROM AUTHOR]

Novel magnetic cross-linked lipase aggregates were fabricated by immobilizing the cross-linked lipase aggregates onto magnetic particles with a high number of -NH2 terminal groups using p-benzoquinone as the cross-linking agent. At the optimal fabrication conditions, 100% of immobilization efficiency and 139% of activity recovery of the magnetic cross-linked lipase aggregates were achieved. The magnetic cross-linked lipase aggregates were able to efficiently resolve ( R, S)-2-octanol, and retained 100% activity and 100% after 10 cycles of reuse, whereas the cross-linked lipase aggregates only retained about 50% activity and 70% due to insufficient cross-linking. These results provide a great potential for industrial applications of the magnetic cross-linked lipase aggregates. Chirality 27:199-204, 2015. © 2014 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Hydroxynitrile lyases are powerful catalysts in the synthesis of enantiopure cyanohydrins which are key synthons in the preparations of a variety of important chemicals. The response surface methodology including three-factor and three-level Box-Behnken design was applied to optimize immobilization of hydroxynitrile lyase purified partially from Prunus dulcis seeds as crosslinked enzyme aggregates (PdHNL-CLEAs). The quadratic model was developed for predicting the response and its adequacy was validated with the analysis of variance test. The optimized immobilization parameters were initial glutaraldehyde concentration, ammonium sulfate saturation concentration, and crosslinking time, and the response was relative activity of PdHNL-CLEA. The optimal conditions were determined as initial glutaraldehyde concentration of 25% w/v, ammonium sulfate saturation concentration of 43% w/v, and crosslinking time of 18 h. The preparations of PdHNL-CLEA were examined for the synthesis of (R)-mandelonitrile, (R)-2-chloromandelonitrile, (R)-3,4-dihydroxymandelonitrile, (R)-2-hydroxy-4-phenyl butyronitrile, (R)-4-bromomandelonitrile, (R)-4-fluoromandelonitrile, and (R)-4-nitromandelonitrile from their corresponding aldehydes and hydrocyanic acid. After 96-h reaction time, the yield-enantiomeric excess values (%) were 100−99, 100−21, 100−99, 83−91, 100−99, 100−72, and 100−14%, respectively, for (R)-mandelonitrile, (R)-2-chloromandelonitrile, (R)-3,4-dihydroxymandelonitrile, (R)-2-hydroxy-4-phenyl butyronitrile, (R)-4-bromomandelonitrile, (R)-4-fluoromandelonitrile, and (R)-4-nitromandelonitrile. The results show that PdHNL-CLEA offers a promising potential for the preparation of enantiopure (R)-mandelonitrile, (R)-3,4-dihydroxymandelonitrile, (R)-2-hydroxy-4-phenyl butyronitrile, and (R)-4-bromomandelonitrile with a high yield and enantiopurity. © 2014 American Institute of Chemical Engineers Biotechnol. Prog, 30:818-827, 2014 [ABSTRACT FROM AUTHOR]

The para-nitrobenzyl esterase (PNBE) was successfully immobilized onto electrospun polystyrene/poly(styrene-co-maleic anhydride) (PS/PSMA) nanofibers as cross-linked enzyme aggregates (CLEA). The maximum amount of PNBE immobilized on the PSPSMA nanofibers was determined to be 56.4 mg/g of nanofibers. The variation of pH and temperature has been observed to affect the enzymatic activity of the free and immobilized PNBE-CLEA. The immobilized PNBE-CLEA has been observed to show improved stability as compared to the native one. PNBE-CLEA has been found to retain more than 63% activity after 60 consecutive reuses. Finally, the immobilized PNBECLEA presented a high decomposition efficiency of cephalexin tosylate PNB ester. The durability determination of PNBE-CLEA immobilized nanofibers showed 70% of de-esterification after 10 cycles. These results demonstrate that PNBE-CLEA immobilized nanofiber could be used as an economical and environment friendly system for the catalytic process of paranitrobenzyl esters in pharmaceutical synthesis. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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]

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]

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]

Copyright of Turkish Journal of Biochemistry / Turk Biyokimya Dergisi is the property of De Gruyter and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

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.

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]

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]

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]