Formation Of Cross-linked Enzyme Aggregates Research Articles

Enzymatic hydrolysates of κ-carrageenan by κ-carrageenase-CLEA immobilized on amine-modified ZIF-8 confer hypolipidemic activity in HepG2 cells

κ-Carrageenase can degrade κ-carrageenan to produce bioactive κ-carrageenan oligosaccharides (KCOs) that have potential applications in pharmaceutical, food, agricultural, and cosmetics industries. Immobilized enzymes gain their popularity due to their good reusability, enhanced stability, and tunability. In this study, the previously characterized catalytic domain of Pseudoalteromonas purpurea κ-carrageenase was covalently immobilized on the synthesized amine-modified zeolitic imidazolate framework-8 nanoparticles with the formation of cross-linked enzyme aggregates, and the immobilized κ-carrageenase was further characterized. The immobilized κ-carrageenase demonstrated excellent pH stability and good reusability, and exhibited higher optimal reaction temperature, better thermostability, and extended storage stability compared with the free enzyme. The KCOs produced by the immobilized κ-carrageenase could significantly decrease the TC, TG, and LDL-C levels in HepG2 cells, increase the HDL-C level in HepG2 cells, and reduce the free fatty acids level in Caco-2 cells. Biochemical assays showed that the KCOs could activate AMPK activity, increase the ratios of p-AMPK/AMPK and p-ACC/ACC, and downregulate the expression of the lipid metabolism related proteins including SREBP1 and HMGCR in the hyperlipidemic HepG2 cells. This study provides a novel and effective method for immobilization of κ-carrageenase, and the KCOs produced by the immobilized enzyme could be a potential therapeutic agent to prevent hyperlipidemia.

Fermentation, immobilization, and application of de-acylase for biosynthesis of anti-fungal intermediates

The enzymatic synthesis of antifungal intermediates is receiving huge attention due to global demand. A de-acylase (EC 3.5.1.98) was produced by recombinant Streptomyces lividans using minimal salt media under optimized conditions. The recombinant S. lividans was subjected to fermentation for 90 h followed by enzyme isolation by KCl extraction method. Further, it was concentrated by tangential flow filter (TFF). Produced de-acylase was immobilized in the form of cross-linked enzyme aggregates (CLEAs) to prepare a robust formulation for further applications. In order to make de-acylase CLEAs, de-acylase was precipitated by ammonium sulphate (100% saturation) and cross-linked by glutaraldehyde (0.5% v/v). The immobilized CLEAs were lyophilized, and was utilized for synthesizing micafungin precursor-2 (FR179642) from the micafungin precursor-1 (FR901379). It was found that the de-acylase CLEAs exhibited more than 96% molar yield which were more efficient than free enzyme (showed nearly 65% molar yield) after 12 h of reaction. In the end, the prepared CLEAs were effectively recycled for five cycles with the retention of 55% residual activity and stored for 24 days without observable loss of activity.

An improved process for the preparation of ethyl-(R)-2-hydroxy-4-phenylbutyrate, (R)-HPB ester by lipase from Thermomyces lanuginosus

Kinetic resolution of rac-ethyl-2-hydroxy-4-phenylbutyrate was carried out using lipase from Thermomyces lanuginosus immobilized in form of cross-linked enzyme aggregates (CLEAs). The reaction was performed in phosphate buffer (50 mM, pH 7.5) at 27 °C and 200 rpm with 10 % w/w substrate and catalyst loading in a moving basket reactor. The immobilized enzyme was recycled up to 5 times without any loss of activity, followed by marginal loss of activity (6 %) up to 10 cycles. The required pure ethyl-(R)-2-hydroxy-4-phenylbutyrate was isolated with 48 % yield and > 99 % e.e. The estimated kinetic parameters were Vmax,app = 1623 ± 81.15 mM/h/g and Km,app = 0.13 ± 0.07 M (correlation coeff. r = 0.94).

Experimental Design Optimization and Decolorization of an Azo Dye by Cross-Linked Peroxidase Aggregates

The immobilization of horseradish peroxidase in the form of cross-linked enzyme aggregates (HRP–CLEAs) was optimized by three parameters full factorial experimental design at two levels and the degradation of an azo dye was tested. The optimal immobilization conditions were estimated as 0.06 mg enzyme/mL (0.96 U), 3 % (v/v, Glutaraldehyde)) cross-linker ratio, and 6 mg /mL Bovine Serume Albumine amount, respectively. The effects of variables were analysed by responce surface plots and the enzyme concentration effect was insignificant for the tested levels. The maximum CLEAs activity was 0.2188 U and the kinetic parameters (Km and Vmax) were 0.0314 mM and 0.1044 mM/min while the calculated values for soluble enzyme were 0.06 mM and 0.468 mM/min, respectively. Hydrogen peroxide has inhibitory effect on enzyme aggregate activities at higher amounts than 0.012 mM. Under optimal conditions, a textile azo dye, Reactive Blue 160 was completely oxidized within 90 h and higher temperatures also showed promising results in dye degradation.

Development and characterization of cross-linked laccase aggregates (Lac-CLEAs) from Trametes versicolor IBL-04 as ecofriendly biocatalyst for degradation of dye-based environmental pollutants

Extracellular laccase from Trametes versicolor IBL-04 was insolubilized in the form of cross-linked enzyme aggregates (CLEAs) for degradation of different recalcitrant dyes. Among various precipitants, polyethylene glycol (PEG) ensured the best activity recovery (72.85%) of laccase-immobilized cross-linked enzyme aggregates (Lac-CLEAs) following ammonium sulfate (63.24%), n-propanol (46.98%), tert-butanol (22.94%), and acetone (14.5%). Lac-CLEAs were catalytically more active and stable over a broader pH range as compared to the free counterpart. Free enzyme lost 70% of its relative activity at 75 °C, whereas the immobilized derivatives, i.e. propanol-Lac-CLEAs, AS-Lac-CLEAs, and PEG-Lac-CLEAs retained 62.69%, 68.48%, and 86.29% of their relative activities, respectively, at this temperature. Though Lac-CLEAs exhibited a reduced affinity towards its substrate, Vmax values were markedly increased compared to the free enzyme. Lac-CLEAs catalyzed 94.57%, 90.63%, and 78.72% degradation of Remazol Brilliant Blue R, crystal violet, and Reactive Black 5, respectively. A dramatic decline in the peaks after treatment confirms the Lac-CLEAs potential to remove dyes. Interestingly, the immobilized system retained more than 60% of degradation efficiency even after eight successive uses. In conclusion, the present findings suggest the potential of Lac-CLEAs to mitigate a wide range of dyes and emerging contaminants.

Immobilized laccase in the form of (magnetic) cross-linked enzyme aggregates for sustainable diclofenac (bio)degradation

Nowadays, major problems worldwide derive from the presence of pharmaceutically active compounds in the wastewater of hospitals and healthcare facilities. To address this issue, biological transformation of diclofenac using native and immobilized laccase is described in our study. Laccase stabilization is demonstrated, using two different types of immobilization: cross-linked enzyme aggregates (CLEAs) and magnetic cross-linked enzyme aggregates (mCLEAs). High diclofenac (DCF) removal capacity of CLEAs laccase and mCLEAs laccase in comparison to native laccase is determined. Under optimal conditions, the removal capacity of native laccase is 11.5 ± 0.3 μg DCF/glaccase, of CLEAs 15.6 ± 0.4 μg DCF/glaccase and of mCLEAs 13.6 ± 0.4 μg DCF/glaccase. Our immobilized laccase also possesses very good reusability, since the half-life of the immobilized laccase in the form of CLEAs is reached after the second cycle of biocatalyst reuse, while the half-life of the mCLEAs laccase can be detected at the fourth cycle. The improved stability of immobilized laccase in supercritical carbon dioxide (SC CO2) is shown. The promising results of DCF removal using CLEAs laccase and mCLEAs laccase show that both immobilized forms of laccase have the potential to be used in cleaner industrial technologies, e.g. wastewater treatment.

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

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

Immobilized Laccase for Sustainable Technological Processes

Enzyme-catalyzed processes are currently a powerful tool in the chemical industry due to the reduction of process time, specificity, eco-friendly characteristics, intake of low energy input, lower cost and nontoxic properties. Laccases are versatile enzymes, which catalyze oxidation reactions coupled to four-electron reduction of molecular oxygen to water. Laccases are used for decolorization and detoxification of industrial effluents and are widely used in wastewater treatment. For improving enzyme utilization in biotechnological processes, the process cost has to be reduced, the enzyme stability during industrial processes should be enhanced and the recycle and reuse step should be favorable. The immobilization of the enzyme is an important step for enhancing enzyme catalytic properties and operational stability. In our study, two different types of immobilization were used for laccase: cross-linked enzyme aggregates (CLEA) and magnetic cross-linked enzyme aggregates (mCLEA). The enzyme activity of synthesized CLEA was 0.237 U/mg laccase and mCLEA 0.637 U/mg laccase, respectively. In the next step, immobilized laccase in the form of CLEA and mCLEA was treated under supercritical conditions. Supercritical fluids (SCFs) comprise a unique class of nonaqueous media for biocatalysis and bioseparation. It is very difficult (almost impossible) to predict the stability and activity of an enzyme in any SCF at a certain pressure. Therefore, the stability of both laccase immobilized forms in supercritical carbon dioxide (SC CO2) at different pressures was studied. The highest residual activity for both laccase immobilized forms were obtained after exposure to SC CO2 at 20 MPa, 35 °C for 5 h. The obtained residual activities were 145 ± 3 % for mCLEA and 128 ± 3 % for CLEA. Improved stability of immobilized laccase in the form of CLEA and mCLEA after SC CO2 treatment indicates the possibility of using laccase in supercritical media. Further application of CLEA laccase and mCLEA laccase could be reflected in the field of hospital wastewater treatment for removal of drugs.

Combined Cross-Linked Enzyme Aggregates of Monoamine Oxidase and Putrescine Oxidase as a Bifunctional Biocatalyst for Determination of Biogenic Amines in Foods

In order to determine total biogenic amines in fermented foods, the combined cross-linked enzyme aggregates of a monoamine oxidase and a putrescine oxidase (combi-CLEAs) and the cross-linked enzyme aggregates (CLEAs) of the fused enzyme of two amine oxidases (MonoAmine Putrescien Oxidase, MAPO) were prepared. The effects of various parameters were examined to optimize the CLEAs formation. Biochemical characterization and stability of free and the CLEAs enzymes were performed. Through optimization of the CLEAs formation condition, the combi-CLEAs and the CLEAs-MAPO were prepared with 82% and 78% of residual activities relative to the activities of the subjected enzymes were in a preparative scale. The optimal pH for tyramine-activities of the CLEAs enzymes were shifted to relatively basic pH, leading to synchronization of the optimal performances of combi-CLEAs over pH for tyramine and putrescine. In addition, thermostability of the CLEAs enzymes were improved with almost double half-lives at 65 °C in comparison to the free enzymes. The catalytic efficiencies of combi-CLEAs for tyramine, histamine and putrescine were reduced by 41%, 56%, and 31%, respectively, and the inhibition potency by the substrate was reduced by two-fold in comparison of the mixed free enzymes. In conclusion, combi-CLEAs are a promising catalyst with the improved stability and the same optimum pH for dual activities in enzymatic determination of biogenic amines in foods.

Preparation of Magnetic Cross-Linked Amyloglucosidase Aggregates: Solving Some Activity Problems

The preparation of Cross-Linked Enzyme Aggregates (CLEAs) is a simple and cost-effective technique capable of generating insoluble biocatalysts with high volumetric activity and improved stability. The standard CLEA preparation consists of the aggregation of the enzyme and its further crosslinking, usually with glutaraldehyde. However, some enzymes have too low a content of surface lysine groups to permit effective crosslinking with glutaraldehyde, requiring co-aggregation with feeders rich in amino groups to aid the formation of CLEAs. The co-aggregation with magnetic particles makes their handling easier. In this work, CLEAs of a commercial amyloglucosidase (AMG) produced by Aspergillus niger were prepared by co-aggregation in the presence of polyethyleneimine (PEI) or starch with aminated magnetic nanoparticles (MNPs) or bovine serum albumin (BSA). First, CLEAs were prepared only with MNPs at different glutaraldehyde concentrations, yielding a recovered activity of around 20%. The addition of starch during the precipitation and crosslinking steps nearly doubled the recovered activity. Similar recovered activity (around 40%) was achieved when changing starch by PEI. Moreover, under the same conditions, AMG co-aggregated with BSA was also synthesized, yielding CLEAs with very similar recovered activity. Both CLEAs (co-aggregated with MNPs or BSA) were four times more stable than the soluble enzyme. These CLEAs were evaluated in the hydrolysis of starch at typical industrial conditions, achieving more than 95% starch-to-glucose conversion, measured as Dextrose Equivalent (DE). Moreover, both CLEAS could be reused for five cycles, maintaining a DE of around 90%. Although both CLEAs had good properties, magnetic CLEAs could be more attractive for industrial purposes because of their easy separation by an external magnetic field, avoiding the formation of clusters during the filtration or centrifugation recovery methods usually used.

Preparation and Characterization of Sugar-Assisted Cross-Linked Enzyme Aggregates (CLEAs) of Recombinant Cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus ( CsCE).

High-efficiency lactulose-producing enzyme of Caldicellulosiruptor saccharolyticus cellobiose 2-epimerase (WT- CsCE) was immobilized in the form of cross-linked enzyme aggregates (CLEAs). Conditions for enzyme aggregation and cross-linking were optimized, and a sugar-assisted strategy with less damage to enzyme secondary structures was developed to improve the activity yield of CLEAs up to approximately 65%. The resulting CLEAs with multiple-layer network structures exhibited an enlarged optimal temperature range (70-80 °C) and maintained higher activity at 50-90 °C. Besides, CLEAs retained more than 95% of their initial activity after 10 successive batches at 60 °C, demonstrating superior reusability. Moreover, CLEAs displayed an equivalent or higher catalytic ability to free WT- CsCE in lactulose biosynthesis, and the final sugar ratios were similar, lactulose 58.8-61.7%, epilactose 9.3-10.2%, and lactose 27.8-30%, with a constant isomerization selectivity of 0.84-0.87 regardless of enzymes used and temperature applied. The proposed strategy is the first trial for enzymatic synthesis of lactulose catalyzed by CLEAs of WT- CsCE.

Protease-based cross-linked enzyme aggregates with improved catalytic stability, silver removal, and dehairing potentials

Proteases have gained special research place due to their broader activity spectrum and applied perspectives for different industrial sectors. The present research focused on three aims, i.e., (1) to identify the best protease producer strain among three different Aspergillus strains, (2) the development of protease-based cross-linked enzyme aggregates (CLEAs) and (3) silver removal and dehairing potentialities of developed CLEAs. A. flavus gave optimum activity (98.50 U/mL) with the culture conditions (pH -7.5, 35 °C, inoculum 2.5 mL and fermentation time 48 h) by applying RSM under CCD. The protease-CLEAs were developed with recovery activity (37.45%) by optimizing conditions through RSM under CCD (80% ammonium sulfate, 65 mM glutaraldehyde, and 0.15 mM BSA). The adequacy of the model was checked by ANOVA, and the interactions among different variables were plotted using 3-D graphs. The characterization profile revealed high pH and thermal stability at pH -9 and 60 °C, respectively. The kinetic study revealed lower KM and higher Vmax values (31.02 μM and 91.16 U/mL, respectively) after CLEAs formation, as compared to the free protease (61.42 μM and 84.45 U/mL, respectively). By applying on X-ray film and animal hides, protease-CLEAs showed the best activity with minimum time as compared to free protease.

Precipitation-Based Nanoscale Enzyme Reactor with Improved Loading, Stability, and Mass Transfer for Enzymatic CO2 Conversion and Utilization

Enzymatic CO2 conversion has gathered a growing attention due to its fast kinetics in converting CO2 to bicarbonate, but the carbonic anhydrase enzymes easily lose their activities in CO2 conversion processes. Here, we propose a “precipitation-based nanoscale enzyme reactor (p-NER)” approach, which stabilizes the activity of carbonic anhydrase, prepared via the two steps of enzyme adsorption into magnetic mesoporous silica and simultaneous enzyme precipitation/cross-linking. The simple addition of enzyme precipitation during cross-linking step resulted in the formation of cross-linked enzyme aggregates (CLEAs) not only inside the mesopores but also on the surface of mesoporous silica. External CLEAs of p-NER contributed to the improvement of enzyme loading (32.9% (w/w)) and mass transfer (KM = 3.68 mM) compared to those of NER (20.1% (w/w) and 4.29 mM, respectively), prepared without enzyme precipitation step and showing no external CLEAs. p-NER was stable under vigorous shaking (200 rpm) with no activity...

Towards an affordable enzymatic production of biopolyols – Comparing the immobilization of lipases by two optimized techniques

The production of biopolyols (sustainable, renewable and biodegradable precursors), is exclusively focused on the conventional chemical synthesis pathways, and currently, no polyols are produced by enzymatic processes at an industrial scale, because they are not competitive when compared to petrochemically based polyols due to the overall cost for their production. This can be mainly attributed to the high costs of commercial enzymes used in this process and their use is limited by their lack of stability during the bioprocess. Immobilization of enzymes gives the opportunity to converge two important features of enzymes: to increase the protein stability and to reuse enzyme as a catalyst. By this study, we were aiming to explore alternatives to commercially available enzymes. We investigated an effective hydrolytic route from epoxidized oils to polyols by applying lipolytic enzymes from Yarrowia lipolytica and Candida cylindracea immobilized onto fumed silica nanoparticles (fsNP) and those immobilized by the formation of cross-linked enzyme aggregates (CLEAs). Oxirane ring hydrolysis of epoxidized rapeseed oil catalyzed by lipase-fsNP and CLEAs was investigated with respect to various parameters (temperature, pH, concentrations, time of sorption and cross-linking, etc.). The highest conversions for oxirane ring hydrolysis were estimated for CLEA-Lip-derivatives (45–56%).

Development and characterization of cross-linked enzyme aggregates of thermotolerant alkaline protease from Bacillus licheniformis

An alkaline protease was produced by B. licheniformis with 132.43±3.4U/mL activity in LSF which was further enhanced by optimizing culture conditions. The optimum enzyme activity (148.9±4.1U/mL) was harvested at pH7.5; temperature, 40°C and inoculum, 1.5mL after 48h incubation. Alkaline protease was immobilization by forming cross linked enzyme aggregates (CLEAs) and the processes of CLEAs formation was also optimized. The protease CLEAs developed using 80% ammonium sulfate, 65mM glutaraldehyde and 0.11mM BSA showed best activity recovery (39.76%). Free protease and CLEAs were characterized and compared. It was observed that CLEAs of protease exhibited broad pH range with best activity at pH10. The immobilized protease was also thermo-tolerant with optimum activity at 65°C temperature. The Vmax and Km of protease-CLEAs were 125.5U/mL and 18.97μM, respectively as compared to 104.9U/mL and 29μM, respectively for free protease. It was concluded that immobilized enzyme in the form of CLEAs is a valuable catalyst for potential industrial applications.

Enhancement of n-3 polyunsaturated fatty acid glycerides in Sardine oil by a bioimprinted cross-linked Candida rugosa lipase.

Considering the advantages of bioimprinting and carrier free immobilization, cross-linked enzyme aggregates (CLEA) were prepared by using bioimprinted Candida rugosa lipase (CRL) with Bovine serum albumin (BSA), Polyethyleneimine and glutaraldehyde. Effect of various factors such as CRL-Oleic acid ratio, CRL-BSA ratio, CRL- Polyethyleneimine ratio, glutaraldehyde loading, cross-linking time etc., on lipase activity recovery and aggregate yield were studied and optimized. This immobilized lipase (CRL-CLEA) was used for the selective hydrolysis of ester linkages of non-PUFA glycerides, with an aim to concentrate EPA and DHA glycerides in the Sardine oil. Imprinting with oleic acid in the presence of ethanol and Tween 60, and further immobilization with co-aggregates and cross-linking agent showed 10.4 times higher degree of hydrolysis compared to free enzyme. As result, 2.83-fold increase of n-3 PUFA content in deacidified oil was obtained by using CRL-CLEA. The resultant oil had negligible di- and triglycerides content, proving higher efficiency in hydrolysing ester bonds of fatty acids, other than n-3 PUFA. Reusability studies showed CRL-CLEA could be reused up to 5 runs without a substantial reduction in its performance. Improvement in degree of hydrolysis, thermostability, efficiency of hydrolysis and reusability were achieved due to bioimprinting and subsequent immobilization of CRL in the form of CLEA.

Immobilized Cerrena sp. laccase: preparation, thermal inactivation, and operational stability in malachite green decolorization

Laccases are polyphenol oxidases with widespread applications in various industries. In the present study, the laccase from Cerrena sp. HYB07 was immobilized with four methods, namely entrapment in alginate, covalently binding to chitosan as well as formation of cross-linked enzyme aggregates (CLEAs) and magnetic CLEAs (M-CLEAs). The activity recovery rates of the immobilized laccases ranged from 29% to 68%. Immobilization elevated the reaction temperature optimum and reduced substrate specificity, but not necessarily the turnover rate. pH stability of immobilized laccases was improved compared with that of the free laccase, especially at acidic pH values. Thermal inactivation of all laccases followed a simple first-order exponential decay model, and immobilized laccases displayed higher thermostability, as manifested by lower thermal inactivation rate constants and longer enzyme half-life time. Operational stability of the immobilized laccase was demonstrated by decolorization of the triphenylmethane dye malachite green (MG) at 60 °C. MG decolorization with free laccase was accompanied by a shift of the absorption peak and accumulation of a stable, colored intermediate tetradesmethyl MG, probably due to lower thermostability of the free laccase and premature termination of the degradation pathway. In contrast, complete decolorization of MG was achieved with laccase CLEAs at 60 °C.

Bio-based degradation of emerging endocrine-disrupting and dye-based pollutants using cross-linked enzyme aggregates.

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 3.0h 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.

Optimizing the preparation conditions and characterization of cross-linked enzyme aggregates of a monoamine oxidase.

Monoamine oxidases are useful in determination of biogenic monoamines, particularly histamine and tyramine. In this study, cross-linked enzyme aggregates (CLEAs) technique was applied to improve the stability of a monoamine oxidase from Arthrobacter aurescens (AMAO). Under the optimized condition (50% of saturated ammonium sulfate, 5 mM glutaraldehyde, 2.0 mg/mL AMAO, 4 h-cross-linking at 25°C, pH 8.0), CLEAs-AMAO was recovered with a yield of 82% based on the subjected total enzyme activity. Both pH activity and stability at alkaline pHs of CLEAs-AMAO were significantly improved compared to those of the free enzyme, resulting in the shift of optimum pH to pH 8.0 and a broader pH profile. The half-life of the CLEAs at 65°C was elongated by 1.7-fold compared to that of the free enzyme, suggesting the thermal stability of AMAO was also improved by the CLEAs formation.

Preparation of cross-linked enzyme aggregates of nitrile hydratase ES-NHT-118 from E. coli by macromolecular cross-linking agent

Cross-linked enzyme aggregates (CLEAs) of nitrile hydratase (NHase) ES-NHT-118 from Escherichia coli were prepared by using ammonium sulfate as precipitating agent followed by cross-linking with dextran polyaldehyde for the first time. In this process, egg white was added as protein feeder for facilitating the formation of CLEAs. The optimal conditions of the immobilization process were determined. Michaelis constants (Km) of free NHase and NHase CLEAs were also determined. The NHase CLEAs exhibited increased stability at varied pH and temperature conditions compared to its free counterpart. When exposed to high concentrations of acrylamide, NHase CLEAs also exhibited effective catalytic activity.