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The presence of antibiotics in water systems and drinking water has detrimental effects on various organisms and poses health risks to both animals and humans. This study describes a sustainable approach using laccases to remove ciprofloxacin from water solutions. Laccases produced by monoculture of Trametes villosa and in co-culture with Pycnoporus sanguineus were immobilized using the method of cross-linked enzyme aggregates (CLEAs). The immobilization process was statistically optimized, and the biocatalysts were characterized and applied for biodegradation of ciprofloxacin at different concentrations. Laccase production was enhanced in co-culture conditions, yielding isoforms of molecular masses ranging from 55 to 45 kDa. Optimum conditions for immobilizing laccases by CLEAs were achieved with ammonium sulfate as a precipitant and glutaraldehyde as a cross-linker. Immobilization improved the thermal stability of the enzymes at 40 °C and 55 °C, and both forms of laccase CLEAs retained approximately 38% of their initial activity after five reuse cycles. Free and immobilized laccases demonstrated comparable efficiency in removing ciprofloxacin from aqueous solutions (at 2.5 mg L−1) with a 53–62% removal rate in the presence of 4-hydroxybenzoic acid as a natural redox mediator. The degradation of ciprofloxacin by laccases also reduced its antimicrobial activity against Escherichia coli. [Display omitted] • Co-culture of Trametes villosa with Pycnoporus sanguineus enhances laccase production. • Laccases immobilized as CLEAS exhibited improved thermal stability and reusability. • Free and immobilized laccases effectively catalyzed the degradation of ciprofloxacin • Enzymatic biodegradation of ciprofloxacin reduced its action against Escherichia coli. [ABSTRACT FROM AUTHOR]

• Optimal design of magnetite cross-linked tyrosinase aggregates was firstly presented. • Multiple nonlinear neuro-regression analysis was performed. • Realistic functional structures were obtained by examining the models. • Deceptive results could be eliminated by this optimization approach. • This novel approach is feasible for another modeling-design-optimization problems. In this study, a new design strategy with a systematic optimization process is proposed for the preparation of magnetite cross-linked tyrosinase aggregates (MCLTA) by using the concentration of magnetite nanoparticle, glutaraldehyde and tyrosinase enzyme as design variables. A comprehensive study on multiple non-linear neuro-regression analysis has been performed as a compelling alternative to the insufficient approaches on modeling-design-optimization of MCLTA. For this aim, the experimental process has been modeled with 13 candidate functional structures by using a hybrid method to test the accuracy of their predictions. R2 training , R2 testing values, and boundedness of the functions have been checked to reveal the realistic ones. Then four different design approaches in terms of three distinct scenarios have been used to optimize the process. The results show that, all models define the process well, depending on R2 training. However, only five and nine models are appropriate based on R2 testing for the first use activity and residual activity, respectively. On the other hand, depending on to be a realistic value, model TON best describes the "first use activity," while the best one is FONT for residual activity. It is also concluded that the scenario types and selection of constraints for design variables affect the optimization results. [ABSTRACT FROM AUTHOR]

We describe the development of cross-linked enzyme aggregates (CLEA) of dextransucrase of Leuconostoc mesenteroides B-512 F. Treatments of enzyme preparation using dextranase were evaluated varying incubation time to remove the dextran layer involving the enzyme molecule, turning it suitable for immobilization. Results showed 21 days of treatment as the best outcome. Subsequently, we tested different water-miscible organic solvents as precipitants, the reaction and centrifugation times, and the concentration of cross-linker agent (glutaraldehyde) in the preparation of CLEA. Optimal conditions were: the use of isopropanol as solvent, 30 min of centrifugation time, 3 h of cross-linking time, and 100 mM of glutaraldehyde. A central composite design was carried out to optimize conditions to obtain the highest enzymatic activity, testing the pH (3.0–7.0) and temperature (20 °C-60 °C). Results showed that dextransucrase CLEA operate at optimal pH of 3.0 and temperature of 60 °C. The operational stability of the immobilized biocatalyst showed up to 30% of residual activity after 10 cycles of reuse, in a solution of 100 mM of sucrose and 600 mM of maltose. The preparation of dextransucrase CLEA is described for the first time and results suggest that this novel immobilized biocatalyst has potential in many industrial applications. [ABSTRACT FROM AUTHOR]

Highlights: [•] Staphylococcus carnosus N-acetylneuraminate aldolase (ScNAL) was a good catalyst for d-mannose. [•] ScNAL thermal stability was improved by cross-linked immobilization to form FastPrep-CLEAs. [•] A 2:1 pyruvate:d-mannose ratio and 37°C provided the highest KDN conversion to date (90%). [•] ScNAL FastPrep-CLEAs showed good operational stability. [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: 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]

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]