Partial purification, characterization and immobilization of a novel lipase from a native isolate of Lactobacillus fermentum

Background and Objectives: Due to the widespread use of lipase enzymes in various industries, finding native lipase pro- ducing microorganisms is of great value and importance. In this study, screening of lipase-producing lactobacilli from native dairy products was performed. Materials and Methods: Qualitative evaluation of lipolytic activity of lipase-producing lactobacilli was performed in differ- ent media containing olive oil. A clear zone observation around the colonies indicated the lipolytic activity. The strain with the highest enzymatic activity was identified. Determination of optimal pH and temperature of lipase activity was measured by spectrophotometry using p-nitrophenyl acetate (ρ-NPA) substrate. Partial purification of lipase enzyme was performed using 20-90% saturation ammonium sulfate. Eventually, lipase was immobilized by physical adsorption on chitosan beads. Results: Among screened lipolytic bacterial strains, one sample (5c isolate) which showed the highest enzymatic activity (5329.18 U/ml) was close to Lactobacillus fermentum. During characterization, the enzyme showed maximum activity in Tris-HCl buffer with pH 7, while remaining active over a temperature range of 5°C to 40°C. The results of the quantitative assay demonstrated that the fraction precipitated in ammonium sulfate at 20% saturation has the highest amount of lipolytic activity, with a specific activity of 22.0425 ± 3.6 U/mg. Purification folds and yields were calculated as 8.73 and 44%, respec- tively. Eventually, the enzyme was immobilized by physical adsorption on chitosan beads with a yield of 56.21%. Conclusion: The high efficiency of enzyme immobilization on chitosan beads indicates the suitability of this method for long-term storage of new lipase from native 5c isolate.

Use of Immobilized Enzyme In Granular Activated Carbon And Chitosan Beads For Phenol Removal From Effluents

Tyrosinase enzyme present in a crude extract was immobilized in granular activated carbon (GAC) and activated chitosan beads (ACB). It was possible to immobilize up to 70.0 % of the enzymes in GAC in the conditions of 10.0 g of support, 15.7 rad/s of agitation and 90 minutes of contact time, and 100.0 % of enzymes in ACB when using 5 g of support, agitation of 15.7 rad/s and contact time of 120 minutes. In enzymatic oxidation tests, tyrosinase immobilized in GAC was able to achieve a final phenol concentration below the limit required by Brazilian law, 0.5 mg/L for phenol solutions with an initial concentration up to 20.0 mg/L while the enzyme immobilized in ACB was able to adapt solutions with initial concentrations of phenol up to 40.0 mg /L. It was possible to reuse the enzyme immobilized in GAC 2 times, maintaining the same phenol removal efficiency, while the enzyme immobilized in ACB maintained up to 98.0 % of its efficiency in 5 cycles of enzymatic oxidation of solutions with 10.0 mg/L of phenol initially. It was possible to maintain the same phenol removal efficiency as immobilized enzymes when stored for up to 2 weeks.

Lipase of Pachira speciosa seeds immobilized in chitosan beads: a recyclable bio-catalytic system

Las lipasas vegetales son biocatalizadores altamente versátiles debido a su quimioselectividad, enantioselectividad y regioselectividad. El propósito fue obtener un sistema biocatalítico reciclable a partir de lipasas de semillas de Pachira speciosa, aplicable a la biotransformación de lípidos. Se obtuvo una lipasa parcialmente purificada de extractos de semillas de P. speciosa, mediante cromatografía de filtración en gel en Sephadex G-100; la actividad específica lipasa (ALe) se determinó por el método de titulación de ácidos grasos libres. Se aplicó un diseño de superficie de repuesta Box-Behnken para establecer las condiciones que maximizan la inmovilización de la lipasa a tres soportes: esferas de quitosano (Q), esferas de alginato de calcio cubiertas con quitosano (Alg-Q) y esferas magnéticas de quitosano-Fe(OH)3 (Q-Fe). La mayor ALe de la enzima libre fue 0,49±0,01U/mg, a 40 °C y pH 9. El porcentaje de inmovilización y la ALe de cada biocatalízador fue: Q = 90,6 % y 3,74±0,3 nKat/mg; Alg-Q = 88,5 % y 3,62±0,1 nKat/mg; EQ-Fe = 76,4 % y 2,88±0,1 nKat/mg. El sistema biocatalítico más estable fue la lipasa inmovilizada en quitosano, con 85 % de rentención de la ALe hasta el tercer ciclo catalítico. Estudios futuros estarán enfocados a establecer los parámetros cinéticos del nuevo biocatalizador.

Highly Porous Hydroxyapatite/Graphene Oxide/Chitosan Beads as an Efficient Adsorbent for Dyes and Heavy Metal Ions Removal

Dye and heavy metal contaminants are mainly aquatic pollutants. Although many materials and methods have been developed to remove these pollutants from water, effective and cheap materials and methods are still challenging. In this study, highly porous hydroxyapatite/graphene oxide/chitosan beads (HGC) were prepared by a facile one-step method and investigated as efficient adsorbents. The prepared beads showed a high porosity and low bulk density. SEM images indicated that the hydroxyapatite (HA) nanoparticles and graphene oxide (GO) nanosheets were well dispersed on the CTS matrix. FT-IR spectra confirmed good incorporation of the three components. The adsorption behavior of the obtained beads to methylene blue (MB) and copper ions was investigated, including the effect of the contact time, pH medium, dye/metal ion initial concentration, and recycle ability. The HGC beads showed rapid adsorption, high capacity, and easy separation and reused due to the porous characteristics of GO sheets and HA nanoparticles as well as the rich negative charges of the chitosan (CTS) matrix. The maximum sorption capacities of the HGC beads were 99.00 and 256.41 mg g−1 for MB and copper ions removal, respectively.

Chitosan Beads Incorporated with Graphene Oxide/Titanium Dioxide Nanoparticles for Removing an Anionic Dye

Dyes present in industrial effluents have been treated by many methods, among which adsorption stands out for its high efficiency, low costs, simple operation processes, and the absence of hazardous byproducts. In this research, two adsorbents were obtained from chitosan beads (CS) cross-linked with glutaraldehyde (GLA), graphene oxide (GO), and titanium dioxide nanoparticles (TiO2) for the adsorption of the anionic dye FD&C Red 40 used as a model pollutant. The optimum removal conditions of FD&C Red 40 dye using CS-TiO2-GLA beads were determined (pH = 1.73, TiO2 amount =279.77 mg, and initial dye concentration = 55.23 mg L−1) with a central composite design with surface response methodology (RSM). The effect of the graphene oxide (GO) in the adsorption properties of CS-TiO2-GLA beads was evaluated, showing a considerable improvement in the removal efficiency of the model dye. The intraparticle diffusion mechanism best described the adsorption kinetics for the two adsorbents. This research demonstrates the potential of chitosan beads incorporated with graphene oxide and titanium dioxide nanoparticles to remove anionic contaminants from wastewater.

Immobilization of Fusarium solani Cutinase onto Magnetic Genipin-Crosslinked Chitosan Beads

Genipin was used as a crosslinking agent to prepare magnetic genipin-crosslinked chitosan beads, which were then used as a carrier for immobilizing recombinant cutinase from Fusarium solani (FSC) to obtain immobilized FSC. The optimal temperature for the immobilized FSC was 55 °C, which was 5 °C higher than that of the free enzyme, whereas its optimal pH was increased from 8.0 to 9.0; this indicates that the immobilized FSC had improved pH and thermal stability. After repeated use for 10 cycles, the activity of the immobilized FSC remained at more than 50%; after being stored at 4 °C for 30 days, its activity was still approximately 88%. We also found that the Km of the immobilized FSC was higher than that of the free enzyme. These results indicate that the performance of FSC was improved after immobilization, which is an important basis for the subsequent application of FSC in industrial production.

Process conditions optimization and study of pH and thermal stability of free and immobilized gutaminase-free recombinant L-asparaginase II of Pactobacterium carotovorum MTCC 1428 from Escherichia coli

L-asparaginase (EC 3.5.1.1) is used for the treatment of acute lymphocytic leukemia and food processing of starch rich foods for reducing the acrylamide formation. In our current efforts, we have immobilized the purified glutaminase-free recombinant Lasparaginase II of Pectobacterium carotovorum MTCC 1428 from Escherichia coli BL21 (DE3) on glutaraldehyde activated chitosan beads. The purified recombinant L-asparaginase II has no partial glutaminase activity which is a pre-requisite to reduce the possibility of side effects during the course of anticancer therapy. In order to study the best conditions for the performance of free enzymes and immobilized enzymes, response surface methodology was used to optimize the pH and temperature of the process conditions. It was found that the most favorable pH and temperature for the free enzyme were pH 7.83 and 47.64°C while for the immobilized enzyme, the optimum pH and temperature levels were found to be 7.88 and 48.07 °C. Furthermore, the thermal and pH studies of free and immobilized enzymes were studied under various combinations of pH and temperature and finally the thermodynamic parameters of free and immobilized glutaminase-free recombinant asparaginase were evaluated.