The Stability Improvement of α-Amylase Enzyme from Aspergillus fumigatus by Immobilization on a Bentonite Matrix

The stability of the α-amylase enzyme has been improved from Aspergillus fumigatus using the immobilization method on a bentonite matrix. Therefore, this study aims to obtain the higher stability of α-amylase enzyme from A. fumigatus; hence, it is used repeatedly to reduce industrial costs. The procedures involved enzyme production, isolation, partial purification, immobilization, and characterization. Furthermore, the soluble enzyme was immobilized using 0.1 M phosphate buffer of pH 7.5 on a bentonite matrix, after which it was characterized with the following parameters such as optimum temperature, Michaelis constant (KM), maximum velocity V max , thermal inactivation rate constant (ki), half-life (t1/2), and the change of energy due to denaturation (ΔGi). The results showed that the soluble enzyme has an optimum temperature of 55°C, KM of 3.04 mg mL−1 substrate, V max of 10.90 μmole mL−1 min−1, ki of 0.0171 min−1, t1/2 of 40.53 min, and ΔGi of 104.47 kJ mole−1, while the immobilized enzyme has an optimum temperature of 70°C, KM of 8.31 mg mL−1 substrate, V max of 1.44 μmole mL−1 min−1, ki of 0.0060 min−1, t1/2 of 115.50 min, and ΔGi of 107.37 kJ mole−1. Considering the results, the immobilized enzyme retained 42% of its residual activity after six reuse cycles. Additionally, the stability improvement of the α-amylase enzyme by immobilization on a bentonite matrix, based on the increase in half-life, was three times greater than the soluble enzyme.

Extraction cellulose from corn-stalk taking advantage of pretreatment technology with immobilized enzyme

In this paper, the corn-stalk cellulose (CSC) was extracted from the corn-stalk pretreated by the immobilized enzyme which was prepared using xylanase and laccase.

ADAPTATION OF THE PROCEDURE OF CO-IMMOBILIZATION OF ENZYMES WITH DIFFERENT MODIFICATIONS OF ZEOLITES ON THE SURFACE OF CONDUCTOMETRIC TRANSDUCERS

A comparative study of different parameters of conductometric biosensors based on urease and glucose oxidase, co-immobilized with different types of zeolites. Urease immobilized on silicalite-2 was shown to have better performance than immobilized urease without zeolites. Conductometric biosensor with glucose oxidase co-immobilized with zeolite NH4+-Beta 25 had similar response values compared to immobilized enzyme without zeolite. Immobilization of zeolites NH4+-BEA 30 and H+-BEA 30 together with urease leads to an increase in the response of the biosensor, while the reproducibility of the signal remains unchanged. Biosensors with zeolites with a higher Si/Al ratio were characterized by increased signals. The use of zeolites modified with methylviologen and silver did not give a positive effect.

DEGRADATION OF REACTIVE DYES USING IMMOBILIZED PEROXIDASE PURIFIED FROM NIGELLA SATIVA

The present study was aimed to exploit the free and immobilized peroxidase from Nigella sativa seeds to degradation of textile dyes polluting the environment and water. The optimum conditions for extracting the enzyme from the Nigella seeds were determined and the highest specific activity of the enzyme was obtained 1750 units / mg protein when extracting the enzyme from the ground seeds at a ratio of 1:20 (w: v) with sodium acetate buffer at 0.2 M and pH 5.0 for 15 minutes. The enzyme was purified using two steps including the concentration by sucrose and gel filtration by using Sephadex G-150. The results shown an increase in final purification folds 2.8 time with an enzyme yield of 35%. The immobilization of peroxidase were done by entrapment method using Ca- alginate and the immobilization ratio was reached to 49%. The removal efficiency of dyes by crude enzyme (free, immobilized) and partial purified peroxidase were studied with textile dyes such as yellow, red, black and blue dyes at optimum conditions pH 5, temperature 37oC after 3 hr. Maximum removal efficiency of dyes observed with crude peroxidase and reached (76.9, 88.7, 91 and 88) % respectively. These results were close to the efficiency of the purified enzyme in removing the four dyes, while the efficiency of the crude immobilized enzyme in removing the dyes was about (70, 81, 72 and 56.4)%, respectively.

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.

Monolithic Papain-Immobilized Enzyme Reactors for Automated Structural Characterization of Monoclonal Antibodies

The characterization of monoclonal antibodies (mAbs) requires laborious and time-consuming sample preparation steps before the liquid chromatography–mass spectrometry (LC-MS) analysis. Middle-up approaches entailing the use of specific proteases (papain, IdeS, etc.) emerged as practical and informative methods for mAb characterization. This work reports the development of immobilized enzyme reactors (IMERs) based on papain able to support mAb analytical characterization. Two monolithic IMERs were prepared by the covalent immobilization of papain on different supports, both functionalized via epoxy groups: a Chromolith® WP 300 Epoxy silica column from Merck KGaA and a polymerized high internal phase emulsion (polyHIPE) material synthesized by our research group. The two bioreactors were included in an in-flow system and characterized in terms of immobilization yield, kinetics, activity, and stability using Nα-benzoyl-L-arginine ethyl ester (BAEE) as a standard substrate. Moreover, the two bioreactors were tested toward a standard mAb, namely, rituximab (RTX). An on-line platform for mAb sample preparation and analysis with minimal operator manipulation was developed with both IMERs, allowing to reduce enzyme consumption and to improve repeatability compared to in-batch reactions. The site-specificity of papain was maintained after its immobilization on silica and polyHIPE monolithic supports, and the two IMERs were successfully applied to RTX digestion for its structural characterization by LC-MS. The main pros and cons of the two supports for the present application were described.