Exploring magnetic and imprinted cross-linked enzyme aggregates of rhamnopyranosidase in microbioreactors.

The goal of this work was the development of magnetic cross link enzyme aggregates (mCLEAs) of rhamnopyranosidase (Rhmnase), prepared by chemical cross-linking with functionalized magnetite nanoparticles for glycompounds biosynthesis in microbioreactors (specially design 24-well microplate and mini-packed bed). Rhamnopyranosidase (EC number 3.2.1.40) present high potential in glycocompounds production, with applications in food and pharmaceutical industries. The influence of precipitants, cross-linkers, temperature and time on (m)CLEAs@Rhmnase development were optimized. Biocatalyst activity was accessed in the hydrolysis of 4',5,7-trihydroxyflavanone-7-rhamnoglucoside and kinetic constants in the deglycosylation reaction were evaluated. Rhmnase operational stability was enhanced in mCLEAs, retaining almost 90% initial activity after 7 cycles of 24 h each. In a mini-packed bed bioreactor a maximum volumetric productivity of 140 µmol/L.h was attained. In this bioreactor the operational stability of mCLEAs@Rhmnase were evaluated at a flow rate of 5 mL/h during 5 days and a residual activity of 95% was observed.

Improved thermostable polyvinyl alcohol electrospun nanofibers with entangled naringinase used in a novel mini-packed bed reactor.

Polyvinyl alcohol (PVA) electrospun nanofibers were produced using an electrospinning technique. Key parameters (e.g. collectors, distance from needle tip to collector, among others) that influence the structure and morphology of fibers were optimized. The naringinase entrapped in PVA nanofibers retained over 100% of its initial activity after 212h of operation, at 25°C. Chemical crosslinking with several boronic acids further increased the hydrolysis temperature (up to 85°C) and yielded nanofibers with thermal stability up to 121°C. A mini packed bed reactor (PBR) developed to establish the feasibility for continuous enzymatic operation, ran for 16days at 45°C. Highest naringenin biosynthesis was attained at a flow rate of 10mLh(-1). Highest volumetric (78molL(-1)h(-1)) and specific (26molh(-1)genzyme(-1)) productivities were attained at 30mLh(-1). The activity of NGase in electrospun nanofibers remained constant for almost 16days of operation at 10mLh(-1).

Operational stability of naringinase PVA lens-shaped microparticles in batch stirred reactors and mini packed bed reactors-one step closer to industry.

The immobilization of naringinase in PVA lens-shaped particles, a cheap and biocompatible hydrogel was shown to provide an effective biocatalyst for naringin hydrolysis, an appealing reaction in the food and pharmaceutical industries. The present work addresses the operational stability and scale-up of the bioconversion system, in various types of reactors, namely shaken microtiter plates (volume ⩽ 2 mL), batch stirred tank reactors (volume <400 mL) and a packed-bed reactor (PBR, 6.8 mL). Consecutive batch runs were performed with the shaken/stirred vessels, with reproducible and encouraging results, related to operational stability. The PBR was used to establish the feasibility for continuous operation, running continuously for 54 days at 45°C. The biocatalyst activity remained constant for 40 days of continuous operation. The averaged specific productivity was 9.07 mmol h(-1) g enzyme(-1) and the half-life of 48 days.

Microtiter plates versus stirred mini-bioreactors in biocatalysis: a scalable approach.

To place the application of miniaturized vessels as microbioreactors on a firm footing, focus has been given to engineering characterization. Studies on this matter have mostly involved carrier-free biological systems, while support-based systems have been overlooked. The present work aims to contribute to fill in such gap. Thus, it intended to establish a robust scaled down approach to identify and optimize relevant operational conditions of naringin hydrolysis by naringinase in PVA lens-shaped particles. The influence of geometric and dynamic (viz. Reynolds number) parameters was evaluated. Naringin hydrolysis in round, flat bottom MTP proved more effective than in square, pyramidal bottom. The bioconversion at MTP and stirred tank reactors scales showed that, given the 12.5-fold scale difference was in agreement between the bioconversion rates. The external mass transfer resistances were negligible as deduced from Damkohler modulus ≤1. The bioconversion was effectively scaled-up 200-fold from shaken microtiter plates to stirred tank reactors.

Hesperidinase encapsulation towards hesperitin production targeting improved bioavailability.

Hesperidin (hesperitin-7-O-rutinoside) and hesperitin (hesperitin-7-O-glucoside) show anti-inflammatory, antimicrobial, antioxidant, and anticarcinogenic effects and prevent bone loss. However, hesperidin has a low bioavailability compared to hesperitin due to the rutinoside moiety attached to the flavonoid. The removal of the rhamnose group to yield the corresponding flavonoid glucoside (hesperetin-7-glucoside) improved the bioavailability of the aglycone, hesperetin, in humans. In line with these assumptions, the aim of this work was the enzymatic production of hesperitin from hesperidin with hesperidinase. Despite the low hesperidin solubility in the reaction medium, the enzymatic bioconversion was carried with hesperidin soluble at lower concentrations (≤0.05 mg ml(-1)) and insoluble for high concentrations (>0.1-50 mg ml(-1)). A twofold increase in maximum reaction rates overtook the expected values, pointing to the enzyme ability to degrade insoluble hesperidin. To improve the bioprocess, hesperidinase was tested soluble and immobilized in calcium alginate (2%), k-carrageenan (2%), and chitosan (2%) beads. The immobilization was carried out by adsorption and encapsulation. Chitosan was cross-linked with glutaraldehyde (1% and 2%) and sodium sulfate (13.5% and 15%) in acetate buffer (0.02 M, pH 4.0). The relation between bioprocessing conditions and hesperidinase stability was studied. A residual activity of 193% was obtained with immobilized hesperidinase compared to the soluble form. A half-life of 770 min was attained with hesperidinase encapsulated in calcium alginate beads. The results presented in this work highlight the potential of hesperidinase encapsulation towards hesperitin production with insoluble substrate. To our knowledge, this work presents for the first time the potential of hesperidinase encapsulation on hydrogels for hesperitin production. This is an important achievement for pharmaceutical and nutraceutical applications of hesperitin because this compound presents a higher bioavailability compared to hesperidin.

High-affinity water-soluble system for efficient naringinase immobilization in polyvinyl alcohol-dimethyl sulfoxide lens-shaped particles.

Polyvinyl alcohol (PVA) is a water-soluble, biocompatible and biodegradable synthetic polymer whose application in the immobilization of biological agents for use in biocatalysis has shown promising results. This study aimed to investigate and optimize the immobilization of naringinase from Penicillium decumbens in PVA networks, targeting for the hydrolysis of naringin. Variables such as the most suitable cross-linker, catalyst, inorganic salt, co-solvents and solidification process were identified as key issues for PVA-based methods to form lens-shaped particles, while retaining high enzyme activity and stability. Major improvements were established for better and more reproducible immobilization conditions, namely, by designing a new immobilization apparatus to produce uniform lens-shaped particles. The common problems of PVA-based entrapment were significantly mitigated, through the use of selected cross-linker, glutaraldehyde (GA), and co-solvent, dimethyl sulfoxide (DMSO), which decreased the toxicity of the immobilization process and allowed the control of membrane porosity, respectively. The relevance of DMSO and GA and their interaction and effect on the swelling ratio, encapsulation efficiency and residual activity of PVA biocatalysts were established. The immobilization of naringinase in PVA under a DMSO concentration of 60%, cross-linked with 1% GA, and particle lens size of 3.5-4.0 mm, width of 100-300 µm and average particle volume of 12.5 ± 0.92 µL, allowed an encapsulation efficiency of 98.6% and an average residual activity of 87% ± 3.6%. The kinetic characterization of the immobilized naringinase showed no changes in pH profile, whereas hydrolytic activity increased up to 60 °C. Immobilization in PVA/DMSO/GA lens-shaped particles enhanced the storage stability of naringinase. Moreover, these naringinase bio-immobilizates retained a conversion rate higher than 78% after 23 runs.

Immobilization of naringinase in PVA-alginate matrix using an innovative technique.

A synthetic polymer, polyvinyl alcohol (PVA), a cheap and nontoxic synthetic polymer to organism, has been ascribed for biocatalyst immobilization. In this work PVA-alginate beads were developed with thermal, mechanical, and chemical stability to high temperatures (<80 degrees C). The combination of alginate and bead treatment with sodium sulfate not only prevented agglomeration but produced beads of high gel strength and conferred enzyme protection from inactivation by boric acid. Naringinase from Penicillium decumbens was immobilized in PVA (10%)-alginate beads with three different sizes (1-3 mm), at three different alginate concentrations (0.2-1.0%), and these features were investigated in terms of swelling ratio within the beads, enzyme activity, and immobilization yield during hydrolysis of naringin. The pH and temperature optimum were 4.0 and 70 degrees C for the PVA-alginate-immobilized naringinase. The highest naringinase activity yield in PVA (10%)-alginate (1%) beads of 2 mm was 80%, at pH 4.0 and 70 degrees C. The Michaelis constant (K(Mapp)) and the maximum reaction velocity (V(maxapp)) were evaluated for both free (K(Mapp) = 0.233 mM; V(maxapp) = 0.13 mM min(-1)) and immobilized naringinase (K(Mapp) = 0.349 mM; V(maxapp) = 0.08 mM min(-1)). The residual activity of the immobilized enzyme was followed in eight consecutive batch runs with a retention activity of 70%. After 6 weeks, upon storage in acetate buffer pH 4 at 4 degrees C, the immobilized biocatalyst retained 90% of the initial activity. These promising results are illustrative of the potential of this immobilization strategy for the system evaluated and suggest that its application may be effectively performed for the entrapment of other biocatalysts.