Immobilization Of Biocatalysts Research Articles

Screening, immobilization and utilization of whole cell biocatalysts to mediate the ethanolysis of babassu oil

Abstract The screening, biomass growth of lipase-producing fungus isolated from different sources and available at URM (University Recife Mycologia), as well as, the immobilization and utilization of the whole cells for the transesterification of babassu oil were investigated. Rhizopus oryzae (URM 3231, 4692), Mucor circinelloides (URM 4140, 4182) and Penicillium citrinum URM 4216 were considered to be good intracellular lipase producers whereas those from Mucor hiemalis URM 4144 and Mucor piriformis URM 4145 were weaker. Fungi biomass containing high lipase activities was immobilized on different biomass support particles (BSPs) and with the exception of Penicillium citrinum URM 4216 all the other fungi strains exhibited high lipase activity (20–50 U g −1 ) when immobilized in situ using polyurethane foam particles. Transesterification activities of the immobilized whole cells were evaluated in the ethanolysis reaction with babassu oil and the highest performance was attained by M. circinelloides URM 4182 giving 83.22 ± 3.68% ester yield in less than 96 h reaction. The biocatalyst operational stability was also assessed and an inactivation profile was found to follow the Arrhenius model, revealing values of 26 days and 2.6 × 10 −2 day −1 , for half-life and a deactivation coefficient, respectively. The purified product (biodiesel) exhibited viscosity (6.63 cSt) close to the value to attend specifications by the ASTM D6751 to be used as biofuel. Results are favorable compared with data already reported in the literature and demonstrated that M. circinelloides URM 4182 whole cells is a cheaper biocatalyst that can be used in the biodiesel synthesis.

Carbon nanotube-modified biocatalytic microelectrodes with multiscale porosity

Macropores were introduced into nanotube matrices via polystyrene bead templates, and the resulting matrix was applied to carbon fiber microelectrodes as a porous medium for immobilization of enzymatic biocatalysts. The macropores were found to increase the electrochemically active surface area by twofold at a nominal polystyrene mass fraction of 73%. The modified electrodes were further coated with biocatalyst hydrogel comprising glucose oxidase, redox polymer, and crosslinker to create a glucose oxidizing bioanode. Glucose oxidation current density also increased two fold after introduction of the macropores. Focused ion beam cut cross-sections reveal complete adsorption of the enzyme-hydrogel matrix into the CNT layer. This templating technique is a promising approach to the maximization of surface area and transport in bioelectrodes.

Immobilization of biocatalysts for enzymatic polymerizations: Possibilities, advantages, applications

Biotechnology also holds tremendous opportunities for realizing functional polymeric materials. Biocatalytic pathways to polymeric materials are an emerging research area with not only enormous scientific and technological promise, but also a tremendous impact on environmental issues. Many of the enzymatic polymerizations reported proceed in organic solvents. However, enzymes mostly show none of their profound characteristics in organic solvents and can easily denature under industrial conditions.Therefore, natural enzymes seldom have the features adequate to be used as industrial catalysts in organic synthesis. The productivity of enzymatic processes is often low due to substrate and/or product inhibition. An important route to improving enzyme performance in non-natural environments is to immobilize them.In this review we will first summarize some of the most prominent examples of enzymatic polymerizations and will subsequently review the most important immobilization routes that are used for the immobilization of biocatalysts relevant to the field of enzymatic polymerizations.

Hydrogen production from biodiesel byproduct by immobilized Enterobacter aerogenes

The recent rapid growth of the biodiesel industry has generated a significant amount of glycerol as a byproduct. As a result, the price of glycerol is currently relatively low, making it an attractive starting material for the production of chemicals with higher values. Crude glycerol can be directly converted through microbial fermentation into various chemicals such as hydrogen. In this study, we optimized immobilization of a facultative hydrogen producing microorganism, Enterobacter aerogenes, with the goal of developing biocatalysts that was appropriate for the continuous hydrogen production from glycerol. Several carriers were tested and agar was found to be the most effective. In addition, it was clearly shown that variables such as the carrier content and cell loading should be controlled for the immobilization of biocatalysts with high hydrogen productivity, stability, and reusability. After optimization of these variables, we were able to obtain reusable biocatalysts that could directly convert the byproduct stream from biodiesel processes into hydrogen in continuous processes.

Immobilization of enzyme biocatalyst on natural halloysite nanotubes

Abstract In this study, we use natural halloysite nanotubes as novel support materials to immobilize enzymes. Two typical industrial enzymes (α-amylase and urease) with different sizes were immobilized in channels of the nanotubes through simple physical adsorption. After 60 min heating, both immobilized enzymes retained more than 80% activity. Stored for 15 days, the immobilized enzymes still showed more than 90% activity. More than 55% initial activity of the enzyme was retained after 7 cycles. The immobilized enzymes exhibited thermal stability, good storage stability and reusability, which indicate that halloysite is a promising support material for enzyme immobilization.

Development of a continuous steroid biotransformation process and product extraction within microchannel system

Results of an integrated approach for the improvement of progesterone 11α-hydroxylation by Rhizopus nigricans, which is one of the key steps in the production of corticosteroid drugs and hormones, are presented. Several issues have been addressed in order to increase process productivity, including biocatalyst immobilization, improvement in the solubility of the lipophilic substrate, setting-up of continuous reaction process and easing downstream processing. After preliminary studies on the effects on Rhizopus nigricans morphology, fungal pellets of a desired size and structure could be obtained. The mycelia grown in spherical agglomerates further enabled its continuous use in the process of biotransformation. The problem of low steroid solubility was addressed by the addition of organic solvents or β-cyclodextrin. In order to develop an integrated biotransformation process, a continuous two-liquid phase extraction within microchannel device has been set up, where very efficient steroid extraction was achieved within a few seconds.

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.

Nickel-Impregnated Silica Nanoparticle Synthesis and Their Evaluation for Biocatalyst Immobilization

In the present investigation, impact of nickel-impregnated silica paramagnetic particles (NSP) as biocatalyst immobilization matrices was investigated. These nanoparticles were synthesized by sol-gel route using a nonionic surfactant block co polymer [poly (ethylene glycol)-block-poly-(propylene glycol)-block-poly (ethylene glycol)]. Diastase enzyme was immobilized on these particles (enzyme-impregnated NSP) as model enzyme and characterized using Fourier-transform infrared spectroscopy and X-ray crystallography. Analysis of enzyme-binding nature with these nanoparticles at different physiological conditions revealed that binding pattern and activity profile varied with the pH of the reaction mixture. The immobilized enzyme was further characterized for its biocatalytic activity with respect to kinetic properties such as Km and Vmax and compared with free enzyme. Paramagnetic nanoparticle-immobilized enzyme showed more affinity for substrate compared to free one. The nature of silica and nickel varied from amorphous to crystalline nature and vice versa upon immobilization of enzyme. To the best of our knowledge, this is the first report of its kind for change of nature from one form to other under normal temperatures upon diastase interaction with NSP.

Cold plasma functionalized TeraHertz BioMEMS for enzyme reaction analysis

In this paper, we describe the development, functionalization and functionality testing of a TeraHertz (THz) Bio-MicroElectroMechanical System (BioMEMS) dedicated to enzyme reaction analysis. The microdevice was fabricated by mixing clean room microfabrication with cold plasma deposition. The first is used to build the microfluidic circuits and the THz sensor, while the later serves for the polymerization of allylamine using a homemade glow discharge plasma reactor for a subsequent immobilization of enzymatic biocatalysts. Thermal stability of the deposited plasma polymer has been investigated by infrared spectroscopy. Fluorescent detection confirmed the efficiency of the immobilization and the enzyme hydrolysis into the BioMEMS microchannels. For the first time, the progression of the hydrolysis reaction over time was monitored by the THz sensor connected to a vectorial network analyzer. Preliminary results showed that sub-THz transmission measurements are able to discriminate different solid films, various aqueous media and exhibit specific transmission behavior for the enzyme hydrolysis reaction in the spectral range 0.06–0.11 THz.

Functional Characterization of Chitin and Chitosan

Chitin and its deacetylated derivative chitosan are natural polymers composed of randomly distributed � -(1-4)- linked D-glucosamine (deacetylated unit) and N-acetyl-D-glucosamine (acetylated unit). Chitin is insoluble in aqueous media while chitosan is soluble in acidic conditions due to the free protonable amino groups present in the D-glucosamine units. Due to their natural origin, both chitin and chitosan can not be defined as a unique chemical structure but as a fam- ily of polymers which present a high variability in their chemical and physical properties. This variability is related not only to the origin of the samples but also to their method of preparation. Chitin and chitosan are used in fields as different as food, biomedicine and agriculture, among others. The success of chitin and chitosan in each of these specific applica- tions is directly related to deep research into their physicochemical properties. In recent years, several reviews covering different aspects of the applications of chitin and chitosan have been published. However, these reviews have not taken into account the key role of the physicochemical properties of chitin and chitosan in their possible applications. The aim of this review is to highlight the relationship between the physicochemical properties of the polymers and their behaviour. A functional characterization of chitin and chitosan regarding some biological properties and some specific applications (drug delivery, tissue engineering, functional food, food preservative, biocatalyst immobilization, wastewater treatment, molecular imprinting and metal nanocomposites) is presented. The molecular mechanism of the biological properties such as biocompatibility, mucoadhesion, permeation enhancing effect, anticholesterolemic, and antimicrobial has been up- dated.

A simple method for biocatalyst immobilization using PVA‐based hydrogel particles

AbstractBACKGROUND: The aim of this study was to evaluate the feasibility of enzyme immobilization in PVA particles through extrusion of LentiKat®Liquid in polyethylene glycol. Inulinase, with invertase activity for sucrose hydrolysis, was used as model system.RESULTS: Inulinase was effectively immobilized in PVA particles. The pH optimum of the enzyme activity was broadened for lower pH values. Mechanical instability of the PVA under prolonged incubation above 55 °C was observed. A 1.8‐fold increase in the apparent KM (Michaelis constant) suggests diffusion limitations as a result of immobilization. The immobilized biocatalyst exhibited considerable operational stability, since a decrease of roughly 10% in the product yield after 24 h biotransformation runs was observed in trials performed at 50 °C, following 20 repeated, consecutive batches.CONCLUSION: The results obtained highlight the potential of PVA‐based particles obtained through extrusion into PEG for the production of suitable biocatalysts for application in large‐scale processes. Copyright © 2008 Society of Chemical Industry

Screening and reaction engineering for the bioreduction of ethyl benzoylacetate and its analogues

Microbial reduction of benzoylacetates is already an established part of the synthetic toolbox to obtain chiral ethyl 3-hydroxy-3-phenylpropionate although bioreduction yields are low to moderate. A 30% increase in the enantioselectivity to 99% ee and a significant improvement in the yields to around 85% were achieved by combining simple screening procedures and a reaction engineering strategy. Three experimental parameters were selected for investigation: the influences of glucose, enzymatic inhibitor and biocatalyst immobilization. The screened yeasts Pichia kluyveri, Pichia stipitis and Candida utilis were found to give better yields and ee's for ethyl benzoylacetate 1a, p-nitrobenzoylacetate 1b and p-methoxybenzoylacetate 1c, respectively, with addition of glucose, α-chloroacetophenone as inhibitor and immobilization of the yeasts in alginate beads. Our results demonstrate that the optimized process can be implemented on a preparative scale without any loss in yield and ee.

Nitrate removal without carbon source feeding by permeabilized Ochrobactrum anthropi SY509 using an electrochemical bioreactor

Biological denitrification is very attractive due to its economical and environmental advantages. However, an external carbon source is required to maintain biological activity in biological denitrification. Permeabilized cells have been used to achieve denitrification that does not require an external carbon source. In this study, permeabilized Ochrobactrum anthropi SY509, which contains the denitrifying enzymes, nitrate reductase, nitrite reductase, nitric oxide reductase and nitrous oxide reductase, was used. NADH participates in denitrifying enzyme reaction as an electron donor. However, in the bioelectrochemical method, a redox molecule was used for electron transfer to the enzyme, and the denitrification was performed bioelectrochemically. This redox molecule functions as an artificial electron transferring agent, and is thus called a mediator. Mediated bioelectrocatalysis is a useful system for many redox biocatalyst reactions as the aforementioned mediator mediates the biocatalytic reaction between electrode and biocatalyst. In this study, neutral red was selected as a mediator. For application to a real wastewater treatment system, immobilization of biocatalyst and mediator on the electrode is essential. Carbon nanopowder was used as a support material to immobilize neutral red. When both the permeabilized cells and the carbon nanopowder with the mediator were immobilized on a graphite felt electrode, mediator leakage was completely prevented and high denitrification efficiency was obtained.

Choline biosensor constructed with chitinous membrane from soldier crab and its application in measuring cholinesterase inhibitory activities

An amperometric flow-injection choline biosensor was assembled utilizing natural chitinous membrane as the supporting material for biocatalyst immobilization, and the membrane was purified from Taiwanese soldier crab, Mictyris brevidactylus. The chitinous membrane (<50.0 μm in thickness) was covalently immobilized with choline oxidase (EC 3.1.1.17 from Alcaligenes sp.) and then attached onto the platinum electrode of an amperometric flow cell. The flow cell served as the choline sensing device of the proposed FIA system. The sensor signal (peak height of the FIAgram) was linearly related to choline concentration ( r = 0.999 for choline up to 5.0 mM) with low detection limit (S/N > 3 for 10.0 μM choline) and high reproducibility (CV < 3% for 1.0 mM choline, n = 7). The system was proved to be useful in measuring cholinesterase inhibitory activities of synthetic chemicals or natural products.

Ionic Liquid-Reconstituted Cellulose Composites as Solid Support Matrices for Biocatalyst Immobilization

Preparation of cellulose-polyamine composite films and beads, which provide high loading of primary amines on the surface allowing direct one-step bioconjugation of active species, is reported using an ionic liquid (IL) dissolution and regeneration process. Films and bead architectures were prepared and used as immobilization supports for laccase as a model system demonstrating the applicability of this approach. Performance of these materials, compared to commercially available products, has been assessed using millimeter-sized beads of the composites and the lipase-catalyzed transesterification of ethyl butyrate.

Immobilization of a whole-cell epoxide-hydrolyzing biocatalyst in sodium alginate−cellulose sulfate−poly(methylene- co-guanidine) capsules using a controlled encapsulation process

A whole-cell epoxide-hydrolyzing biocatalyst designed as uniform polyelectrolyte complex capsules was developed. Nocardia tartaricans bacterial cells with cis-epoxysuccinate (CES) hydrolase activity were used as a model microbial strain. Stereospecific hydrolysis of CES catalyzed by CES hydrolase in encapsulated cells yields enantiomerically pure l-(+)-tartrate. An air-stripping device with multiloop reactor was used for controlled production of capsules containing bacterial cells with the standard deviation in diameter below 4% and in membrane thickness below 7%. Capsules formed by polyelectrolyte complexation of sodium alginate and cellulose sulfate as polyanions, poly(methylene- co-guanidine) as polycation, CaCl 2 and NaCl (SA-CS/PMCG) provide a favorable microenvironment for encapsulated cells. Biotransformation was monitored by RP-HPLC, electrospray ionization mass spectrometry and optical rotation. The results are discussed in view of data previously obtained by cell entrapment in hardened calcium pectate gelled beads (CPG). Encapsulation of the whole -cell biocatalyst in SA-CS/PMCG capsules leads to (i) about two-fold increase, from 91.5 to 208.2 U/mg, in the CES hydrolase activity, (ii) a decreased time required for total biotransformation, from 5.5 to 3 h, and (iii) a significantly improved relative increase in CES hydrolase activity during 51 days of storage, from about 3-fold to about 20-fold, compared to the CPG beads.

A mini-review of Chinese contributions to Journal of Molecular Catalysis B: Enzymatic

Since the birth of Journal of Molecular Catalysis B: Enzymatic in 1995, 13 papers contributed from Chinese authors have been published in this journal by the end of year 2001. These papers were summarized in this mini-review by dividing their contents into several subjects, including immobilization of biocatalysts, enzymatic polymerization, asymmetric biocatalysis, non-aqueous biotransformation, and overexpression and stabilization of enzymes. Though the number of annually published papers by Chinese authors in this journal is still very limited, it is increasing rapidly in the recent years.

Carrageenan: A Food-Grade and Biocompatible Support for Immobilisation Techniques

Immobilisation of both enzymes and whole-cell systems is of major importance in the improvement of the stability, activity and reusability of these biocatalysts. This review describes the use of the naturally occurring polysaccharide carrageenan as a support for the immobilisation of biocatalysts. Carrageenan is a food-grade and biocompatible support material extracted from red seaweeds. Before focusing on the use of carrageenan as an immobilisation support, an overview is given of the present uses of biocatalysts in industrial processes. The basic concepts of enzyme and whole-cell immobilisation are discussed, as well as the background of carrageenan as a biopolymer. Several examples of enzymes and whole-cell systems immobilised in carrageenan are discussed. A list of the most relevant patents in this field is presented as well as a list of enzymes and cell systems immobilised in carrageenan as described in the literature.

The potential of polymeric cryogels in bioseparation.

This is a review discussing the production and properties of cryogels (from the Greek kappa rho iota sigma (kryos) meaning frost or ice), immobilization of ligands in cryogels and the application of affinity cryogels in bioseparation. Cryotropic gel formation proceeds in a non-frozen liquid microphase existing in the macroscopically frozen sample. Due to the cryoconcentration of gel precursors in the non-frozen liquid microphase, cryogelation is characterised by a decrease in the critical concentration of gelation and an increase in gelation rates compared with traditional gelation at temperatures above freezing point. Cryogels can be obtained through the formation of both physically and covalently cross-linked heterogeneous polymer networks. Interconnected systems of macropores and sponge-like morphology are typical for cryogels, allowing unhindered diffusion of solutes of practically any size. Most of the water present in spongy cryogels is capillary bound and can be removed mechanically by squeezing. The properties of cryogels can be regulated by the temperature of cryogelation, the time the sample is kept in a frozen state and freezing/thawing rates, by the nature of the solvent and by the use of soluble and insoluble additives. The unique macroporous morphology of cryogels, in combination with osmotic, chemical and mechanical stability, makes them attractive matrices for chromatography of large entities such as protein aggregates, membrane fragments, viruses, cell organells and even whole cells. Special attention is given to immunosorption of viruses on cryogel-based sorbents. As chromatographic materials, cryogels can be used both in bead form and as spongy cylindrical blocks (monoliths) synthesized inside the chromatographic column. The macroporous nature of cryogels is also advantageous for their application as matrices in the immobilization of biocatalysts operating in both aqueous and organic solvents. New potential applications of cryogels are discussed.

Immobilization of Rhodococcus AJ270 and Useof Entrapped Biocatalyst for the Productionof Acrylic Acid

Immobilization of Rhodococcus AJ270 and Useof Entrapped Biocatalyst for the Productionof Acrylic Acid