Reuse Of Biocatalyst Research Articles

Generation of a Highly Stable Reusable Biocatalyst by Entrapment of an Oligomeric Enzyme in Ultra-Large-Pore Mesoporous Silica

Phenylalanine ammonia lyase (PAL, E.C.4.3.1.24), was entrapped in ultra-large-pore mesoporous silica (ULPS, 23 nm pore diameter) generating a recyclable, separable biocatalyst. The entrapped ULPS-PAL materials showed excellent stabilization, even after significant exposure to prolonged heating. Additionally, the entrapped ULPS-PAL materials showed extremely high catalytic activity in the deamination of l-phenylalanine to trans-cinnamic acid in aqueous solution and were recovered and recycled up to five times without any observable loss in activity. This approach is simple and capitalizes on the facile synthesis and easy recoverability of mesoporous silicas to generate a stable, reusable PAL-based biocatalyst.

Lipase Immobilized on Graphene Oxide As Reusable Biocatalyst

Graphene oxide (GO) has shown promising applications as supports for heterogeneous catalysts. This study reports the functionalization of GO by (3-mercaptopropyl) trimethoxysilane to immobilize lipase. The immobilization is carried out by the site-specific covalent binding between thiol groups of lipase and thiolsulfonate groups on modified GO. For the hydrolysis of p-nitrophenylpalmitate, the obtained GO–lipase shows catalytic performance (93.4%) comparable with that of the free lipase, and the reusability after 10 times is 69.9%. Compared with the free lipase, the GO–lipase exhibits good pH, thermal, and storage stability, which are important in practical applications.

Synthesis, properties, and application in peptide chemistry of a magnetically separable and reusable biocatalyst

Enzyme-catalyzed chemical processes are selective, very productive, and generate little waste. Nevertheless, they may be optimized using enzymes bound to solid supports, which are particularly important for protease-mediated reactions since proteases undergo fast autolysis in solution. Magnetic nanoparticles are suitable supports for this purpose owing to their high specific surface area and to be easily separated from reaction media. Here we describe the immobilization of bovine α-chymotrypsin (αCT) on silica-coated superparamagnetic nanoparticles (Fe3O4@silica) and the characterization of the enzyme-nanoparticle hybrid (Fe3O4@silica-αCT) in terms of protein content, properties, recovery from reaction media, application, and reuse in enzyme-catalyzed peptide synthesis. The results revealed that (i) full acid hydrolysis of the immobilized protease followed by amino acid analysis of the hydrolyzate is a reliable method to determine immobilization yield; (ii) despite showing lower amidase activity and a lower K cat/K m value for a specific substrate than free αCT, the immobilized enzyme is chemically and thermally more stable, magnetically recoverable from reaction media, and can be consecutively reused for ten cycles to catalyze the amide bond hydrolysis and ester hydrolysis of the protected dipeptide Z-Ala-Phe-OMe. Altogether, these properties indicate the potential of Fe3O4@silica-αCT to act as an efficient, suitably stable, and reusable catalyst in amino acid, peptide, and protein chemistry as well as in proteomic studies.

Enzymatic dynamic kinetic resolution of racemic N-formyl- and N-carbamoyl-amino acids using immobilized L-N-carbamoylase and N-succinyl-amino acid racemase.

Taking advantage of the catalytic promiscuity of L-carbamoylase from Geobacillus stearothermophilus CECT43 (BsLcar) and N-succinyl-amino acid racemase from Geobacillus kaustophilus CECT4264 (GkNSAAR), we have evaluated the production of different optically pure L-α-amino acids starting from different racemic N-formyl- and N-carbamoyl-amino acids using a dynamic kinetic resolution approach. The enzymes were immobilized on two different solid supports, resulting in improved stability of the enzymes in terms of thermostability and storage when compared to the enzymes in solution. The bienzymatic system retained up to 80% conversion efficiency after 20 weeks at 4 °C and up to 90% after 1 week at 45 °C. The immobilization process also resulted in a great enhancement of the activity of BsLcar toward N-formyl-tryptophan, showing for the first time that substrate specificity of L-carbamoylases can be influenced by this approach. The system was effective for the biosynthesis of natural and unnatural L-amino acids (enantiomeric excess (e.e.) >99.5%), such as L-methionine, L-alanine, L-tryptophan, L-homophenylalanine, L-aminobutyric acid, and L-norleucine, with a higher performance toward N-formyl-α-amino acid substrates. Biocatalyst reuse was studied, and after 10 reaction cycles, over 75% activity remained.

Efficient Magnetic Recycling of Covalently Attached Enzymes on Carbon-Coated Metallic Nanomagnets

In the pursuit of robust and reusable biocatalysts for industrial synthetic chemistry, nanobiotechnology is currently taking a significant part. Recently, enzymes have been immobilized on different nanoscaffold supports. Carbon coated metallic nanoparticles were found to be a practically useful support for enzyme immobilization due to their large surface area, high magnetic saturation, and manipulatable surface chemistry. In this study carbon coated cobalt nanoparticles were chemically functionalized (diazonium chemistry), activated for bioconjugation (N,N-disuccinimidyl carbonate), and subsequently used in enzyme immobilization. Three enzymes, β-glucosidase, α-chymotrypsin, and lipase B were successfully covalently immobilized on the magnetic nonsupport. The enzyme-particle conjugates formed retained their activity and stability after immobilization and were efficiently recycled from milliliter to liter scales in short recycle times.

Optimized enzymatic synthesis of the food additive polyglycerol polyricinoleate (PGPR) using Novozym® 435 in a solvent free system

Polyglycerol polyricinoleate (PGPR) is used as an emulsifier in the food industry, especially in chocolate coatings and chocolate bars. PGPR improves the characteristics of molten chocolate by reducing yield stress, facilitating the coating of confectionery pieces, while limiting the amount of cocoa butter involved.The enzymatic synthesis of PGPR catalyzed by lipases presents several advantages over chemical synthesis, including enzyme specificity and the mild conditions needed, thereby avoiding undesirable side-reactions and by-products. A novel process to synthesize PGPR using a biocatalyst, Novozym® 435, is presented. Novozym® 435 is appropriate for catalyzing both the reactions involved in this process. A PGPR fulfilling European specifications for this food additive as well as recommendations set out in the Food Chemical Codex, was obtained using a discontinuous vacuum reactor with a dry nitrogen flow. In addition, the biocatalyst reuse would decrease costs. Moreover, it was confirmed that the ability to obtain PGPR in a one-step reaction significantly shortens the time required.

PVA/chitosan–glutaraldehyde cross-linked nitrile hydratase as reusable biocatalyst for conversion of nitriles to amides

• Facile method for immobilization of nitrile hydratase using PVA/chitosan. • Characterization by FTIR, SEM, AFM, DSC–TGA and XRD. • High catalytic activity, thermal stability and increased tolerance to the varied pH. • Hydration of 3-cyanopyridine with excellent reusability. • Kinetic parameters of free and immobilized NHase from Lineweaver–Burk plot. The present work aims at development of facile method for immobilization of Rhodococcus rhodochrous ATCC BAA-870 nitrile hydratase (NHase) using poly(vinylalcohol) (PVA)/chitosan biocompatible complex and investigation of its potential application for biocatalytic production of amide from nitrile. The morphology, structure and properties of immobilized nitrile hydratase were characterized by FTIR, SEM, AFM, DSC–TGA and XRD. The morphological characterization indicates that the PVA/chitosan–GA cross-linked NHase forms porous structure with smooth and uniform surface. The biocatalyst preparation is thermally stable compared to its free counterpart when subjected to thermal degradation studies. The immobilized NHase exhibited greatly effective catalytic activity, thermal stability and increased tolerance to the varied pH conditions for nitrile hydration of 3-cyanopyridine. Furthermore, the operational stability of the biocatalyst was improved significantly by immobilization, and it retained good activity up to eight successive batches of reaction. Kinetic parameters of free and immobilized NHase were determined from Lineweaver–Burk plot.

Efficient desymmetrization of 4,6-di-O-benzyl-myo-inositol by Lipozyme TL-IM

The enantioselective enzymatic desymmetrization of 4,6-di-O-benzyl-myo-inositol, a myo-inositol derivative, was effectively catalyzed by Thermomyces lanuginosus lipase (TL-IM). The product 1D-1-O-acetyl-4,6-di-O-benzyl-myo-inositol, a useful precursor to inositol phosphates, was obtained in excellent yield and enantiomeric excess. Through the investigation of the effects of solvent, biocatalyst load, and temperature, a more economical procedure resulted. The feasibility of biocatalyst reuse was also shown.

Efficient Biocatalysis with Immobilized Enzymes or Encapsulated Whole Cell Microorganism by Using the SpinChem Reactor System

The chambers of Trivial Pursuit: Locking immobilized enzymes or encapsulated whole cells into a compartment that is connected to the stirrer enables efficient reactions and facilitates the reuse of biocatalysts by using this SpinChem system. R-ATA=(R)-amine transaminase.

ChemInform Abstract: Potential of Different Enzyme Immobilization Strategies to Improve Enzyme Performance.

AbstractReview: about 150 refs.

Inclusion bodies of fuculose‐1‐phosphate aldolase as stable and reusable biocatalysts

Fuculose-1-phosphate aldolase (FucA) has been produced in Escherichia coli as active inclusion bodies (IBs) in batch cultures. The activity of insoluble FucA has been modulated by a proper selection of producing strain, culture media, and process conditions. In some cases, when an optimized defined medium was used, FucA IBs were more active (in terms of specific activity) than the soluble protein version obtained in the same process with a conventional defined medium, supporting the concept that solubility and conformational quality are independent protein parameters. FucA IBs have been tested as biocatalysts, either directly or immobilized into Lentikat beads, in an aldolic reaction between DHAP and (S)-Cbz-alaninal, obtaining product yields ranging from 65 to 76%. The production of an active aldolase as IBs, the possibility of tailoring IBs properties by both genetic and process approaches, and the reusability of IBs by further entrapment in appropriate matrices fully support the principle of using self-assembled enzymatic clusters as tunable mechanically stable and functional biocatalysts.

Lipase incorporated ionic liquid polymers as active, stable and reusable biocatalysts

The aim of this study was to develop ionic liquid (IL) polymer materials incorporating enzymes that can be used as active, stable and reusable biocatalysts. To this goal, Candida rugosa lipase has been microencapsulated in surfactant aggregates formed in an IL monomer or the solution of an IL monomer/IL and then incorporated into polymer frameworks through the free radical polymerization of an IL (1-vinyl-3-ethylimidazolium bis(trifluoromethyl-sulfonyl) amide) ([veim][Tf(2)N]). The activity, stability and reusability of such IL polymer materials containing lipase were evaluated using lipase-catalyzed hydrolysis of p-nitrophenyl butyrate (p-PNB) as a model reaction. Lipase encapsulated within ionic liquid polymer materials remained active and exhibited excellent stability in aqueous solutions. More importantly, these biopolymer materials retained most of their activity after five reaction cycles, in which biopolymers were recovered from the reaction mixture simply by centrifugation. This study promulgates a direction toward the design of IL - an interesting class of tunable and designable solvents - based polymer materials containing biomolecules via a combination of polymer and supramolecular chemistry for diverse applications.

Potential of Different Enzyme Immobilization Strategies to Improve Enzyme Performance

AbstractEnzyme biocatalysis plays a very relevant role in the development of many chemical industries, e.g., energy, food or fine chemistry. To achieve this goal, enzyme immobilization is a usual pre‐requisite as a solution to get reusable biocatalysts and thus decrease the price of this relatively expensive compound. However, a proper immobilization technique may permit far more than to get a reusable enzyme; it may be used to improve enzyme performance by improving some enzyme limitations: enzyme purity, stability (including the possibility of enzyme reactivation), activity, specificity, selectivity, or inhibitions. Among the diverse immobilization techniques, the use of pre‐existing supports to immobilize enzymes (via covalent or physical coupling) and the immobilization without supports [enzyme crosslinked aggregates (CLEAs) or crystals (CLECs)] are the most used or promising ones. This paper intends to give the advantages and disadvantages of the different existing immobilization strategies to solve the different aforementioned enzyme limitations. Moreover, the use of nanoparticles as immobilization supports is achieving an increasing importance, as the nanoparticles versatility increases and becomes more accessible to the researchers. We will also discuss here some of the advantages and drawbacks of these non porous supports compared to conventional porous supports. Although there are no universal optimal solutions for all cases, we will try to give some advice to select the optimal strategy for each particular enzyme and process, considering the enzyme properties, nature of the process and of the substrate. In some occasions the selection will be compulsory, for example due to the nature of the substrate. In other cases the optimal biocatalyst may depend on the company requirements (e.g., volumetric activity, enzyme stability, etc).

Immobilization of Delftia tsuruhatensis in macro-porous cellulose and biodegradation of phenolic compounds in repeated batch process

Delftia tsuruhatensis BM90, previously isolated from Tyrrhenian Sea and selected for its ability to degrade a wide array of phenolic compounds, was immobilized in chemically modified macro porous cellulose. The development of bacterial adhesion on the selected carrier was monitored by scanning electron microscopy. Evident colonization started already after 8h of incubation. After 72h, almost all the carrier surface was covered by the bacterial cells. Extracellular bacterial structures, such as pili or fimbriae, contributed to carrier colonization and cell attachment. Immobilized cells of D. tsuruhatensis were tested for their ability to biodegrade a pool of 20 phenols in repeated batch process. During the first activation batch (72h), 90% of phenols degradation was obtained already in 48h. In the subsequent batches (up to 360h), same degradation was obtained after 24h only. By contrast, free cells were slower: to obtain almost same degradation, 48h were needed. Thus, process productivity, achieved by the immobilized cells, was double than that of free cells. Specific activity was also higher suggesting that the use of immobilized D. tsuruhatensis BM90 could be considered very promising in order to obtain an efficient reusable biocatalyst for long-term treatment of phenols containing effluents.

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.

Multilayer enzyme-coupled magnetic nanoparticles as efficient, reusable biocatalysts and biosensors

Herein we report the development of a highly active, magnetically retrievable and reusable biocatalyst using multilayer enzyme coupled-magnetic nanoparticles (MNPs) prepared by layer-by-layer assembly using two well-studied enzymes, horseradish peroxidase (HRP) and glucose oxidase (GOX), as a model enzyme system. We show that by combining the use of a biocompatible linker as well as biospecific immobilisation, the first layer enzyme in our HRP(1)-MNP system retains the native activity of the enzyme in solution, and the overall catalytic activity of the multilayer enzyme system, HRP(x)-MNP, increases linearly with the increasing number of enzyme layers. Furthermore, the HRP(x)-MNP system can be conveniently retrieved by using an external magnetic field and reused for 10 consecutive cycles without apparent reduction of catalytic activity. We also report the development of a novel coupled bienzyme, GOX/HRP(x)-MNP, system that can perform bi-enzymatic reactions to couple the colourless GOX-catalyzed reaction to the chromophoric HRP-catalyzed reaction via H(2)O(2) production. This model bienzyme-MNP system can be used for simple, rapid colorimetric quantification of micromolar glucose.

Self-Assembled Enzyme Capsules in Ionic Liquid [BMIM][BF4] as Templating Nanoreactors for Hollow Silica Nanocontainers

Most of the self-assembly studies have hitherto explored the aqueous media as fluid phase for self-assembly of amphiphilic biomacromolecules, wherein architectural modification of biomolecules is generally a prerequisite for self-assembly of modified biomolecules. We demonstrate for the first time that ionic liquids can act as nonaqueous designer solvents to self-assemble amphiphilic biomacromolecules without requiring their prior modification. To this end, we show that enzyme (phytase) molecules self-assembled in the presence of an appropriate ionic liquid, resulting in the formation of enzyme capsules. Phytase capsules synthesized using this approach were further used as templating nanoreactors for the synthesis of enzyme-containing hollow silica nanocontainers. In situ immobilized phytase enzyme in the silica nanocontainers, when subjected to enzyme-reusability application, establishes them as excellent reusable biocatalysts.

Cross-linked enzyme aggregates of recombinant Pseudomonas putida nitrilase for enantioselective nitrile hydrolysis

The cross-linked enzyme aggregate (CLEA) method is used for the dual purpose of combining both the purification and immobilization of enzyme in one step. The present work involved the preparation of a carrier-free, highly active reusable biocatalyst (nitrilase) which encounters least mass-transfer limitations with higher thermal and storage stability. The effect of type of aggregating agent, its concentration as well as that of cross-linking agent was studied. Nitrilase aggregates were prepared using ammonium sulphate (35%) precipitation followed by cross-linking with glutaraldehyde (125mM) which rendered 70% activity retention. The various cross-linking parameters were optimized in order to increase the activity retention. Stability in terms of temperature, reusability and leaching were also examined. The CLEA preparation showed residual nitrilase activity on repeated use. A highly stable CLEA of nitrilase was finally prepared with maximum activity recovery.

Rapid Production of Nitrilase Containing Silica Nanoparticles Offers an Effective and Reusable Biocatalyst for Synthetic Nitrile Hydrolysis

Rapid and efficient immobilization of nitrilase within silica nanoparticles overcomes many hurdles associated with biocatalysis. A water-miscible dendrimer catalyzes the condensation of silicic acid to silica dioxide, entrapping electrostatically bound nitrilase molecules. Michaelis−Menten kinetics shows encapsulated nitrilase functions similarly to free nitrilase in solution. Additionally, HPLC analysis demonstrates that simple benchtop separation and recycling of the biocatalyst over 10 individual reactions are achieved without significant loss of enzyme and/or function. These findings broaden the use of nitrilases in the production of fine chemicals as well as general syntheses by overcoming some of the traditional barriers associated with enzyme reagents and nitrile conversion.

Enzyme encapsulation in microparticles composed of polymerized ionic liquids for highly active and reusable biocatalysts

Horseradish peroxidase (HRP) is encapsulated in polymerized ionic liquid microparticles (pIL-MP), which are prepared by polymerization of 1-vinyl-3-ethylimidazolium bromide in the presence of the crosslinker N,N'-methylenebis(acrylamide) in a concentrated water-in-oil (W/O) emulsion. pIL-MP encapsulating HRP chemically-modified with comb-shaped polyethylene glycol (PM(13)-HRP) exhibit excellent activity for guaiacol oxidation in an aqueous solution. The PM(13)-HRP in pIL-MP shows more than 2-fold higher activity than that of the enzyme encapsulated in a polyacrylamide microparticle. The catalytic activity declines with an increase in the crosslinker concentration of the pIL-MP, probably due to suppression of substrate diffusion. The activity of PM(13)-HRP in pIL-MP depends on the external environment of the gel (i.e. pH and temperature). The pIL-MP are easily recovered from the reaction mixture by centrifugation, which makes it possible to recycle the biocatalyst for repeated oxidation reactions.