Haralambos Stamatis

Development of effective nanobiocatalytic systems through the immobilization of hydrolases on functionalized carbon-based nanomaterials

In this study we report the use of functionalized carbon-based nanomaterials, such as amine-functionalized graphene oxide (GO) and multi-walled carbon nanotubes (CNTs), as effective immobilization supports for various lipases and esterases of industrial interest. Structural and biochemical characterization have revealed that the curvature of the nanomaterial affect the immobilization yield, the catalytic behavior and the secondary structure of enzymes. Infrared spectroscopy study indicates that the catalytic behavior of the immobilized enzymes is correlated with their α-helical content. Hydrolases exhibit higher esterification activity (up to 20-fold) when immobilized on CNTs compared to GO. The covalently immobilized enzymes exhibited comparable or even higher activity compared to the physically adsorbed ones, while they presented higher operational stability. The enhanced catalytic behavior observed for most of the hydrolases covalently immobilized on amine-functionalized CNTs indicate that these functionalized nanomaterials are suitable for the development of efficient nanobiocatalytic systems.

Enzymes in non-conventional phases

The use of enzymes for technical applications has become increasingly important in different areas of biotechnology such as the food or pharmaceutical industries. Various processes have been developed using soluble or immobilized enzymes mainly in aqueous reaction phases. However, the catalytic activity of enzymes not only in water, but also in other solvents was first investigated at the beginning of the century (Bourquelot, E. and Bridel, M., 1911). Apart from water as the exclusive solvent and reaction phase, many kinds of solvent systems for enzymatic reactions became the subject of intensive research over the last 10-15 years (Buckland, Dunnill and Lilly, 1975; Klibanov, Samokhin, Martinek and Berezin, 1977; Nakanishi and Matsuno, 1986; Chen and Sih, 1989; Klibanov, 1990; Blanch, 1992; Carrea et al., 1992).

Graphene-based nanobiocatalytic systems: recent advances and future prospects.

Graphene-based nanomaterials are particularly useful nanostructured materials that show great promise in biotechnology and biomedicine. Owing to their unique structural features, exceptional chemical, electrical, and mechanical properties, and their ability to affect the microenvironment of biomolecules, graphene-based nanomaterials are suitable for use in various applications, such as immobilization of enzymes. We present the current advances in research on graphene-based nanomaterials used as novel scaffolds to build robust nanobiocatalytic systems. Their catalytic behavior is affected by the nature of enzyme-nanomaterial interactions and, thus, the availability of methods to couple enzymes with nanomaterials is an important issue. We discuss the implications of such interactions along with future prospects and possible challenges in this rapidly developing area.

Lipase immobilization on smectite nanoclays: Characterization and application to the epoxidation of α-pinene

The immobilization of lipase B from Candida antarctica on smectite group nanoclays (Laponite, SWy-2 and Kunipia), as well as on their organically modified derivatives, was investigated. A combination of techniques, namely X-ray diffraction, thermal analysis, X-ray photoelectron and FT-IR spectroscopy, was used for characterization of the novel immobilized biocatalyst. Structural and biochemical characterization have revealed that the hydrophobic microenvironment created by the organo-modified clays induces minor changes on the secondary structure of the enzyme, resulting in enhanced catalytic behaviour in hydrophobic media. The immobilized lipase on such modified nanoclays can be effectively applied for the indirect epoxidation of alpha-pinene using hydrogen peroxide as substrate. The amount of alpha-pinene epoxide produced in a single-step biocatalytic process is up to 3-fold higher than that of free enzyme or enzyme immobilized in non-modified clays. Moreover, lipase immobilized in modified clays retains up to 90% of its initial activity, even after 48h of incubation in the presence of oxidant, and up to 60% after four reaction cycles, while other forms of the enzyme retain less than 10%.

Antimicrobial activity of acidic xylo-oligosaccharides produced by family 10 and 11 endoxylanases

Acidic oligosaccharides were obtained from birchwood xylan by treatment with a Thermoascus aurantiacus family 10 and a Sporotrichum thermophile family 11 endoxylanases. The main difference between the products liberated by xylanases of family 10 and 11 concerned the length of the products containing 4-O-methyl-D-glucuronic acid. The xylanase from T. aurantiacus liberate from glucuronoxylan an aldotetrauronic acid as the shortest acidic fragment in contrast with the enzyme from S. thermophile, which liberated an aldopentauronic acid. Acidic xylooligosaccharides were separated from the hydrolysate by anion-exchange and size-exclusion chromatography (SEC) and the primary structure was determined by 13C NMR spectroscopy. The acidic xylo-oligosaccharides were tested against three Gram-positive and three Gram-negative aerobically grown bacteria, as well as against Helicobacter pylori. Aldopentauronic acid was proved more active against the Gram-positive bacteria and against H. pylori.

Purification and characterization of a Fusarium oxysporum feruloyl esterase (FoFAE-I) catalysing transesterification of phenolic acid esters

An extracellular feruloyl esterase (FoFAE-I) from the culture filtrates of Fusarium oxysporum F3 was purified to homogeneity by ion-exchange, hydrophobic interaction and gel filtration chromatograp ...

Microemulsion-based organogels as matrices for lipase immobilization

Organogels based on water-in-oil microemulsions can be formed using various natural polymers such as gelatin, agar or cellulose derivatives. Enzymes entrapped in the water core of the microemulsion can keep their activity and enhance their stability within the gel matrix. The importance of the microemulsion based organogels (MBGs) leans on their numerous potential biotechnological applications. An important example is the use of various lipase microemulsion systems for hydrolytic or synthetic reactions. In this review, several MBGs are being evaluated as immobilization matrices for various enzymes. The main subject focuses on the parameters that affect the use of MBGs as media for bioorganic reactions using lipases as catalysts.

Efficient enzymatic preparation of hydroxycinnamates in ionic liquids enhances their antioxidant effect on lipoproteins oxidative modification

Biocatalytic lipophilization of hydroxycinnamic acids was performed in several BF(4)(-) and PF(6)(-) imidazolium ionic liquids using immobilized lipases. The influence of various reaction parameters on the performance of the biocatalytic process was pointed out, using as model reaction the esterification of ferulic acid. The biocatalytic lipophilization strongly depended on the ion composition of ionic liquids used. Conversions and initial reaction rates were significantly higher in PF(6)(-) as compared with BF(4)(-) ionic liquids and commonly used organic solvents. The high enzyme stability and the relative solubility of substrate versus product in PF(6)(-) ionic liquids can account for the improved synthesis of lipophilic ferulates. These lipophilic derivatives, when used at a concentration of up to 400-fold lower than the parental compound, efficiently inhibited the oxidation of isolated LDL, HDL and total serum in vitro. Moreover, it has been shown for the first time that the lipophilic ferulates improve the antioxidant efficiency of the HDL(3c) towards LDL in vitro oxidation.

Lipase-mediated epoxidation of α-pinene

This work describes the lipase-mediated synthesis of α-pinene oxide at ambient temperature. The immobilized lipase from Candida antarctica (Novozyme 435) is used to generate peroxyoctanoic acid directly from octanoic acid and hydrogen peroxide. The peroxy acid formed is then applied for in situ oxidation of α-pinene. High conversion of α-pinene to α-pinene oxide (approximately 70%) was achieved when using a two-phase system of toluene and water. Various parameters affecting the conversion of α-pinene to α-pinene oxide were studied.

Biocatalysis using microemulsion-based polymer gels containing lipase

Abstract Natural gelling agents such as gelatin, agar and κ-carrageenan have been tested for the formation of lecithin microemulsion-based gels as well as hydrogels (without surfactant and oil). The results presented in this work provide information concerning the utility of these solid gels as lipase immobilization matrices. It was found that lipase from Pseudomonas cepacia keeps its catalytic function after entrapment in the gels, catalyzing the esterification reaction of propanol with lauric acid in various hydrocarbons at room temperature. Various parameters which affect the lipase catalytic behavior such as the nature and the concentration of the gelling agent, as well as the concentration of the biocatalyst and the mole ratio of the substrates have been examined. High yields (80%) were obtained with agar and κ-carrageenan organogels in isooctane. The remaining lipase activity, in repeated syntheses was found to depend on the nature of the biopolymer used for the formation of the organogels. Gelatin and agar microemulsion-based gels showed the highest operational stability.