Immobilization of Candida bombicola cells on free-standing organic-gold nanoparticle membranes and their use as enzyme sources in biotransformations.

Preparation of chemically functionalized biocompatible surfaces is of current interest, with application in the immobilization of various bioactive species such as DNA, enzymes, whole cells, etc. We report herein the one-step synthesis of a self-supporting gold nanoparticle membrane, its surface modification, and application in the immobilization of Candida bombicola (yeast) cells. The gold nanoparticle membrane is prepared by the spontaneous reduction of aqueous chloroaurate ions by a diamine at a liquid-liquid interface. The gold nanoparticles in the polymeric membrane may be capped with octadecylamine (ODA) molecules, thereby rendering the nanoparticle membrane hydrophobic. Exposure of the hydrophobized organic-gold nanoparticle membrane to C. bombicola yeast cells results in their binding to the membrane, possibly through nonspecific interactions such as hydrophobic interactions between the yeast cell walls and the ODA molecules. The enzyme cytochrome P450 present in the yeast cells immobilized on the organic-gold nanoparticle membrane was then used in the transformation of the arachidonic acid (AA) to sophorolipids followed by acid hydrolysis to form 20-hydroxyeicosatetraneoic acid (20-HETE). The organic-gold nanoparticle membrane-C. bombicola bioconjugate could be easily separated from the reaction medium and reused a number of times.

Enhancing the reusability of endoglucanase-gold nanoparticle bioconjugates by tethering to polyurethane microspheres.

The synthesis of polyurethane microsphere-gold nanoparticle "core-shell" structures and their use in the immobilization of the enzyme endoglucanase are described. Assembly of gold nanoparticles on the surface of polymer microspheres occurs through interaction of the nitrogens in the polymer with the nanoparticles, thereby precluding the need for modifying the polymer microspheres to enable such nanoparticle binding. Endoglucanse could thereafter be bound to the gold nanoparticles decorating the polyurethane microspheres, leading to a highly stable biocatalyst with excellent reuse characteristics. The immobilized enzyme retains its biocatalytic activity and exhibits improved thermal stability relative to free enzyme in solution. The high surface area of the host gold nanoparticles renders the immobilized enzyme "quasi free", while at the same time retaining advantages of immobilization such as ease of reuse, enhanced temporal and thermal stability, etc.

Immobilization and biocatalytic activity of fungal protease on gold nanoparticle-loaded zeolite microspheres.

Gold nanoparticles are excellent biocompatible surfaces for the immobilization of enzymes. However, separation of the gold nanoparticle-enzyme bioconjugate material from the reaction medium is often difficult. In this study, we investigate the assembly of the gold nanoparticles on the surface of the amine-functionalized zeolite microspheres in the formation of zeolite-gold nanoparticle "core-shell" structures and, thereafter, the use of this structure in immobilization of fungal protease. The assembly of gold nanoparticles on the zeolite surface occurs through the amine groups present in 3-aminopropyltrimethoxysilane (3-APTS). The fungal proteases bound to the massive "core-shell" structures were easily separated from the reaction medium by mild centrifugation and exhibited excellent reuse characteristics. The biocatalytic activity of fungal protease in the bioconjugate was marginally enhanced relative to the free enzyme in solution. The bioconjugate material also showed significantly enhanced pH and temperature stability and a shift in the optimum temperature of operation.

Invertase-lipid biocomposite films: preparation, characterization, and enzymatic activity.

The formation of biocomposite films of the industrially important enzyme invertase and fatty lipids under enzyme-friendly conditions is described. The approach involves a simple beaker-based diffusion protocol wherein invertase diffuses into the cationic lipid octadecylamine during immersion of the lipid film in the enzyme solution. Entrapment of invertase in the octadecylamine film is highly pH-dependent, underlining the role of attractive electrostatic interactions between the enzyme and the lipid in the biocomposite film formation. The kinetics of formation of the enzyme-lipid biocomposites has been studied by quartz crystal microgravimetry (QCM) measurements. The stability of the enzyme in the lipid matrix was confirmed by fluorescence spectroscopy and biocatalytic activity measurements. The biocatalytic activity of the invertase-lipid biocomposite films was comparable to that of the free enzyme in solution and showed marginally higher temperature stability. Particularly exciting was the excellent reuse characteristics of the biocomposite films, indicating potential industrial application of these films.

Water-dispersible tryptophan-protected gold nanoparticles prepared by the spontaneous reduction of aqueous chloroaurate ions by the amino acid.

The synthesis of water-dispersible amino-acid-protected gold nanoparticles by the spontaneous reduction of aqueous chloroaurate ions by tryptophan is described. Water-dispersible gold nanoparticles may also be obtained by the sequential synthesis of the gold nanoparticles by borohydride reduction of chloroauric acid followed by capping with tryptophan. Comparison of the proton NMR spectroscopic signatures from the tryptophan-protected gold nanoparticles obtained by the two processes indicated that the indole group in tryptophan is responsible for reduction of the aqueous chloroaurate ions. The reduction of the metal ions is accompanied by oxidative polymerization of the indole group of the tryptophan molecules and, consequently, some degree of cross-linking of the gold nanoparticles.

Free-standing nanogold membranes as scaffolds for enzyme immobilization.

We demonstrate herein the formation of a free-standing gold nanoparticle membrane and its use in the immobilization of the enzyme, pepsin. The nanogold membrane is synthesized by the spontaneous reduction of aqueous chloroaurate ions at the liquid-liquid interface by the bifunctional molecule bis(2-(4-aminophenoxy)ethyl) ether (DAEE) taken in chloroform. This process results in the formation of a robust, malleable free-standing nanogold membrane consisting of gold nanoparticles embedded in a polymeric background. Recognizing that gold nanoparticles are excellent candidates for immobilization of enzymes, we have immobilized pepsin on the nanogold membrane, leading to a new class of biocatalyst. A highlight of the new pepsin-nanogold biocatalyst is the ease with which separation from the reaction medium may be achieved. The catalytic activity of pepsin in the bioconjugate was comparable to that of the free enzyme in solution. The pepsin-nanogold membrane bioconjugate material exhibited excellent biocatalytic activity over 10 successive reuse cycles as well as enhanced pH, temperature, and temporal stability.

Candida bombicola cells immobilized on patterned lipid films as enzyme sources for the transformation of arachidonic acid to 20-HETE.

Preparation of biocompatible surfaces for immobilization of enzymes and whole cells is an important aspect of biotechnology due to their potential applications in biocatalysis, biosensing, and immunological applications. In this report, patterned thermally evaporated octadecylamine (ODA) films are used for the immobilization of Candida bombicola cells. The attachment of the cells to the ODA film surface occurs possibly through nonspecific interactions such as hydrophobic interactions between the cell walls and the ODA molecules. The enzyme cytochrome P450 present in the immobilized yeast cells on the ODA film surface was used for the transformation of the arachidonic acid to 20-hydroxyeicosatetraenoic acid (20-HETE). The assembly of cells on the hydrophobic ODA surface was confirmed by quartz crystal microgravimetry (QCM), Fourier transform infrared spectroscopy (FTIR), and scanning electron microscopy (SEM). SEM images confirmed the strong binding of the yeast cells to the ODA film surface after biocatalytic reactions. Moreover, the biocomposite films could be easily separated from the reaction medium and reused.

Improved performance of preordered fungal protease-stearic acid biocomposites: enhanced catalytic activity, reusability, and temporal stability.

In an earlier report on fungal protease (F-prot)-fatty acid biocomposite film formation [Gole et al. Anal. Chem. 2000, 72, 4301], it was observed that the biocatalytic activity of the immobilized enzyme was comparable to that of the free enzyme in solution. However, a somewhat negative aspect of the protocol was the steady loss in activity during reuse and storage of the biocomposite film. In this paper, we address the latter issues and demonstrate successful attempts toward the realization of efficient biocomposite films with enhanced biological activity, temporal stability, and excellent reusability. The improved performance of the F-prot-stearic acid biocomposite is accomplished by preordering the fatty acid film by incorporation of Pb(2+) ions into the lipid matrix prior to enzyme immobilization. The lead cation induces lamellar ordering in the lipid film and thus facilitates diffusion of the F-prot molecules into the lipid matrix and accessibility of the substrate molecules (hemoglobin, Hb) to the entrapped F-prot enzyme molecules. The preordering consequently leads to effective control of the "mass transport" problem and might be responsible for the enhanced biological activity ( approximately 36%) of the enzyme molecules in the biocomposite in comparison with the free enzyme in solution, as well the excellent reusability of the composite film. In addition to biocatalytic activity measurements, the formation and characterization of the F-prot-lead stearate biocomposite films was done by quartz crystal microgravimetry and X-ray diffraction.

Penicillin G acylase-fatty lipid biocomposite films show excellent catalytic activity and long term stability/reusability.

The formation of biocomposite films of the pharmaceutically important enzyme penicillin G acylase (PGA) and fatty lipids under enzyme-friendly conditions is described. The approach involves a simple beaker-based diffusion protocol wherein the enzyme diffuses into the lipid film during immersion in the enzyme solution, thereby leading to the formation of a biocomposite film. The incorporation of the enzyme in both cationic as well as anionic lipids suggests the important role of secondary interactions such as hydrophobic and hydrogen bonding in the enzyme immobilization process. The kinetics of formation of the enzyme-lipid biocomposites has been studied by quartz crystal microgravimentry (QCM) measurements. The stability of the enzyme in the lipid matrix was confirmed by Fourier transform infrared spectroscopy (FTIR) and biocatalytic activity measurements. Whereas the biological activity of the lipid-immobilized enzyme was marginally higher than that of the free enzyme, the biocomposite film exhibited increased thermal/temporal stability. Particularly exciting was the observation that the biocomposite films could be reused in biocatalysis reactions without significant loss in activity, which indicates potentially exciting biomedical/industrial application of these films.