Immobilization of Candida bombicola Cells on Free-Standing Organic-Gold Nanoparticle Membranes and Their Use as Enzyme Sources in Biotransformations

Abstract

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.

Purification by silica gel chromatography using dialysis tubing and characterization of sophorolipids produced from Candida bombicola grown on glucose and arachidonic acid

“…The partial mass spectrum of the hydroxylated fatty acids released from the sophorolipids was compared with standard 20-HETE mass spectrums (data not shown). Figure Phadtare et al 2004). The mass spectrum thus clearly indicated the hydroxyl group at the C20 position and the compound was 20-hydroxy-5Z,8Z,11Z,14Z-eicosatetraenoic acid (20-HETE).…”

“…Liberated hydroxyfatty acids were purified on 500 mg Aminopropyl Sep-Pak Cartridges (Waters) as described by Prabhune et al (2002) and Phadtare et al (2004). The isolated hydroxyeicosatetraenoic acids were methylated with diazomethane and silylated according to MacLauf and Rigaud (1982) using trimethylchlorosilane.…”

“…A Shimadzu GCMS QP 5050 automated quadrupole mass spectrometer operating in the electron impact mode was used for GC MS analysis as described by Phadtare et al (2004).…”

Plasmon-Enhanced Enzymatic Reactions: A Study of Nanoparticle-Enzyme Distance and Nanoparticle Loading-Dependent Enzymatic Activity

“…Moreover, these hybrid systems can enhance one or both of the functions of its components; for example, it was previously shown that plasmonic nanoparticles enhance the electro-catalytic response of a glucose oxidase system (glucose biosensor)[1] and the detection of various analytes of interest, such as hydrogen peroxide and uric acid [2], thiocholine [3], carcinoembryonic antigen [4] and DNA [5]. Several strategies for the immobilization of enzyme on plasmonic structures have been developed: layer-by-layer assembly[6], self-assembled monolayers[7], protein-antigen interactions[8], covalent attachment[9] and sol-gels[10]. Despite the existence of wide range of studies on enzyme/nanoparticle hybrids, the comparison of enzyme immobilization strategies and the effect of nanoparticle loading on surfaces on enzymatic activity were never reported.…”

Hybrid Systems Biomolecule-Polymeric Nanoparticle: Synthesis, Properties and Biotechnological Applications

“…This research group has recently shown that Yarrowia lipolitica [156] and Candida bombicola yeast cells [157] can be immobilized on patterned hydrophobic regions of thermally evaporated fatty lipid films. In the latter case, the enzyme cytochrome P450 present in the yeast cells was used to catalyze in situ ö and ö-1 hydroxylation of arachidonic acid (AA).…”