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Cell-free synthesis has been adopted in the bioconversion process due to its known advantages, such as fast production rate, high product content, and no substrate/product inhibition effect. In this study, the cell-free supernatant of Pseudomonas aeruginosa was used to improve the production of 7,10‐dihydroxy‐8(E)‐octadecenoic acid (DOD) from oleic acid. DOD production using cell-free supernatant demonstrated reduction in bioconversion duration and higher product concentration than conventional method using whole cell culture. The maximum DOD concentration (6.41 g/L) was obtained after 36 h of biotransformation using 1 % v/v oleic acid as a substrate with a productivity of 0.178 g/L/h and a yield of 74.8 %. DOD concentration, productivity, and yield using cell-free supernatant were 2.12, 7.12, and 2.22 times higher, respectively, than using the conventional whole cell culture method. Of the carbon and nitrogen sources used in pre-culture, galactose and sodium glutamate along with diammonium phosphate were found to be the most effective for DOD production. An incubation temperature of 27 °C and pH 8.0 were found to be most favorable for DOD production. In addition, sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis demonstrated the presence of enzymes related to DOD production in the cell-free supernatant, which was substantiated by performing DOD production experiment using the supernatant enzymes extracted from protein gel bands with oleic acid as a substrate. To the best of our knowledge, this is the first report on DOD production using a cell-free supernatant and verifying the existence of the relevant enzymes in the cell-free supernatant. Compared to whole cell process, cell-free DOD production holds several advantages, including higher DOD productivity which could be beneficial for large-scale production.

Diisononyl adipate is a plasticizer which has excellent property of low temperature resistance and is permitted in food contact materials. In this study, diisononyl adipate was synthesized from adipic acid and isononyl alcohol in a solvent-free system via immobilized lipase-catalyzed esterification. Liquid Eversa lipase from Thermomyces lanuginosus was immobilized on Lewatit VP OC 1600 carrier. The effects of temperature, substrate molar ratio, water activity of enzyme, vacuum, and enzyme loading on the reaction efficiency were investigated. Application of vacuum played a key role to achieve a 100% conversion. The optimal temperature, molar ratio (adipic acid to isononyl alcohol), water activity of enzyme, vacuum were 50 °C, 1:3, 0.75, 13.3 kPa, and 10% (based on weight of total substrate). Under these conditions, 100% conversion was achieved within 6 h.

Acetyl esterases are important in the complete degradation of acetylated polysaccharides, such as pectins and xylans. We isolated the gene encoding acetyl xylan esterase (AfAXE) from a genomic λ library of Aspergillus ficuum. We cloned the corresponding cDNA by RT-PCR. The Afaxe gene contained two introns, one TATAA box, and two CAAT-like boxes. The transcription initiation site was 61 bp upstream of the start codon. The deduced amino acid sequence consisted of a putative 28-amino acid leader peptide and a mature protein with an estimated molecular mass of 29.5 kDa. The nearest homolog of the cloned gene was acetyl xylan esterase of A. niger . The cloned gene was placed in a Pichia expression vector and expressed in Pichia pastoris . The culture filtrate of the transformant liberated acetyl moieties from p -nitrophenyl acetate and its activity reached 75.8 IU/ml, which was over 100-fold greater than the activity of the native enzyme expressed in A. ficuum . The cloned enzyme catalyzed the release of acetic acid from acetylated hardwood xylan, confirming that the cloned gene encoded an acetyl xylan esterase of A. ficuum . The native and recombinant acetyl xylan esterases were purified from the culture filtrates of A. ficuum and P. pastoris , respectively. Both enzymes had approximately the same optimal temperature (37 °C) and pH (7.0). The recombinant protein had greater tolerance for alkaline conditions (pH≥7.0), but was less thermostable above 55 °C.

Hydroxy fatty acids have gained important attentions because of their special properties such as higher viscosity and reactivity compared with other non-hydroxy fatty acids. Previously we reported that a novel bacterial strain Pseudomonas aeruginosa PR3 converted linoleic acid to the equimolar mixture of two compounds, 9,10,13- trihydroxy-11( E )-octadecenoic acid (9,10,13-THOD) and 9,12,13-trihydroxy-10( E )-octadecenoic acid (9,12,13-THOD) which showed anti-fungal activities (Kim, H. Gardner, H.W., Hou, C.T., J. Ind. Microbiol. Biotechnol. 2000, 25, 109–115). In this study we report for the first time the effect of several metal ions as catalytic agents of lipid peroxidation on the production of total THODs by PR3. Among eight different metal ions tested, Fe +2 and Cu +2 were effective to produce THODs. However bacterial growth was not significantly affected by the existence of metal ions tested. Fe +2 ion requirement was specific to THOD production from linoleic acid but not to other incorporation of dihydroxyl group on oleic acid and ricinoleic acid by PR3.

7,10-Dihydroxy-8(E)-octadecenoic acid (DOD), one of hydroxy fatty acids, was successfully produced from oleic acid and natural vegetable oils containing oleic acid by a bacterial strain Pseudomonas aeruginosa (PR3). However, biological properties of DOD remained unknown so far. In this study, as a trial to determine the biological properties of DOD molecule, antibacterial activities of DOD against plant pathogenic bacteria were determined qualitatively and quantitatively. DOD presented strong antibacterial activities against all the bacterial strains tested with MIC value being in the range of 125-1000μg/ml and there was no sensitivity preference detected between Gram-positive and Gram-negative strains.