Thesis (Ph.D.). / Written for the Dept. of Food Science and Agricultural Chemistry. Title from title page of PDF (viewed 2008/05/12). Includes bibliographical references.

Aspergillus niger and Penicillium candidum were grown and harvested on days 6 and 8, which corresponded to their maximal biomasses and lipoxygenase (LOX) activities. The extracts were enriched with ammonium sulfate precipitation at 30 to 70% and 20 to 60% of saturation, respectively. The LOX activity was assayed with linoleic, linolenic and arachidonic acids as substrates. Both enriched microbial LOXs demonstrated preferential substrate specificities towards free fatty acids, over acyl esters of linoleic acid. The LOXs had the highest catalytic efficiency values (ratio of V max to Km) for linolenic acid biocatalysis. Major and minor pH optima at 5.0 and 10.5 were observed for A. niger, whereas for P. candidum they were at 6.0 and 8.5. Normal phase high-performance liquid chromatography (NP-HPLC) and gas-liquid chromatography/mass spectrometry (GC/MS) characterization of end products revealed that both LOXs produced the 10-hydroperoxide of linoleic acid (10-HPOD) at 15 to 16% of total isomers detected, respectively. Chiral studies of the P. candidum LOX catalyzed hydroperoxides revealed an excess in the production of (S) stereo-isomers resultant from linoleic, linolenic and arachidonic acids bioconversion. Penicillium camemberti was grown and harvested at its maximal biomass and LOX activity. The microbial extract was ultrafiltered (30 kDa NMWCO) and KCI (7.5 ppm) was added prior to lyophilization for the stabilization of enzyme activity. The LOX and hydroperoxide lyase (HPL) activities were assayed using linoleic acid and the 10-HPOD as substrates, respectively. The post-lyophilization residual activities were 93% and 223% for LOX and HPL, respectively. The long-term storage stability (-80°C) of the extract (KCI 7.5 ppm) was ~100% after 8 and 4 weeks for LOX and HPL, respectively. The investigated stabilizing chemical additives included glycine, mannitol, glycerol, sucrose and polyethylene glycol. The lowest Kinactivation values were observed with glycine with 0.136 and 0.0296 for LOX and HPL, respectively. Thermostability studies indicated that 5 and 10% (w/v) mannitol and glycine effectively stabilized LOX and HPL, respectively. Immobilization of an enzymatic extract from P. camemberti containing LOX and HPL activities was performed on EupergitRTMC and EupergitRTMC250L-iminodiacetate (IDA), respectively. The free and immobilized extracts both possessed LOX activity with a pH optimum of 6.0, whereas pH 6.0 and 4.0 were the optima of the HPL activity for free and immobilized extract, respectively. Optimal LOX reaction temperatures were 30 and 55°C for the free and immobilized extract, respectively, whereas 45 and 30°C were determined for the HPL activity of the free and immobilized extract, respectively. Long-term stability (-80°C) of the immobilized extract containing LOX and HPL activity showed residual activities of 82.6 and 93.8% after 4 and 8 weeks, respectively.

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Biomasses of Penicillium camemberti, Penicillium roqueforti and Geotrichum candidum strains were grown and harvested after a culture incubation period that corresponded to the maximal dry weight of mycelium as well as to lipoxygenase (LOX) activity. The crude enzymatic extracts were recovered from the homogenized mycelium cells and the partially purified LOX extracts were obtained by ammonium sulfate precipitation. Using linoleic acid as substrate, the partially purified LOX extracts from P. camemberti, P. roqueforti and G. candidum exhibited a major activity, at pH 6.50, 5.50 and 3.75, respectively, and a minor one at pH 8.00. The partially purified LOX extracts exhibited an overall preferential specificity towards free fatty acids, including linoleic, linolenic and arachidonic acids, than that for fatty acid acylglycerols, including mono-, di- and tri-linolein. The Km and Vmax values for partially purified LOXs were investigated. Normal phase high-performance liquid chromatography (NP-HPLC) and gas-liquid chromatography/mass spectrometry (GC/MS) analyses showed that the LOX activity of the partially purified LOX extracts converted mainly linoleic acid into the corresponding 9- and 13-hydroperoxides (HPODs); however, the production of a relatively important proportion of 10-HPOD, ranging from 4 to 9% of the total HPODs, was also demonstrated with the partially purified LOX extracts from Penicillium sp. The chiral phase HPLC analysis demonstrated the production, by the partially purified LOX extracts from Penicillium sp., of both (R)- and (S)-enantiomers of HPODs, with a slightly higher proportion of the (S)-enantiomers. The crude enzymatic extracts from Penicillium sp. were incubated, at pH 6.50, with individual racemic mixture of 9(R,S)-, 10(R,S)-, 12( R,S)- and 13(R,S)-HPODs, prepared by photooxidation and separated by NP-HPLC. The experimental results indicated that only 10( S)-HPOD isomer was cleaved by a hydroperoxide lyase activity that resulted by the for

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