Candida rugosa lipase
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Isolation of Erucic Acid from Mustard Seed Oil by Candida rugosa lipase
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| Author |
Miss Raviwan Tinoi
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| M.S. |
Biotechnology |
| Examining Committee |
Asst. Prof. Dr. Nuansri Rakariyatham
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Chairman |
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Assoc. Prof. Dr. Duang Buddhasukh
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Member |
|
Dr. Hataichanoke Niamsup
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Member |
Mustard seed oil was subjected to alkali hydrolysis, in order to isolate fatty acid. Mustard seed oil was hydrolyzed at 80°C by 20% sodium hydroxide for 60 minutes and analyzed by gas chromatography to obtain maximum free fatty acid which was 44.08% of mustard seed oil. The methylation fatty acids took 15 minutes and 94% of methyl ester produced was recovered. Then, mustard seed oil was hydrolyzed by alkali in optimized condition (20% NaOH at 80°C). The amount of fatty acids was determined by gas chromatography in methyl ester forms. Eleven kinds of fatty acid were found (52.52% of mustard seed oil) mainly oleic acid, linoleic acid and erucic acid (16.03, 13.16 and 10.78% of mustard seed oil or 30.49, 24.97 and 20.50% of total fatty acid, respectively). The amount of fatty acid determined by
titration with sodium hydroxide was found to be 55.49% of mustard seed oil.
Isolation of fatty acid from mustard seed oil by commercial lipase from Candida rugosa was investigated. The mustard seed oil was hydrolyzed by 50 units of Candida rugosa lipase at room temperature (31 ±1°C) for 20 hours and analyzed by gas chromatography. About ten fatty acids were identified (18.90% of mustard seed oil) and the amount of oleic acid, linoleic acid and erucic acid were found to be 6.16, 3.35 and 4.85% of mustard seed oil, respectively (32.59, 17.71 and 25.64% of total fatty acid, respectively). The amount of fatty acid as determined by titration with sodium hydroxide was found to be 20.69% of mustard seed oil .
Mustard seed oil was also hydrolyzed by Aspergillus sp. lipase which was produced from Aspergillus sp. growing in mustard cake extracted medium. Optimum condition for Aspergillus sp. lipase hydrolysis was at 37°C , pH 7.0 for 30 minutes. It was found that maximum activity of 5.08 units could be obtained from 1 ml of enzyme extracts. Mustard seed oil was hydrolyzed by Aspergillus sp. lipase in optimized condition and analyzed by gas chromatography. About eight kinds of fatty acid were identified (7.32% of mustard seed oil) and the amount of oleic acid, linoleic acid and erucic acid were found to be 3.20, 1.21 and 0.64% of mustard seed oil, respectively (or 43.73 , 16.51 and 8.70% of total fatty acid, respectively). The amount of fatty acid determined by titration with sodium hydroxide was 9.34% of mustard seed oil.
Mustard seed oil was hydrolyzed by lipases in 4 kinds of detergent solution i.e. sodium dodecyl sulfate, sodium dodedylbenzene sulfonate, tetraethylammonium chloride and dodecyltrimethylammonium bromide. It was found that, as determined by gas chromatography, yield of fatty acids using by lipases with tetraethylammonium chloride and dodecyltrimethylammonium bromide increased about 60% of mustard seed oil.
Erucic acid was isolated from mixed fatty acids hydrolyzed by Candida rugosa and Aspergillus sp. The optimum condition for isolation was -11°C using aqueous ethanol (ratio 3 : 1) . It was found that amount of erucic acid from Candida rugosa and Aspergillus sp. lipase hydrolysis was 31.74 and 27.51% of total fatty acid, respectively.