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Synthesis Of Linoleic Acid Hydroperoxide Catalyzed By Soybean Lipoxygenase Under Aerobic Condition

Posted on:2005-07-13Degree:DoctorType:Dissertation
Country:ChinaCandidate:K CaiFull Text:PDF
GTID:1101360125960724Subject:Light Industry and Chemical Technology and Engineering
Abstract/Summary:PDF Full Text Request
Lipoxygenase (LOX) contains a nonheme iron in it's catalytic center. It can catalyze the oxidation of polyunsaturated fatty acids containing the cis,cis-1,4-pentadiene moiety to the corresponding hydroperoxides (HPOD) of conjugated (trans, cis)-dienes, which are important intermediates of synthesizing drugs and chemicals. Soybean lipoxygenase as a model in this paper, extraction, factors affecting enzyme activity, reaction course, exogenous kinetic control, analysis and primary application of HPOD, stabilization of LOX were investigated. The main points were as follows:Influence of exogenous Fe3+, Fe2+ on LOX catalysis was studied as viewed from chemicobiology for the first time. A certain amount of exogenous Fe3+, Fe2+ or linoleic acid hydroperoxide could speed up the oxidation process of linoleic acid catalyzed by soybean lipoxygenase. Absorbance band between 300~400 nm in UV spectrum of soybean lipoxygenase was strengthened once upon adding of Fe3+ but only changed slightly with adding of Fe2+. Potential function of exogenous Fe3+, Fe2+ or linoleic acid hydroperoxide in aerobic catalysis mechanism of soybean lipoxygenase is proposed as electron transfer from the inside electron donor to the exogenous electron accepter, which speeds up the conversion from Fe2+ to Fe3+ inside the active center of LOX. Since the conversion from Fe2+ to Fe3+ inside the active center of LOX is the control step in aerobic catalysis of LOX, the fact can be successfully explained that the above exogenous matters accelerate overall rate of aerobic catalysis. Modified scheme of aerobic oxidation of linoleic acid by LOX was proposed.Kinetic analysis of LOX catalysis showed there was no lag phase in reaction progress curve when substrate concentration was less than 14.25 μmol/L. Km and vmax were calculated from dual-reciprocal curve. Initial rate reached maximum when substrate concentration was 0.1 mmol/L, and decreased with higher concentration which did not fit in with Michaelis-Menten any more and showed marked substrate inhibition.LOX was extracted from defatted soybean flour with several methods and LOX activity was measured. The total enzyme activity 6.71×107 U/10 g (defatted soybean flour) obtained by water-leaching is highest and the corresponding specific enzyme activity is 8.61×105 U/mL (enzyme preparation). Specific activity of LOX 9.42×107 U/g solid obtained by base-solubilizing and acid-depositing followed by two salt-out steps is highest of all. Free LOX is quite unstable at room temperature. It is found that some salts, sugar, polyol or organic electrolytes does well to the stability and thermostability of LOX. A simple and effective method for stabilization of LOX were found. After adding 1%(w/w) sodium citrate and 1‰ sodium benzoate, the activity of LOX keeps 90% of the original after 60 days, and 73% after 120 days. The enzyme preparation procedures of LOX using atlapulgite or talc as carrier are optimized and scaled up. When kept at 0~4℃ for 20 days the enzyme preparation loses activity by 19.2% for atlapulgite or 17.7% for talc, and remain constant in the followed two months. When the enzyme preparation of LOX beads was kept in glycerol(φ(glycerol)=75%) for 33 days, four months and six months, the beads maintain 100%,90%,77% of original respectively.Synthesis of linoleic acid hydroperoxide with linoleic acid catalyzed by soybean lipoxygenase in aqueous phase was studied. The optimal conditions were 4.11g/L of linoleic acid in buffer solution of pH 9 reacting for 120 min at 10℃. Some external factors can promote the lipoxygenase catalysis. Addition of AEO4 shortened the reaction time to 30 min with a 12% increase of yield with a ratio of m(LA)∶m(surfactant)=1∶1. With optimized condition for 4 g/L,30g/L,43 g/L of LA scale, yield was increased to 92%,84% and 63%, respectively. Under the same conditions, the enzyme preparation using atlapulgite or talc as carrier accelerated the reaction. With LC-EMS analysis, the product contains linolenic acid-HPOD, linoleic acid-HPOD and oxo-dienoic ac...
Keywords/Search Tags:Soybean lipoxygenase, Enzymatic catalysis, Linoleic acid hydroperoxide, Kinetic control, stabilization
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