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Optimization of Fermentation Conditions of Recombinant Lipoxygenase for the Production in Wheat Flou

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Tutor: LuZhaoXin
School: Nanjing Agricultural College
Course: Of Food Science
Keywords: recombinant lipoxygenase,culture optimization,enzyme properties,wheat flour
CLC: TS201.25
Type: Master's thesis
Year:  2011
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Abstract:
Lipoxygenase that extensively exists in plants and microorganisms and has the role of gluten and bleaching can be used as flour improver(potassium bromate) and potential alternatives of benzoyl peroxide. Thus, it becomes the hot spot of research and development about green food additives. Compared with the traditional flour improver, lipoxygenase as a novel flour improver has the advantanges of safety¡¢nutrition and healthy. However, the production of lipoxygenase that extracted from various plants(such as soybean) and its purity was low, so its large-scale applying had not been breakthrough. The study primarily optimizated the fermentation conditions of recombinant E.coli that cloned and expressed from Anabaena sp. PCC 7120 and studied enzyme activity of recombinant lipoxygenase with its applying in the wheat flour in addition. Main study results are as following:1.Lipoxygenase was fused with the expression vector pET-32a, and transformed into Escherichia coli BL21 (DE3), and finally obtained the recombinant pET-32a-ana-Lox. The induced culture conditions were optimizated in orthogonal experiment. With the induction by 1g/L of lactose, OD600 0.8,16¡æfor 16 h, the lipoxygenase activity reached up to 812 U/mL.2.The fusion protein ligand of BL21/pET-32a-ana-Lox was removed by Nickel column, and the electrophoretically pure Lox protein was obstained. The comparative activity of BL21/pET-32a-ana-Lox was up to 13.7U¡Á103/mg and its purity multiples was up to 23.3; the recovery rate of enzyme activity was 61.9%; the molecular weight of lipoxygenase was about 66 kDa.The optimal pH of the recombinase was 6.0 and the optimal temperature was 45¡æ, the heat stability of recombinant was not high.The activity of BL21/pET-32a recombinase was raised by Fe2+ and weakerly raised by Mg2+¡¢Ca2+3.The lipoxygenase can oxidate -SH of water soluble proteins and SDS soluble proteins in the wheat flour that reached up to the lowest value by 10 U/g ana-Lox and 4 U/g ana-Lox and inhibit the reduction of -S-S- that reached up to the highest value by 10 U/g ana-Lox and 4 U/g ana-Lox.Meanwhile, the lipoxygenase can promote the physical or chemical crosslinking in the water soluble protein and SDS soluble protein and rise to the highest extraction amount of albumin¡¢globulin¡¢gliadin¡¢glutenin in the wheat flour by ana-Lox about 6 U/g¡¢6 U/g.4 U/g.4 U/g in respectively. Compared with KBrO3, the lipoxygenase was easy to over oxidizing SDS soluble protein(gliadin. glutenin) as the oxidation rate of Lox was more faster, so the quality of wheat flour was decreased. The lipoxygenase played the degradation effect on the¦Â-carotene in the wheat flour. The quality of¦Â-carotene reached to the lowest value by 8U/g ana-Lox. The temperature played an important role in the oxidation about Lox to¦Â-carotene. The degradation rate of¦Â-carotene was more higher at 40¡æ. Some series indexes of break measurements(such as hardness¡¢springiness¡¢chewiness¡¢cohesiveness) achieved the highest level by ana-Lox about 4 U/g.8 U/g.8 U/g.8 U/g in respectively.
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