| Lipoxygenase （EC1.13.11.12, LOX）, a kind of dioxygenase, can catalyzepolyunsaturated fatty acids containing cis, cis-1,4-pentadiene structure to form hydroperoxidewith conjugated double bond. LOX is widely used in food industry, especially in the flourprocessing. In addition, LOX is also used in producing pharmaceutical, paper, textile,plasticizer and so on. However, the current commercial LOXs are mostly from plantscontaining a variety of isozymes, which influence control and detection in the applicationprocess greatly. LOXs from microorganism are more suitable due to their inexistence ofisozymes.This study aims to improve the production and thermal stability of LOX in therecombinant bacteria. The culture conditions of LOX fermentation in3L fermentor wereoptimized. The thermodynamic properties of LOX were further studied, and the thermalstability of LOX was strengthened by additives and molecular modification. The maincontents are as follows:1. Firstly, the culture conditions of LOX fermentation by E. coli in3L fermentor werestudied. The effects of additives, different initial concentrations of induced bacteria andglycerol on LOX producing were also examined. The peak production was got when inducedat the bacteria concentration OD600was10with1%(w v-1) of glycine in the TB medium with8g L-1initial glycerol concentration. Compared with the production achieved on shake flasklevel, the maximum LOX production reached9.3U mL-1after an induction of40h, whichwas increased by9.4%. Besides,20%of the induction time was saved. Production intensitywas improved to the world’s highest level.2. The thermodynamic properties of purified LOX were analyzed. The inactivation rate（k）, half-life （t1/2） and reaction activation energy （E） of LOX at different temperature werestudied and a comparison with LOXs from other sources was given. By adding additives, theeffects of sugars, polyols and salts on thermal stability of LOX were examined respectively.As the heating temperature increased from25oC to55oC, thermal inactivation rate （k） ofLOX increased from0.0407min-1to0.2627min-1while the half-life （t1/2） value weredecreased to3.25min from17.08min. As the reaction activation energy E is usually lowerthan those from other sources, LOX of this study is easier to be inactivated. Most of thesugars and polyols were effective to enhance the thermal stability of LOX. And xylitol (1mol L-1) is the best additive which increases the half-life at45oC by nearly three timescompared with the blank sample. However, almost all the salts showed negative effects onthis part except sodium citrate, which increases half-life by36%.3. The multi-repeat insertions of L6sequence to the junction of N-terminal cover domainand C-terminal catalytic domain were conducted, three mutants （L6Ⅰ, L6Ⅱ, L6Ⅲ） wereformed. And the influence of such modifications on specific activity, thermal stability,hydrophobic and Kmof LOX were studied. As shown in the results, all the three kinds ofmutants have little difference with wild-type LOX in secondary and tertiary structure, themutants showed better affinity to the substrate. The optimal reaction temperature of L6Ⅱ andL6Ⅲ was increased by5oC and10oC, and the specific activity were only28.7%,57.9%and 47.6%of the wild-type LOX. The hydrophobicity of the three mutants were all enhanced bynearly300%compared with the wild-type. |
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