Molecular cloning,expression, and enzymatic characterization of <Emphasis Type="Italic">Solanum tuberosum</Emphasis> hydroperoxide lyase |
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Authors: | Email author" target="_blank">Wanmeng?MuEmail author Qinghai?Xue Bo?Jiang Yufei?Hua |
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Affiliation: | (1) State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, 214122, Jiangsu, China |
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Abstract: | A cDNA encoding hydroperoxide lyase (HPL) was isolated from Solanum tuberosum, cloned into pQE-30 vector, and expressed in E. coli. The recombinant protein was purified by nickel affinity chromatography and showed an approximate molecular weight of 54 kDa
by SDS–PAGE analysis, which was similar to the predicted value based on the putative amino acid sequences (53.9 kDa). 13-Hydroperoxy-linolenic
acid (13-HPOT) was the preferred substrate for the enzyme compared with 13-hydroperoxy-linoleic acid (13-HPOD). The corresponding
volatile products were 2(E)-hexenal and n-hexanal tested by headspace-gas chromatography, respectively. The enzyme was optimally
active at 25 °C and pH 6.5. The K
m, V
max, and the catalytic efficiency (V
max/K
m) for 13-HPOT were 56.6 μM, 71.3 units/mg, and 1.26 units/mg · μM, respectively. Activity of the recombinant potato HPL increased
when Triton X-100, sodium chloride, or potassium chloride was added in the reaction mixture, while calcium chloride decreased
activity of the recombinant enzyme. |
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Keywords: | |
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