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Molecular cloning, expression, and enzymatic characterization of Solanum tuberosum hydroperoxide lyase

Mu, Wanmeng, Xue, Qinghai, Jiang, Bo, Hua, Yufei
European food research & technology 2012 v.234 no.4 pp. 723-731
Solanum tuberosum, acids, affinity chromatography, amino acid sequences, calcium chloride, complementary DNA, enzyme activity, enzyme substrates, genetic vectors, molecular cloning, molecular weight, nickel, pH, potassium chloride, potatoes, recombinant proteins, sodium chloride
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.