Cloning, Expression, Enzymatic Characterization And Immobilization Of Hydroperoxide Lyase

Hydroperoxide lyase (HPL) is a membrane bound heme protein that cleaves hydroperoxides of polyunsaturated fatty acids such asα-linolenic or lenoleic acids to form volatile short chain aldehydes andω-oxoacids. Volatile aldehydes (n-hexanal, 2(E)-hexenal) produced by HPL are important constituents of the characteristic flavors of fruits, vegetables and green leaves. They are widely used in cosmetic industries and as food additives to reconstitute the'fresh green'smell of fruits or vegetables lost during industrial processing.Primers for RT-PCR were designed according to the nucleotide sequence of hydroperoxide lyase mRNA of Solanum tuberosum (GenBank accession number: AJ310520). Total RNA was extracted from immature potato leaves using Trizol reagent. Total RNA was reverse transcribed to obtain the first strand cDNA. Aliquots of the cDNA were used directly as a template for PCR amplification of the cDNA encoding HPL from potato leaves. The cDNA was then ligated into pMD 19-T simple vector. Sequencing showed that open reading frame of the cDNA is composed of 1,443 bp encoding a putative protein of 480 amino acid residues with a calculated molecular mass of 53.9 kDa.The cDNA was cloned into an expression vector pQE-30 and expressed in Escherichia coli M15. The recombinant protein was purified to electrophoretical homogeneity by affinity chromatography using Chelating Sepharose Fast Flow resin. The purified recombinant potato HPL had 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 purified recombinant potato HPL compared to 13-hydroperoxy-linoleic acid (13-HPOD). The corresponding volatile products were 2(E)-hexenal and n-hexanal tested by headspace-gas chromatography, respectively. The purified recombinant enzyme was optimally active at 25 oC and pH 6.5, showing a Km value of 56.6μM with 13-HPOT substrate, a Km value of 71.7μM with 13-HPOD substrate. Activity of the purified 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 potato HPL.A mehod for immobilizing Hydroperoxide lyase (HPL) from Amaranthus tricolor with sodium alginate, gelatin and glutaraldehyde was presented. The optimum conditions for the immobilization were as follows: The concentration (w/v) of sodium alginate, gelatin and CaCl2 was 1.25%, 0.5% and 8%, respectively. The concentration (v/v) of the crosslinking agent (glutaraldehyde) was 1% and the crosslinking time was 20 min. Compared with the free HPL, the optimal temperature and optimal pH of the immobilized enzyme increased to some extent, while the thermal stability, pH stability and storage stability increased greatly.