Gene Cloning Of Orange PME

Pectin Methylesterase (EC:3.1.1.11),which belongs to saponifcational enzyme,can randomly hydrolyze the methyl ester bond of pectin. It is distributed in all of plants and some of microorganisms. PME is one of enzymes that widely used in industry,especially in beverage industry. Recently ,the preparation of LM-pectin by PME is more and more regarded by scholars.The research of PME gene is hotspot overseas,but fresh reports in China.PME in citrus peel has been researched in this article.The main results were as follows:1. PME gene from citrus peels was cloned using RT-PCR.We obtained a length of 1797bp gene, and its ORF is 1785bp ,coding 594 amino acid. The sequence analysis indicated that this gene has 5 potential N-glycosylation sites. Signal peptide is consisted of 19 amino acid residues,the most likely cleavage site is likely to situated in between 45 and 46(VVT-II). Hydrophobic analysis indicated that between sites 20 and 50 is a typical hydrophobic region, and it stands a good chance a span membrane region. But hydrophobic analysis indicated this PME gene is a highly hydrophile protein. PME gene has four highly conserved Cys residues, we presumed that they engaged in disulfide bridges and have a important role in PME structural.2. The clone gene is different with other PMEs from citrus sinensis in anagenesis. So may be it has a different characteristic with others. And this is happened to havethe same view that CsPME4 is a TS-PME. From phylogenetic tree we found that the similarity TS-PME is probable exist in Linum usitatissimum (flax) ,too.3. PME was abtained from citrus peels by salt saturation. Under different temperature and different pH ,the PME activity was measured by pH-stat standard method. The results were as follows: its optimal temperature was about 50°C,and under this temperature the PME has a good stability: there is still have above 80% activity after 50min heat handle at 50 °C .When above 50°C,the activity decreased obviously. Its optimal pH is about 8.A11 indicated that PME's optimal working condition are 50°C, pH=8o...