Study On Construction And Activity Of Site-Directed Mutants Of Linoleate Acid Isomerase From Lactobacillus Plantarum P-8

Conjugated linoleic acid is a kind of natural unsaturated fatty acid which has many physiological functions. Nowadays, it has been widely applied in food, health products, drug, feed industry and others fields. Linoleic acid isomerase(LAI) may transform linoleic acid to conjugated linoleic acid. It has received the widespread attention to get a single and high active isomers of CLA by catalyzed LAI. But the function, the structure domains and the catalytic reaction mechanism of LAI in cell biology have not been a complete explanation, to study the relationship between the structure and function of LAI is urgently needed.In this study Linoleic Acid Isomerase of Lactobacillus plantarum P-8 as the research object, at online tools and software such as ?http://smart.embl-heidelberg.de/and vecterNTI and http://swissmodel.expasy.org/to analysis the bioinfoirmation of linoleic acid isomerase and predict the sites of the enzyme activity related, to study on PCR mediated the site-directed mutagenesis of multiple sites. By comparing the activity change of LAI and its mutants, to determine whether the site is necessary, lay a foundation for further study of the structure and function of LAI. The conclusion is as follow:(1) Through the software tools to predict the tertiary structure of LAI sequence, preliminary identified the five sites which may be closely related to the activity of LAI, thay are respectively the 68th glycine,107th arginine,125th lysine,172th histidine, lysine and 222th tyrosine.(2) Successfully construct mutant G68A (glycine is mutation into alanine), R107L (arginine is mutation into leucine) and H172P (histidine is mutation into proline).(3) Compared with wild type LAI, mutant G68A almost loss of enzyme activity. The enzyme activity of mutant R107L is decreased,the enzyme activity of mutant R107L of H172P increased. Those tree all are the essential amino acids of LAI.