Identification Of The Strain For Phospholipase D Production And Primary Investigation Of The Gene

Due to the monotonous structure,low content and less kinds of natural phospholipids,the promising application of phospholipids in food and medicine industry was severely delayed.Modified phospholipids that conquered the shortages of natural ones received considerable attention in recent years.phospholipase D is a special type of ester bond hydrolase,which can not only hydrolyze phospholipids to generate phosphatidic acid and hydroxy compounds but also bind some hydroxyl-containing compounds to the phospholipids containing acyl groups.Owing to the esterification function of phospholipase D,many rare phospholipids and semi-synthetic phospholipids were produced to meet the growing customers' requirement.Phospholipase D is widely present in microorganisms,plants,and animals.And phospholipase D derived from microorganisms in particular exhibits good transesterification and hydrolysis abilities.We screened a phospholipase D-producing strain,named as P1 by the colony hydrolyzation circle method.Morphological study showed that strain P1 is gram-negative,facultative anaerobic,and short rod-shaped bacteria with spore,long 1.4-2.4 ?m,width0.4-0.6 ?m.The P1 is yellow-white round colonies with rough surface,ridgy center and neat edges.Use 16 S rRNA sequence analysis,P1 belongs to the Bacillus,and the similar rates of type strain Bacillus cereus is 99.73%.Physiological and biochemical tests show that strain P1's optimal growth occurs at range pH 6.0-7.0,30 ? and without NaCl addition.Chemical profiling and mocular characteristic showed that the major phospholipid acids of strain P1's are phosphatidylethanolamine and phosphatidylcholine,and the(G+C)mol% is 42.01 mol%.We also built up the optimized Rayleigh's salt method containing wavelength 526 nm,Reynolds salt concentration 3%,and reaction time 10 min for the determination of phospholipase D activity.At last we obtained the long gene of phospholipase D produced by strain P1 and heterologously expressed in E.coli.