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This paper aims to study the use of a wide range of important industrial concern enzyme, the Streptomyces (Stv. Cinnamoneum) sources of phospholipase D. Its variety of phospholipids having a hydrolyzable and phospholipid transfer activity, can be efficiently, the continuous conversion phosphatidylcholine (phosphatidylcholine, PC) to generate the phosphatidyl ethanolamine (phosphatidylethanolamine, PE). Accordingly, the phospholipase D is a promising catalyst can be synthesized every physiological function of a variety of novel phospholipid polar head group, are widely used in food, cosmetics and pharmaceutical industries. However, phospholipase D in low-yield rate of microorganisms fetters serious in-depth study of the theory and application of enzymatic. Before building secretory phospholipase D expression system, many researchers have tried to phospholipase D expression in cell endocrine colon or Pichia pastoris as a host strain. However, studies have found that the phospholipase D, these host cells have a strong cytotoxic activity of phospholipase D, would immediately lead to serious cytolysis Once expressed, this is likely to be the activity of phospholipase D in the host cell membrane phospholipid degradation due. Therefore, it is necessary to study the feasibility of phospholipase D secretion in prokaryotic host expression, for the industrial production of certain theoretical basis. This study itself carries the signal peptide of the pET-26b (), pEZZ18 plasmid as an expression vector, E. coli w Rosetta, E. coli JM105 has as a host bacterium, STV. Cinnamoneum source of phospholipase D gene as amplified fragment was constructed prokaryotic expression system and issue a soluble phospholipase D activity in periplasmic expression. And further optimization of the various experimental parameters such as the concentration of the inducing agent, the glucose concentration and the medium composition, etc. may influence the expression of phospholipase D production, activity. Eventually, the build pEZZ18-PLD / JM105, prokaryotic expression system pET26b () - PLD / W the Rosetta add glucose final concentration was 0.5%, temperatures were 18 ° C, 28 ° C, IPTG concentration of 0.5 mM were the OD600 value 0.75,0.85 when the expression levels of the target protein PLD highest were 0.8mg / L, 1.1 mg / L; activity is 0.62 × 103 U / L, 0.86 × 103 U / L, respectively. The phospholipase D prokaryotic expression system constructed in this study the expression of phospholipase D production, activity has been further improved, though not ideal, but still provide some reference and reference for the phospholipase D basic theoretical research and phospholipids industrial production.
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