Breeding Of High-yield Phospholipase D-producing Strains By Protoplasts Fusion

Phospholipase D,ubiquitous in the biological world, is the important cellular phospholipid metabolism enzyme. It could catalyze hydrolysis of lecithin ?PC? to generate phosphatide acid ?PA? and free choline by phosphodiester bonds. PLD can also catalyze phosphatide transfer reaction in the synthesis of rare phospholipid in the presence of alcohols. PLD plays a role of emulsifying, wetting and dispersion in the food industry. It is also an important accessory in antineoplastic agents. Streptomyces microorganism is the commonly used production strains of phospholipase D. Due to the low activity of wild strains, obtaining high yield strain by screening and improvement is an important way to improve the enzyme activity of phospholipase D.The article used the Streptomyces sp. LD0501 as the original strain, which is preserved in the laboratory and could be used to produce PLD. The author optimized the preparation of protoplasts and explored the optimum conditions of protoplast fusion, which used UV-23 and UV-p3-45 obtained by the means of UV mutagenesis single spore UV mutagenesis and protoplast as two parental strains of protoplast fusion. Obtain a fusion strains inheriting good traits of two parental strains by screening. Optimize the main factor carbon source in the fermentation medium used to obtain high-yield fusion strains. Then, provide theoretical basis for the PLD industrialization. The main contents and results are as follows:1. This article studied the influencing factors on Streptomyces sp.LD0501 protoplasts dissociation through single factor experiment. It was found that suitable conditions for the protoplast:glycine concentration in the seed medium was 5.0 g/L, bacteria age was 72 h, the concentration of 3 mg/mL lysozyme enzyme at 30? under 75 min. By means of four factors and three levels of test design through the center combination in Design-expert 8.0.6, the optimum reaction conditions was determined, which included that the enzymolysis time 76 h, enzymolysis temperature 30.2?,lysozyme concentration 2.9 g/L, glycine concentration in the seed medium was 5.0 g/L.2. Mutagenesis measures were taken on protoplasts and single spore suspensions prepared from original strains. Then, screen these strains to obtain two high-yield strains. The activity ?3.26 U/mL? of high-yield strain UV-23 was low, but it grew faster. High-yield strain UV-p3-45 grew slowly, but PLD hydrolysis vitality ?4.29 U/mL? was the highest. Use these two strains as two parental strains of protoplast fusion. Inactivate protoplast by the means of high temperature and ultraviolet radiation. The results showed that at 55? water bath treatment, the optimal thermal inactivation time for UV-23 and UV-p3-45 were 75 min and 60 min. Under 15 W UV lamp irradiation at a distance of 30 cm, the optimal UV inactivation time for UV-23 and UV-p3-45 were 120 s. Polyethylene glycol ?PEG? was used to induce protoplast fusion. The optimal conditions of protoplast fusion obtained by single factor experiment were:molecular mass of PEG was 4000, concentration of PEG was 50%, concentration of Ca²⁺ was 3 g/L, the integration time was 6 min, the fusion temperature was 30?.3. Protoplast fusion and screen for fusion strains. Finally, the author obtained three high yield strains F-10, F-21and F-33, which grew faster and inherited good traits of two parental strains. Then, study the genetic stability of fusion strains to prove its good genetic stability.4. Compare the three high-yielding strains with parental strains in fermentation metabolism characteristics, it could be found that the fusion strains had shorter fermentation cycle and gained obvious advantages in the metabolism of glucose and capacity of producing PLD. In addition, the study found the optimal complex carbon sources for F-10, F-21 and F-33 fusion strains were glucose and glycerol, the optimal nitrogen source were soybean powder and peptone.