Screening Of High-yield Phospholipase D Strain And Optimization Of The Condition For Fermentation

Phospholipase D (PLD; EC.3.1.4.4) is an enzyme that catalyzes PLs hydrolysis to release a phosphatidic acid (PA) and a choline. While in the presence of an additional alcohol, PLD preferentially catalyzes the transphosphatidylation reaction, in which a polar-head modified phosphatidyl alcohol is synthesized by the substrate PLs that act as the phosphatidyl-moiety donors. The transphosphatidylation reaction carried out by the PLD enzyme should be, therefore, a powerful tool for achieving PL modifications economically. As a fairly ubiquitous enzyme, PLD is widely distributed in bacteria, fungi, plant and vertebrate species. Particularly, the PLD enzymes produced by Streptomyces species appear to much high activity .However, the wild strain of Streptomyces produces lower unit PLD, limiting its industrial production greatly. In this paper, a rapid determination of PLD enzyme based on the mechanism of the PLD reaction was firstly built with the TLC-Pp-NP method. The selection of the optimum parent strain producing PLD was then conducted with an efficient and convenient biological screening method. The obtained strain was mutagenized by UV light and screened in the shake flask culture.A primary optimization for medium composition in shake flask showed that the best carbon source was glucose and the best complex nitrogen source was the combination of beef and peptone. Their optimum initial concentrations were 10.00 g/l, 5.00 g/l and 5.00 g/l, respectively. The best inorganic salt included NaCl, MgSO₄·7H₂O and CaCl₂ with 3.00 g/l, 1.00 g/l and 3.00 g/l of optimum initial concentrations, respectively, in which CaCl₂ can improve the activity unit of PLD for 7-8 times. The best surfactant, Tween60 with a concentration of 16.30g/l enhanced the PLD activity to the final unit of 5210u/l which was higher 10 times than control.The effects of culturing conditions including temperature, pH, rotatory velocity, culture volume and inoculum volume on PLD production in shake flask were investigated. The optimum conditions for the PLD production were 30℃, 150-200rpm, 6.8-7.0 of initial pH, 20% of the culture volume, 1-2% of inoculum and fermentation lasting 6 days.