Fermentation Conditions And Induced Mutation Of Producing Phospholipase D By Streptomyces Chromofuscus

Phospholipase D (PLD) is an enzyme that hydrolyzes the terminal phosphodiester bond of various phospholipids to generate phosphatidyl acid (PA) and a corresponding alcohol. PLD also catalyzes a transphosphatidylation reaction when alcohol is present as a nucleophilic donor. The transphosphatidylation reaction is important in the synthesis of scarce phospholipids and novel artificial phospholipids. PLD are widespread in plants, mammals and microroorganisms, such as bacteria and yeast. Streptomyces species can produce PLD with high hydrolysis and transphosphatidylation activity.In this paper, we describe the production of PLD from Streptomyces chromofuscus. First, fermentation conditions and optimum medium of PLD production by Streptomyces chromofuscus were optimized. Then, to produce more PLD with high activity, mutagensis of strain Streptomyces chromofuscus by UV light, LiCl and cold plasma at atmospheric pressure was carried out.Fermentation conditions and optimum medium of PLD production by Streptomyces chromofuscus were optimized. The results of single-factor experiment showed that the optimum shaking-flask fermentation conditions were: the fermentation period 7 days, fermentation temperature 28℃, initial pH 6.0, inoculums ratio 3% (v/v), the culture volume 10mL/100mL conical flask, shaking speed 200r/min. A primary optimization for medium composition in shake flask showed that the best nitrogen source was peptone and the best carbon source starch soluble. Their optimum initial concentrations were 20.0g/L and 25.0g/L. The best complex inorganic salt inclues were MgSO4·7H2O 0.5g/L and CaCO3 1.0g/L. And the best surfactant is Tween 80 with a concentration of 20.0g/L. The fermentation period, fermentation temperature and the culture volume were important to attain high enzyme activity. And the addition of CaCO3, Tween 80 and soluble starch soluble played a critical role in the fermentation of production of PLD. Based on the optimum conditions of fermentation, the enzyme activity of PLD produced was increased by 2.2 times as the original conditions.A strain, which could produce higher activity of PLD, was obtained by an induced mutation technology of UV light, LiCl and cold plasma at atmospheric pressure. The results indicated that a better mutant strain named PU-4 was obtained after the original strain of Streptomyces chromofuscus was mutagensised by UV combined LiCl. Then PU-4 was selected as the starting strain for the second mutation by cold plasma at atmospheric pressure and LiCl to produce a positive mutant PUP-8. PLD from PUP-8 was available with enzyme activity of 0.878U/mL and 56% higher than the original srtain (0.562U/mL). The mutant strains PUP-8 had good genetic stability.Based on the fermentation conditions and induced mutation of producing phospholipase D by Streptomyces Chromofuscus, the enzyme activity of PLD produced was increased by 4 times as the original conditions.