【作者】 赵紫薇；

【导师】 杨天奎；

【作者基本信息】 河南工业大学 ， 粮食、油脂及植物蛋白工程， 2010， 硕士

【摘要】 磷脂酶D（PLD）能催化水解磷脂分子中的磷酰氧P-O键生成磷脂酸和羟基化合物,而且当另一种含羟基化合物存在时,磷脂酶D能催化其与磷脂分子末端极性基团发生转磷酯化反应,生成一种新的磷脂。利用磷脂酶D的转磷酯化反应,可制备许多功能良好的稀有磷脂和磷脂衍生物。磷脂酶D广泛存在于各种动植物和微生物（细菌、酵母等）体内,尤其是产自链霉菌的磷脂酶D,表现出较好的水解和转磷酯活性。本文以微生物链霉菌作为生产磷脂酶D的菌种,发酵制备磷脂酶D。首先对色褐链霉菌产磷脂酶D的发酵工艺进行优化,得到其最佳发酵条件;然后利用紫外线、氯化锂及大气压冷等离子体对此菌株进行诱变改良。采用单因素实验,对色褐链霉菌发酵产磷脂酶D的培养条件和培养基组成进行了优化。实验结果表明:摇瓶最佳发酵培养条件为:发酵周期7天,发酵温度28℃,发酵培养基初始pH6.0,接种量3%（v/v）,发酵培养基装液量10mL/100mL三角瓶,摇床转速200r/min;通过对发酵培养基组成的优化,确定了最佳氮源为蛋白胨,最佳碳源为可溶性淀粉,其最适添加浓度分别为20.0g/L、25.0g/L,最佳无机盐组合为MgSO₄·7H₂O 0.5g/L和CaCO₃ 1.0g/L,最佳表面活性剂为Tween（吐温） 80,最适添加浓度为20.0g/L;其中发酵周期、发酵温度、发酵培养基装液量对发酵产酶影响较大,而且可溶性淀粉、CaCO₃以及Tween 80的添加对发酵产酶起到了良好的促进作用。优化后色褐链霉菌发酵产磷脂酶D的酶活比原发酵条件（0.177U/mL）提高了2.2倍,达到0.563U/mL。通过对色褐链霉菌进行诱变选育并得到一株高产磷脂酶D的突变株。首先通过紫外线和氯化锂（LiCl）复合诱变,对色褐链霉菌进行改良,并筛选得到一株名为PU-4的突变菌,然后再对这株菌进行大气压冷等离子体和氯化锂复合诱变,并筛选得到一株名为PUP-8的高产磷脂酶D菌株,其发酵产酶酶活达到0.878U/mL,比出发菌株酶活（0.562U/mL）提高了56%,且通过传代实验,验证了此菌的遗传稳定性。通过对色褐链霉菌产磷脂酶D的发酵条件优化及诱变育种研究,使得其发酵产酶酶活达到0.878U/mL,比出发菌株发酵条件优化前酶活（0.177U/mL）提高了4倍,取得了良好的效果。更多还原

【Abstract】 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.更多还原