磷脂酶D制备及催化合成磷脂酰丝氨酸工艺研究-生物化工专业论文.docxVIP

摘 摘 要 磷脂酶D是作用于磷脂分子中的磷酰氧键P．0的一类酶，它可以催化两种 反应：(1)水解磷脂上的末端磷酸酯键生成磷脂酸和羟基化合物；(2)当有另一 种含羟基的化合物存在时，可催化磷脂酰基与其结合，形成新的磷脂，即磷脂酰 基转移反应。磷脂酶D广泛存在于各种植物中，在动物和微生物中也有分布。 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。通过对 链霉菌进行自然选育得到了一株磷脂酶D产量较高的链霉菌，以此为出发菌株采 用紫外诱变育种方法进行菌种改良，诱变筛选菌株的产磷脂酶D活力提高27％。 另外，对发酵制备的酶液进行初步的分离纯化处理，使得发酵液中磷脂酶D的活 力提高了2．8倍。 通过研究磷脂酶D催化合成磷脂酰丝氨酸工艺，从实验中得出了基本的反应 条件：含酶水相中丝氨酸浓度0．19／ml，调节pH为5．5；水相与含有大豆磷脂的 乙醚相以1：2的体积比混合，在28(2的条件下搅拌反应，磷脂酰丝氨酸的收率 达57％。同时，本文还对两相体系中磷脂酶D的催化反应动力学进行了初步研究， 确定了在磷脂酶D在水一有机相体系中的反应模型。 关键词g磷脂酶D；磷脂酰基转移反应；磷脂酰丝氨酸；链霉菌；非水相催化 The The Preparation of PLD and Study on the Synthetic Technique of Phosphatidylserine Abstract Phospholipases D(PLDs)are the enzymes that catalyse：(a)the hydrolysis of the terminal phosphodiester bond of glycerophospholipids，producing phosphatidic acid analogs and a free alcohol and(b)the exchange of the polar headgroup of the phosphorlipid，forming a new phosphoester bond，in the presence of an alcoholic nucleophile(transphosphatidylation reactions)．PLDs are widespread in plants，but they also exist in animals，fungi and bacteria． In this paper,PLD are produced from Streptomyces by ferment．A strain，which call produce higher unit PLD，was obtained by a isolating and screening method．Then， this fungi Was mutagenized by UV ligh and a strain Was selected as producing PLD increased 27％．The culture supernatants of selected strains were filtrated by a ultrafiltration membrane and the unit PLD Was increased more than 2．8 times by ultrafiltration． In the study of phosphatidylserine synthesis process，the optimal reaction conditions is concluded：the serine(0．1 g／m1)is dissolved into the culture supematants containing PLDs and adjust the pH value of 5．5．Then this solution is mixed with aether dissolving soybean phosphatide by 1：2 proportion．These materials are reacted under the agitation condition in 28。C．Finally,a mixture that contains 57％ phosphatidylserine Was obtained．Besides，a new reaction model for hydrolysis and transphosphatidylation recactions in aqueous-organic solven