色褐链霉菌产磷脂酶D的发酵条件优化及诱变育种研究
|
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MgSO_4·7H_2O 0.5g/L和CaCO_3 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、CaCO_3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。
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.
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MgSO_4·7H_2O 0.5g/L和CaCO_3 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、CaCO_3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。
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.
引文
[1] Wang X.. Plant phospholipases[J]. Annu. Rev. Plant Physiol. Plant Mol. Biol., 2001, 52: 211–231.
[2]闫旭宇,李玉中,李玲,等.植物中的磷脂酶D信号转导[J].植物生理学通讯, 2006, 42(6): 1183–1189.
[3]卢行芳,卢荣.天然磷脂产品的加工及应用[M].北京:化学工业出版社. 2004, 1–7, 19,20,125–136, 216, 223.
[4]谷利伟,曹宪荣,赵金兰.磷脂的酶法改性技术进展[J].中国油脂, 1999, 24(6): 60–62.
[5]杨治彪.磷脂酶D制备及催化合成磷脂酰丝氨酸工艺研究: [硕士学位论文].西安:西北大学, 2007.
[6]袁海英,陈力耕,何新华,等.磷脂酶D在果实发育和成熟过程中的作用[J].园艺学报, 2005, 32(5): 933–938.
[7]兰云军,邹祥龙,银德海,等.磷脂的基本性质及应用领域[J].中国皮革, 2003, 32(21): 34–36.
[8]郭浩.磷脂酶D高产菌株的选育及发酵条件优化: [硕士学位论文].西安:西北大学, 2007.
[9]吴伟.链霉菌发酵生产磷脂酶及酶解大豆卵磷脂的条件的优化: [硕士学位论文].武汉:华中农业大学, 2005.
[10] Paola D’Arrigo, Stefano Servi. Using phospholipases for phospholipid modification[J]. Trends in Biotechnology, 1997, 15(3): 90–96.
[11] Jae Kwang Song, Jeong Jun Han, Joon Shick Rhee. Phospholipases: Occurrence and production in microorganisms, assay for high-throughput screening, and gene discovery from natural and man-made diversity[J]. J. Am. Oil Chem. Soc., 2005, 82(10): 691–705.
[12] Tadashi Hatanaka, Tomofumi Negishi, Megumi Kubota-Akizawa, et al. Study on thermostability of phospholipase D from Streptomyces sp.[J]. Biochimicaet Biophysica Acta, 2002, 1598(2): 156–164.
[13]王瑞霞,高庆荣,崔德才,等. PLD基因的基本功能及在植物中的利用研究现状[J].西北植物学报, 2004, 24(8): 1573–1542.
[14]刘洪展,郑风荣. PLD的生化特性及在信号传导中的作用[J].生命科学研究, 2005, 9(4): 14–17.
[15] Wolfgang Frank, Teun Munnik, Katja Kerkmann, et al. Water deficit triggers phospholipase D activity in the resurrection plant craterostigma plantagineum[J]. Plant Cell. 2000, 12: 111–124.
[16] Wang X.. Multiple forms of phospholipase D in plants: the gene family, catalytic and regulatory properties, and cellular functions[J]. Progress in Lipid Research, 2000, 39(2): 109–149.
[17]王亚雄.磷脂酶D与肿瘤的关系[J].国际免疫学杂志, 2006, 29(2): 89–92.
[18]陈石良,许正宏,孙微,等.磷脂酶D的研究进展[J].工业微生物, 1999, 29(4): 47–50.
[19] S.B. Ryu, X. Wang. Expression of phospholipase D during castor bean leaf senescence[J]. Plant Physiology, 1995, 108(2): 713–719.
[20] Yozo Nakazawa, Hiroaki Sato, Masataka Uchino, et al. Purification, characterization and cloning of phospholipase D from peanut seeds[J]. The Protein Journal. 2006, 25(3): 212–223.
[21]倪培德,裘爱泳,江涛.大豆磷脂酶D的研究[J].中国粮油学报, 1991, 6(3):36–41.
[22]袁海英,陈力耕,李疆,等.草莓果实磷脂酶D活性的测定[J].果树学报, 2005, 22(5): 578–581.
[23]李斌,路福平,史文玉,等.色褐链霉菌PLD基因的克隆与表达[J].化学与生物工程, 2007, 24(5): 48–51.
[24] Masa Aki Shinonaga, Yoshihide Kawamura, Kikuo Shimbo, et al. Continuous production of phospholipase D by Streptomyces lydicus D-121 immobilized with corss-linked chitosan beads[J]. Journal of Fermentation and Bioengineering, 1996, 81(4): 310–314.
[25] C. Ogino, M. Kanemasu, Y. Hayashi, et al. Over-expression system for secretory phospholipase D by Streptomyces lividans[J]. Applied Microbiology and Biotechnology, 2004, 64(6): 823–828.
[26]王兴国,裘爱泳,陶文沂,等.高碱基转移活性磷脂酶D生产菌种的筛选及酶活性质研究[J].无锡轻工大学学报, 1996, 15(3): 209–212.
[27]张梁.一种磷脂酶D的生产方法.中国, CN101182498, 2008.
[28]师文静.磷脂酶D制备及催化反应的初步研究: [硕士学位论文].西安:西北大学, 2007.
[29]赵彭花.磷脂酶D的共价及聚集交联固定化技术研究: [硕士学位论文].西安:西北大学, 2009.
[30]胡博新,顾鸽青,朱裕辉,等.链霉菌磷脂酶D的分离纯化及部分酶学性质[J].中国医药工业杂志, 2008, 39(9): 655–658.
[31] Yugo Iwasaki, Naoto Mishima, Kousaku Mizumoto, et al. Extracellular production of phospholipase D of Sstreptomyces antibioticus using recombinant Escherichia coli[J]. Journal of Fermentation and Bioengineering, 1995, 79(5): 417–421.
[32] Giacomo Carrea, Paola D'Arrigo, Valentino Piergianni, et al. Purification and properties of two phospholipases D from Streptomyces sp.[J]. Biochimica et Biophysica Acta, 1995, 1255: 273–279.
[33] Carlo Zambonelli, Paola Morandi, Maria Antoniett Vanoni, et al. Cloning and expression in Escherichia coli of the gene encoding Streptomyces PMF PLD, a phospholipase D with high transphosphatidylation activity[J]. Enzyme and Microbial Technology, 2003, 33: 676–688.
[34] Tairo Hagishita, Masanobu Nishikawa, Tadashi Hatanaka. Isolation of phospholipase D producing microorganisms with high transphosphatidylation activity[J]. Biotechnology Letters, 2000, 22: 1587–1590.
[35] Jaya Ram Simkhada, Seung Sik Cho, Hyo Jung Lee, et al. Purification and biochemical properties of phospholipase D(PLD57) produced by Streptomyces sp. CS-57[J]. Arch Pharm Res, 2007, 30(10): 1302–1308.
[36] Jei Oh Yon, Ji Seon Lee, Bo Geum Kim, et al. Immobilization of Streptomyces phospholipase D on a Dowex Macroporous Resin[J]. Biotechnology and Bioprocess Engineering, 2008, 13: 102–107 .
[37] Chiaki Ogino, Masayuki Kanemasu, Masashi Fukumoto, et al. Continuous production of phospholipase D using immobilized recombinant Streptomyces lividans[J]. Enzyme and Microbial Technology, 2007, 41: 156–161.
[38]安红,宁伟明,张宏波.磷脂化学及应用技术[M].北京:中国计量出版社. 2006, 1–3, 23–26, 85, 90, 114, 155, 211–216.
[39]王兴国,裘爱泳,陶文沂,等.棒薄层色谱氢火焰法定量测定磷脂组成[J].无锡轻工大学学报, 1995, 14(1): 31–37.
[40] S.L. Melton. Analysis of soybean lecithins and beef phospholipids by HPLC with an evaporative light scattering detector[J]. J. Am. Oil Chem. Soc., 1992, 69(8): 784–788.
[41]张永刚,曹栋,史苏佳,等.磷脂酶D催化转碱基中磷脂的分析[J].广西农业生物科学, 2008, 27(2): 158–160.
[42]黄勇,吴兴中, Qureshi Imran Ahmad,等.用酶联比色发测定肝脏磷脂酰胆碱专一性磷脂酶D及其应用[J].上海医科大学学报, 1997, 24(5): 343–346.
[43]唐执文,周永红.氯化胆碱含量测定方法探讨[J].中国饲料, 2002(10):18–20.
[44] Siddiqui RA., Exton JH.. Phospholipid base exchange activity liver plasma membranes[J]. Bio. Chem., 1992, 267(9): 5755–5760.
[45] Kobayashi M., Kanfer JN.. Solubilization and purification of rat tissue phospholipase D[J]. Method Enzymol, 1991, 197:575–578.
[46]李媛媛,薛静,李莹倩,等.离子色谱法测定婴幼儿乳粉中的胆碱含量[J].食品科技, 2008, 33(12):267–270.
[47]薛静.离子色谱法测和高效液相色谱法测定乳粉中胆碱含量的研究: [硕士学位论文].长春:东北师范大学, 2008.
[48]钱峰,裘爱泳.磷脂改性现状[J].粮食与油脂, 2006(9): 7–9.
[49]崔德才,温孚江.磷脂酶D(PLD)在植物信号转导中的作用[J].山东农业大学学报(自然科学版), 2000, 31(2): 115–119.
[50]孟庆飞,温其标.磷脂酶法改性[J].河南工业大学学报(自然科学版), 2005, 26(5): 89–93.
[51]王兴国,裘爱泳,淘文祈,等.大豆磷脂的酶法改性[J].中国粮油学报, 1996, 11(2): 46–51.
[52]宋晚平,任静,王磊,等.一种粉末磷脂酰丝氨酸的制备方法.中国, CN101230365A, 2008.
[53]于刚,徐伟,栾东磊,等.亚临界R134a流体中酶法制备富含多不饱和脂肪酸的磷脂酰丝氨酸[J].高技术通讯, 2008, 18(10): 1075–1080.
[54]王兴国.磷脂研究的历史和现状[J].中国油脂, 1999, 24(5): 44–47.
[55]张永刚.磷脂酶D制备磷脂酰甘油的酶学特性研究: [硕士学位论文].无锡:江南大学, 2008.
[56] Lekh Raj Juneja, Naruhiro Hibi, Tsuneo Yamane, et al. Repeated batch and continuous operations for phosphatidylglycerol synthesis from phosphatidylcholine with immobilized phospholipase D[J]. Applied Microbiology and Biotechnology, 1987, 27(2): 146–151.
[57] Rina Satoa, Yutaka Itabashi, Tadashi Hatanaka, et al. Asymmetric in vitro synthesis of diastereomeric phosphatidylglycerols from phosphatidylcholine and glycerol bybacterial phospholipase D[J]. Lipids, 2004, 39(10): 1013–1018.
[58] Rina Sato, Yutaka Itabashi, Akira Suzuki, et al. Effect of temperature on the stereoselectivity of phospholipase D toward glycerol in the transphosphatidylation of phosphatidylcholine to phosphatidylglycerol[J]. Lipids, 2004, 39(10): 1019–1023.
[59] George J. Piazza, William N. Marmer. Conversion of phosphatidylcholine to phosphatidylglycerol with phospholipase D and glycerol[J]. J. Am. Oil Chem. Soc., 2007, 84: 645–651.
[60] Yukihiro Yamamoto, Masashi Hosokawa, Hideyuki Kurihara, et al. Preparation of phosphatidylated terpenes via phospholipase D-mediated transphosphatidylation [J]. J. Am. Oil Chem. Soc., 2008, 85: 313–320.
[61] Lekh Raj Juneja, Toru Kazuoka, Naomi Goto, et al. Conversion of phosphatidylcholine to phosphatidylserine by various phospholipase D in the presence of L- or D-serine[J]. Biochimca et Biophysica Acta, 1989, 1003(3): 277–283.
[62] Lekh Raj Juneja, Eri Taniguchi, Shoichi Shimizu, et al. Increasing productivity by removing choline in conversion of phosphatidylcholine to phosphatidylserine by phospholipase D[J]. Journal of Fermentation and Bioengineering, 1992, 73(5): 357–361.
[63] De Ferra Lorenzo, Massardo Pietro, Piccolo Oreste, et al. Process for the industrial preparation of phosphatidylserine. America, US5700668(A), 1997.
[64] Masashi Hosokawa, Tatuya Shimatani, Teruyuki Kanada, et al. Conversion to docosahexaenoic acid-containing phosphatidylserine from squid skin lecithin by phospholipase D-mediated transphosphatidylation[J]. J. Agric. Food Chem., 2000, 48(10): 4550–4554.
[65] Yugo Iwasaki, Yukiko Mizumotoa, Takahiro Okadab, et al. An aqueous suspension system for phospholipase D-mediated synthesis of PS without toxic organic solvent[J]. J. Am. Oil Chem. Soc., 2003, 7(80): 653–657.
[66] Meyer Randal. Method of producing phosphatidylserine. America, US7049107(B1), 2006.
[67]曹军卫,马辉文.微生物工程[M].北京:科学工业出版社. 2002, 24, 25, 80–86, 89–121.
[68]肖冬光.微生物工程原理[M].北京:中国轻工业出版社. 2004, 37–40, 61–71, 200–207.
[69]程殿林.微生物工程技术原理[M].北京:化学工业出版社. 2007, 18–21, 58–61, 92, 114–121.
[70]周德庆.微生物学教程(第二版)[M].北京:高等教育出版社. 2002, 213.
[71]侯英敏.等离子体诱变生产1,3-丙二醇菌种的研究: [硕士学位论文].大连:大连理工大学, 2006.
[72]李爽.等离子体诱变生产1,3-丙二醇菌种的研究: [硕士学位论文].大连:大连理工大学, 2007.
[73]姜玉,宋伟民.大气压冷等离子体杀菌作用及其机制的研究进展[J].国外医学卫生学分册, 2006, 33(5): 318–322.
[74]董晓宇,李爽,侯英敏,等.大气压冷等离子体诱变产1,3-丙二醇菌株Klebsiella pneumonia[J].过程工程学报, 2008, 8(3): 555–560.
[75] Herrmann H.W., Henins I., Park J., et al. Decontamination of chemical and biological warfare(CBW) agents using an atmospheric pressure plasma jet(APPJ)[J]. Physics of Plasmas, 1999, 6(5): 2284–2289.
[76] Thomas C. Montie, Kimberly Kelly-Wintenberg, J. Reece Roth. An overiew of reseacrh using the one amtosphere uniform glow discharge Plasma(OAUGDP) for sterilization of surfaces and materials[J]. IEEE Transactions on Plasma Science, 2000, 28(l): 41–50.
[77]何炜静.等离子体诱变筛选降解高浓度对苯二酚的突变株及其降解特性研究: [硕士学位论文].合肥:合肥工业大学, 2009.
[78]吴跃进,王学栋,刘贵富,等.离子束注入水稻诱变效应的研究[J].安徽农业科学, 1989(2): 10–13.
[79]余增亮,何建国,邓建国,等.离子注入水稻诱变育种的机理初探[J].安徽农业科学, 1989(1): 12–16.
[80]于红.大气压下等离子体失活微生物的机理研究: [博士学位论文].大连:大连理工大学, 2006.
[81]凌代俊,曹金样.低温等离子体诱导产生的一种“空洞”形细胞[J].核技术, 2003, 26(4): 284–286.
[82]蒋世春,吴建平,白骅.应用等离子体辐射技术选育柔红霉素产生菌-天兰淡红链霉菌[J].辐射研究与辐射工艺学报, 2001, 19(2): 133–137.
[83]施跃峰,桑金隆,竺莉红,等.等离子体处理选育之江菌素产生菌[J].核农学报, 2002, 16(4): 208–211.
[84]郁军,裘爱泳,刘元法.磷脂研究进展[J].西部粮油科技, 2000, 25(1): 26–29.
[85]郭秋华,何小维,黄强.大豆磷脂的酶法改性研究进展[J].食品工业科技, 2007, 28(10): 243–246.
[86] Ogino C., Kanemasu M., Hayashi Y., et al. Over-expression system for secretory phospholipase D by Streptomyces lividans[J]. Appl Microbiol Biotechnol, 2004, 64(7): 823–828.
[87]陈立梅,汪旭,李启云,等.链霉菌诱变育种方法综述[J].吉林农业科学, 2006, 31(2): 62–65.
[88]付学琴,魏赛金,黄文新,等.南昌链霉菌发酵条件的研究[J].江西农业大学学报, 2007, 29(3): 4573–460.
[89]韩友文.胆碱和氯化胆碱[J].饲料博览, 2004(3):45–46.
[90]田洪涛.现代发酵工艺原理与技术[M].北京:化学工业出版社, 2007. 46–48.
[91]陈希.产双酶假单胞杆菌的诱变选育及发酵条件的优化: [硕士学位论文].长沙:中南林业科技大学, 2006.
[92]栾雨时,包永明.生物工程实验技术手册[M].北京:化学工业出版社, 2005, 129, 149.
[93]陈志高,沈建新,胡叶碧.紫外线和氯化锂对核糖苷链霉菌原生质体诱变作用的探讨[J].中国医药工业杂志, 2001, 32(4): 150–152.
[94]黄永春,曹仁林,彭祎.紫外诱变吸水链霉菌选育高产农用抗生素菌株[J].安徽农业科学, 2008, 36(16): 6847–6848.
[95]季宇彬,朱兴杰,徐昶儒,等.微生物诱变育种方法的研究进展[J]. 496–502
[96]李戈.链霉菌LA5高产菌株诱变育种研究: [硕士学位论文].儋州:华南热带农业大学, 2007.
[97]诸葛健,沈微.工业微生物育种学[M].北京:化学工业出版社. 2006, 77–79.
[98] Laroussi M.. Non-thermal decontamination of biological media by atmospheric pressure plasmas: review , analysis, and prospects[J]. IEEE Transactions on Plasma Science, 2002, 30(4): 1409–1415.
[99] Lindsey F. Gaunt, Clive B. Beggs, George E. Georghiou. Bactericidal action of the reactive species produced by gas-discharge nonthermal plasma at atmospheric pressure: a review[J]. IEEE Transactions on Plasma Science, 2006, 34(4): 1257–1269.
[100] S.B. Farr, T. Kogoma. Oxidative stress responses in Escherichia coli and Salmonella typhimurium[J]. Microbiol. Rev., 1991, 55(4): 561–585.
[101] Lawrence J. Marnett. Oxyradicals and DNA damage[J]. Carcinogenesis, 2000, 21(3): 361–370.
[102]华泽钊,刘宝林,左建国.药品和食品的冷冻干燥[M].北京:科学出版社. 2006, 2–5, 10, 11.
[2]闫旭宇,李玉中,李玲,等.植物中的磷脂酶D信号转导[J].植物生理学通讯, 2006, 42(6): 1183–1189.
[3]卢行芳,卢荣.天然磷脂产品的加工及应用[M].北京:化学工业出版社. 2004, 1–7, 19,20,125–136, 216, 223.
[4]谷利伟,曹宪荣,赵金兰.磷脂的酶法改性技术进展[J].中国油脂, 1999, 24(6): 60–62.
[5]杨治彪.磷脂酶D制备及催化合成磷脂酰丝氨酸工艺研究: [硕士学位论文].西安:西北大学, 2007.
[6]袁海英,陈力耕,何新华,等.磷脂酶D在果实发育和成熟过程中的作用[J].园艺学报, 2005, 32(5): 933–938.
[7]兰云军,邹祥龙,银德海,等.磷脂的基本性质及应用领域[J].中国皮革, 2003, 32(21): 34–36.
[8]郭浩.磷脂酶D高产菌株的选育及发酵条件优化: [硕士学位论文].西安:西北大学, 2007.
[9]吴伟.链霉菌发酵生产磷脂酶及酶解大豆卵磷脂的条件的优化: [硕士学位论文].武汉:华中农业大学, 2005.
[10] Paola D’Arrigo, Stefano Servi. Using phospholipases for phospholipid modification[J]. Trends in Biotechnology, 1997, 15(3): 90–96.
[11] Jae Kwang Song, Jeong Jun Han, Joon Shick Rhee. Phospholipases: Occurrence and production in microorganisms, assay for high-throughput screening, and gene discovery from natural and man-made diversity[J]. J. Am. Oil Chem. Soc., 2005, 82(10): 691–705.
[12] Tadashi Hatanaka, Tomofumi Negishi, Megumi Kubota-Akizawa, et al. Study on thermostability of phospholipase D from Streptomyces sp.[J]. Biochimicaet Biophysica Acta, 2002, 1598(2): 156–164.
[13]王瑞霞,高庆荣,崔德才,等. PLD基因的基本功能及在植物中的利用研究现状[J].西北植物学报, 2004, 24(8): 1573–1542.
[14]刘洪展,郑风荣. PLD的生化特性及在信号传导中的作用[J].生命科学研究, 2005, 9(4): 14–17.
[15] Wolfgang Frank, Teun Munnik, Katja Kerkmann, et al. Water deficit triggers phospholipase D activity in the resurrection plant craterostigma plantagineum[J]. Plant Cell. 2000, 12: 111–124.
[16] Wang X.. Multiple forms of phospholipase D in plants: the gene family, catalytic and regulatory properties, and cellular functions[J]. Progress in Lipid Research, 2000, 39(2): 109–149.
[17]王亚雄.磷脂酶D与肿瘤的关系[J].国际免疫学杂志, 2006, 29(2): 89–92.
[18]陈石良,许正宏,孙微,等.磷脂酶D的研究进展[J].工业微生物, 1999, 29(4): 47–50.
[19] S.B. Ryu, X. Wang. Expression of phospholipase D during castor bean leaf senescence[J]. Plant Physiology, 1995, 108(2): 713–719.
[20] Yozo Nakazawa, Hiroaki Sato, Masataka Uchino, et al. Purification, characterization and cloning of phospholipase D from peanut seeds[J]. The Protein Journal. 2006, 25(3): 212–223.
[21]倪培德,裘爱泳,江涛.大豆磷脂酶D的研究[J].中国粮油学报, 1991, 6(3):36–41.
[22]袁海英,陈力耕,李疆,等.草莓果实磷脂酶D活性的测定[J].果树学报, 2005, 22(5): 578–581.
[23]李斌,路福平,史文玉,等.色褐链霉菌PLD基因的克隆与表达[J].化学与生物工程, 2007, 24(5): 48–51.
[24] Masa Aki Shinonaga, Yoshihide Kawamura, Kikuo Shimbo, et al. Continuous production of phospholipase D by Streptomyces lydicus D-121 immobilized with corss-linked chitosan beads[J]. Journal of Fermentation and Bioengineering, 1996, 81(4): 310–314.
[25] C. Ogino, M. Kanemasu, Y. Hayashi, et al. Over-expression system for secretory phospholipase D by Streptomyces lividans[J]. Applied Microbiology and Biotechnology, 2004, 64(6): 823–828.
[26]王兴国,裘爱泳,陶文沂,等.高碱基转移活性磷脂酶D生产菌种的筛选及酶活性质研究[J].无锡轻工大学学报, 1996, 15(3): 209–212.
[27]张梁.一种磷脂酶D的生产方法.中国, CN101182498, 2008.
[28]师文静.磷脂酶D制备及催化反应的初步研究: [硕士学位论文].西安:西北大学, 2007.
[29]赵彭花.磷脂酶D的共价及聚集交联固定化技术研究: [硕士学位论文].西安:西北大学, 2009.
[30]胡博新,顾鸽青,朱裕辉,等.链霉菌磷脂酶D的分离纯化及部分酶学性质[J].中国医药工业杂志, 2008, 39(9): 655–658.
[31] Yugo Iwasaki, Naoto Mishima, Kousaku Mizumoto, et al. Extracellular production of phospholipase D of Sstreptomyces antibioticus using recombinant Escherichia coli[J]. Journal of Fermentation and Bioengineering, 1995, 79(5): 417–421.
[32] Giacomo Carrea, Paola D'Arrigo, Valentino Piergianni, et al. Purification and properties of two phospholipases D from Streptomyces sp.[J]. Biochimica et Biophysica Acta, 1995, 1255: 273–279.
[33] Carlo Zambonelli, Paola Morandi, Maria Antoniett Vanoni, et al. Cloning and expression in Escherichia coli of the gene encoding Streptomyces PMF PLD, a phospholipase D with high transphosphatidylation activity[J]. Enzyme and Microbial Technology, 2003, 33: 676–688.
[34] Tairo Hagishita, Masanobu Nishikawa, Tadashi Hatanaka. Isolation of phospholipase D producing microorganisms with high transphosphatidylation activity[J]. Biotechnology Letters, 2000, 22: 1587–1590.
[35] Jaya Ram Simkhada, Seung Sik Cho, Hyo Jung Lee, et al. Purification and biochemical properties of phospholipase D(PLD57) produced by Streptomyces sp. CS-57[J]. Arch Pharm Res, 2007, 30(10): 1302–1308.
[36] Jei Oh Yon, Ji Seon Lee, Bo Geum Kim, et al. Immobilization of Streptomyces phospholipase D on a Dowex Macroporous Resin[J]. Biotechnology and Bioprocess Engineering, 2008, 13: 102–107 .
[37] Chiaki Ogino, Masayuki Kanemasu, Masashi Fukumoto, et al. Continuous production of phospholipase D using immobilized recombinant Streptomyces lividans[J]. Enzyme and Microbial Technology, 2007, 41: 156–161.
[38]安红,宁伟明,张宏波.磷脂化学及应用技术[M].北京:中国计量出版社. 2006, 1–3, 23–26, 85, 90, 114, 155, 211–216.
[39]王兴国,裘爱泳,陶文沂,等.棒薄层色谱氢火焰法定量测定磷脂组成[J].无锡轻工大学学报, 1995, 14(1): 31–37.
[40] S.L. Melton. Analysis of soybean lecithins and beef phospholipids by HPLC with an evaporative light scattering detector[J]. J. Am. Oil Chem. Soc., 1992, 69(8): 784–788.
[41]张永刚,曹栋,史苏佳,等.磷脂酶D催化转碱基中磷脂的分析[J].广西农业生物科学, 2008, 27(2): 158–160.
[42]黄勇,吴兴中, Qureshi Imran Ahmad,等.用酶联比色发测定肝脏磷脂酰胆碱专一性磷脂酶D及其应用[J].上海医科大学学报, 1997, 24(5): 343–346.
[43]唐执文,周永红.氯化胆碱含量测定方法探讨[J].中国饲料, 2002(10):18–20.
[44] Siddiqui RA., Exton JH.. Phospholipid base exchange activity liver plasma membranes[J]. Bio. Chem., 1992, 267(9): 5755–5760.
[45] Kobayashi M., Kanfer JN.. Solubilization and purification of rat tissue phospholipase D[J]. Method Enzymol, 1991, 197:575–578.
[46]李媛媛,薛静,李莹倩,等.离子色谱法测定婴幼儿乳粉中的胆碱含量[J].食品科技, 2008, 33(12):267–270.
[47]薛静.离子色谱法测和高效液相色谱法测定乳粉中胆碱含量的研究: [硕士学位论文].长春:东北师范大学, 2008.
[48]钱峰,裘爱泳.磷脂改性现状[J].粮食与油脂, 2006(9): 7–9.
[49]崔德才,温孚江.磷脂酶D(PLD)在植物信号转导中的作用[J].山东农业大学学报(自然科学版), 2000, 31(2): 115–119.
[50]孟庆飞,温其标.磷脂酶法改性[J].河南工业大学学报(自然科学版), 2005, 26(5): 89–93.
[51]王兴国,裘爱泳,淘文祈,等.大豆磷脂的酶法改性[J].中国粮油学报, 1996, 11(2): 46–51.
[52]宋晚平,任静,王磊,等.一种粉末磷脂酰丝氨酸的制备方法.中国, CN101230365A, 2008.
[53]于刚,徐伟,栾东磊,等.亚临界R134a流体中酶法制备富含多不饱和脂肪酸的磷脂酰丝氨酸[J].高技术通讯, 2008, 18(10): 1075–1080.
[54]王兴国.磷脂研究的历史和现状[J].中国油脂, 1999, 24(5): 44–47.
[55]张永刚.磷脂酶D制备磷脂酰甘油的酶学特性研究: [硕士学位论文].无锡:江南大学, 2008.
[56] Lekh Raj Juneja, Naruhiro Hibi, Tsuneo Yamane, et al. Repeated batch and continuous operations for phosphatidylglycerol synthesis from phosphatidylcholine with immobilized phospholipase D[J]. Applied Microbiology and Biotechnology, 1987, 27(2): 146–151.
[57] Rina Satoa, Yutaka Itabashi, Tadashi Hatanaka, et al. Asymmetric in vitro synthesis of diastereomeric phosphatidylglycerols from phosphatidylcholine and glycerol bybacterial phospholipase D[J]. Lipids, 2004, 39(10): 1013–1018.
[58] Rina Sato, Yutaka Itabashi, Akira Suzuki, et al. Effect of temperature on the stereoselectivity of phospholipase D toward glycerol in the transphosphatidylation of phosphatidylcholine to phosphatidylglycerol[J]. Lipids, 2004, 39(10): 1019–1023.
[59] George J. Piazza, William N. Marmer. Conversion of phosphatidylcholine to phosphatidylglycerol with phospholipase D and glycerol[J]. J. Am. Oil Chem. Soc., 2007, 84: 645–651.
[60] Yukihiro Yamamoto, Masashi Hosokawa, Hideyuki Kurihara, et al. Preparation of phosphatidylated terpenes via phospholipase D-mediated transphosphatidylation [J]. J. Am. Oil Chem. Soc., 2008, 85: 313–320.
[61] Lekh Raj Juneja, Toru Kazuoka, Naomi Goto, et al. Conversion of phosphatidylcholine to phosphatidylserine by various phospholipase D in the presence of L- or D-serine[J]. Biochimca et Biophysica Acta, 1989, 1003(3): 277–283.
[62] Lekh Raj Juneja, Eri Taniguchi, Shoichi Shimizu, et al. Increasing productivity by removing choline in conversion of phosphatidylcholine to phosphatidylserine by phospholipase D[J]. Journal of Fermentation and Bioengineering, 1992, 73(5): 357–361.
[63] De Ferra Lorenzo, Massardo Pietro, Piccolo Oreste, et al. Process for the industrial preparation of phosphatidylserine. America, US5700668(A), 1997.
[64] Masashi Hosokawa, Tatuya Shimatani, Teruyuki Kanada, et al. Conversion to docosahexaenoic acid-containing phosphatidylserine from squid skin lecithin by phospholipase D-mediated transphosphatidylation[J]. J. Agric. Food Chem., 2000, 48(10): 4550–4554.
[65] Yugo Iwasaki, Yukiko Mizumotoa, Takahiro Okadab, et al. An aqueous suspension system for phospholipase D-mediated synthesis of PS without toxic organic solvent[J]. J. Am. Oil Chem. Soc., 2003, 7(80): 653–657.
[66] Meyer Randal. Method of producing phosphatidylserine. America, US7049107(B1), 2006.
[67]曹军卫,马辉文.微生物工程[M].北京:科学工业出版社. 2002, 24, 25, 80–86, 89–121.
[68]肖冬光.微生物工程原理[M].北京:中国轻工业出版社. 2004, 37–40, 61–71, 200–207.
[69]程殿林.微生物工程技术原理[M].北京:化学工业出版社. 2007, 18–21, 58–61, 92, 114–121.
[70]周德庆.微生物学教程(第二版)[M].北京:高等教育出版社. 2002, 213.
[71]侯英敏.等离子体诱变生产1,3-丙二醇菌种的研究: [硕士学位论文].大连:大连理工大学, 2006.
[72]李爽.等离子体诱变生产1,3-丙二醇菌种的研究: [硕士学位论文].大连:大连理工大学, 2007.
[73]姜玉,宋伟民.大气压冷等离子体杀菌作用及其机制的研究进展[J].国外医学卫生学分册, 2006, 33(5): 318–322.
[74]董晓宇,李爽,侯英敏,等.大气压冷等离子体诱变产1,3-丙二醇菌株Klebsiella pneumonia[J].过程工程学报, 2008, 8(3): 555–560.
[75] Herrmann H.W., Henins I., Park J., et al. Decontamination of chemical and biological warfare(CBW) agents using an atmospheric pressure plasma jet(APPJ)[J]. Physics of Plasmas, 1999, 6(5): 2284–2289.
[76] Thomas C. Montie, Kimberly Kelly-Wintenberg, J. Reece Roth. An overiew of reseacrh using the one amtosphere uniform glow discharge Plasma(OAUGDP) for sterilization of surfaces and materials[J]. IEEE Transactions on Plasma Science, 2000, 28(l): 41–50.
[77]何炜静.等离子体诱变筛选降解高浓度对苯二酚的突变株及其降解特性研究: [硕士学位论文].合肥:合肥工业大学, 2009.
[78]吴跃进,王学栋,刘贵富,等.离子束注入水稻诱变效应的研究[J].安徽农业科学, 1989(2): 10–13.
[79]余增亮,何建国,邓建国,等.离子注入水稻诱变育种的机理初探[J].安徽农业科学, 1989(1): 12–16.
[80]于红.大气压下等离子体失活微生物的机理研究: [博士学位论文].大连:大连理工大学, 2006.
[81]凌代俊,曹金样.低温等离子体诱导产生的一种“空洞”形细胞[J].核技术, 2003, 26(4): 284–286.
[82]蒋世春,吴建平,白骅.应用等离子体辐射技术选育柔红霉素产生菌-天兰淡红链霉菌[J].辐射研究与辐射工艺学报, 2001, 19(2): 133–137.
[83]施跃峰,桑金隆,竺莉红,等.等离子体处理选育之江菌素产生菌[J].核农学报, 2002, 16(4): 208–211.
[84]郁军,裘爱泳,刘元法.磷脂研究进展[J].西部粮油科技, 2000, 25(1): 26–29.
[85]郭秋华,何小维,黄强.大豆磷脂的酶法改性研究进展[J].食品工业科技, 2007, 28(10): 243–246.
[86] Ogino C., Kanemasu M., Hayashi Y., et al. Over-expression system for secretory phospholipase D by Streptomyces lividans[J]. Appl Microbiol Biotechnol, 2004, 64(7): 823–828.
[87]陈立梅,汪旭,李启云,等.链霉菌诱变育种方法综述[J].吉林农业科学, 2006, 31(2): 62–65.
[88]付学琴,魏赛金,黄文新,等.南昌链霉菌发酵条件的研究[J].江西农业大学学报, 2007, 29(3): 4573–460.
[89]韩友文.胆碱和氯化胆碱[J].饲料博览, 2004(3):45–46.
[90]田洪涛.现代发酵工艺原理与技术[M].北京:化学工业出版社, 2007. 46–48.
[91]陈希.产双酶假单胞杆菌的诱变选育及发酵条件的优化: [硕士学位论文].长沙:中南林业科技大学, 2006.
[92]栾雨时,包永明.生物工程实验技术手册[M].北京:化学工业出版社, 2005, 129, 149.
[93]陈志高,沈建新,胡叶碧.紫外线和氯化锂对核糖苷链霉菌原生质体诱变作用的探讨[J].中国医药工业杂志, 2001, 32(4): 150–152.
[94]黄永春,曹仁林,彭祎.紫外诱变吸水链霉菌选育高产农用抗生素菌株[J].安徽农业科学, 2008, 36(16): 6847–6848.
[95]季宇彬,朱兴杰,徐昶儒,等.微生物诱变育种方法的研究进展[J]. 496–502
[96]李戈.链霉菌LA5高产菌株诱变育种研究: [硕士学位论文].儋州:华南热带农业大学, 2007.
[97]诸葛健,沈微.工业微生物育种学[M].北京:化学工业出版社. 2006, 77–79.
[98] Laroussi M.. Non-thermal decontamination of biological media by atmospheric pressure plasmas: review , analysis, and prospects[J]. IEEE Transactions on Plasma Science, 2002, 30(4): 1409–1415.
[99] Lindsey F. Gaunt, Clive B. Beggs, George E. Georghiou. Bactericidal action of the reactive species produced by gas-discharge nonthermal plasma at atmospheric pressure: a review[J]. IEEE Transactions on Plasma Science, 2006, 34(4): 1257–1269.
[100] S.B. Farr, T. Kogoma. Oxidative stress responses in Escherichia coli and Salmonella typhimurium[J]. Microbiol. Rev., 1991, 55(4): 561–585.
[101] Lawrence J. Marnett. Oxyradicals and DNA damage[J]. Carcinogenesis, 2000, 21(3): 361–370.
[102]华泽钊,刘宝林,左建国.药品和食品的冷冻干燥[M].北京:科学出版社. 2006, 2–5, 10, 11.