琼胶降解菌F-6筛选、培养条件及对条斑紫菜(Porphyra yezoensis)细胞解离作用的研究  

SCREENING AND CULTIVATION OF AGAROLYTIC BACTERIUM F-6 AND THE DECOMPOSING EFFECT OF AGARASE ON CELLS OF PORPHYRA YEZOENSIS

作　　者：付万冬[1] 韩宝芹[1] 王常红[1] 刘万顺[1] 

机构地区：[1]中国海洋大学海洋生命学院,青岛266003

出　　处：《海洋与湖沼》2007年第4期343-350,共8页Oceanologia Et Limnologia Sinica

基　　金：国家"十五"科技攻关项目;2001BA708B04-07号。

摘　　要：利用琼胶作为唯一的碳源从青岛太平角海域的海水和红藻样品中筛选分离得到一株高产琼胶酶的海洋菌F-6,对其最适的产酶条件,利用琼胶酶分离条斑紫菜的原生质体和原生质体的培养进行研究。结果表明,通过单次单因子和正交实验确定琼胶降解菌株F-6最适产酶培养基配方为(W/V):琼胶0.7%;酵母粉0.3%;蛋白胨0.5%;NaCl2.0%;K2HPO40.1%;CaCl20.02%;MgSO4·7H2O0.05%;FeSO4·7H2O0.002%;起始pH=7.5,最适的发酵产酶条件为:26℃培养36h。经条件优化后,粗酶液的酶活高达631.6U/ml。发酵液经过6000g离心30min得到粗酶液,粗酶液经过8k分离膜超滤,加入1mol/L渗透剂,0.22μm滤膜过滤除菌后降解条斑紫菜组织块,成功地分离得到大量的紫菜原生质体,其产率为3×106个原生质体/g新鲜紫菜组织。原生质体经初步培养发育成愈伤组织,其成活率为60%。The authors isolated F-6, a marine agarolytic bacterium able to produce agarase effectively from red algae in coastal seawater in Qingdao, Shandong of China, using agarose medium as the only-carbon source. The experiment was carried out from Sep. 2004 to Mar. 2005 for studying the decomposition effect by agarase on Porphyra yezoensis cells. With one-factor-at-a-time method and orthogonal experiment, the optimal fermentation condition of strain F-6 was determined. Besides, the decomposition effect by agarase on P. yezoensis cells was studied. Main procedures of experiment of decomposition effect were 1） centrifuged the fermentation broth at 6000g for 30min to obtain supernatant for culture; 2） ultrafiltrated the supernatant and then mixed with 1mol/L permeabilization agents; 3） filtrated the mixture for removing bacteria by 0.22μm separate film; 4） decomposed P. yezoensis tissue for producing protoplasts; 5） poured the collected protoplasts into hyperosmotic and sterilized in sea water with 1% PESI; 6） cultivated the protoplasts in 20℃ in light scheme of 12h/day at 1500-2000 lx until the salt concentration was gradually declined to normal sea water level. We also examined the healthy condition of the protoplasts, typical development of cells and their survival rate. The optimal fermentation condition of strain F-6 was agar 0.7%; yeast powder 0.3%; peptone 0.5%; NaCl 2.0%; K2HPO4 0.1%; MgSO4·7H2O 0.05%; FeSO4·7H2O 0.002%; CaCl2 0.02%; and initial pH 7.5. The optimal temperature and duration were 26℃ and 36h, respectively, at which the highest agarase activity reached 631.6U/ml. The experiments of decomposition effect of agarase on P. yezoensis cells and protoplast cultivation showed that the agarase could easily decompose P. yezoensis tissue into protoplasts at production rate of about 3×10^6 protoplasts per gram of the tissue. The optimal duration of enzymatic hydrolysis to P. yezoensis was 3.5h. The permeabilization agent of glucose （1mol/L） could obviously improve the production rat

关 键 词：琼胶降解菌 琼胶酶 培养条件优化 条斑紫菜 原生质体 

分 类 号：Q556.3[生物学—生物化学]