琼胶酶高产菌株的筛选、酶的分离纯化及红藻原生质体的酶法制备
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摘要
海藻的原生质体制备被广泛应用于生理学、生物化学、遗传学研究,也是海产品养殖、育种中的重要技术。而且,从原生质体出发,可以制备多种对于科研和生产非常有价值的复合物。目前,海藻原生质体的生产主要是使用海洋细菌所产解壁酶进行酶法制备,这些酶包括纤维素酶、果胶酶、木聚糖酶和琼胶酶等。
红藻门包括多种形态、性质各异的海藻,其细胞壁成分随着生长时期、生理状态、培养条件的不同而处于动态变化中。因此找到一种能有效降解红藻细胞壁的酶以大量制备原生质体,就成为开展红藻相关研究的关键。红藻细胞壁共有的主要成分是琼胶——包括琼脂糖和琼脂胶,而琼胶酶就是专一降解这种多糖的水解酶。
本文从在烟台沿海采集的多种红藻表面分离到一株高产琼胶酶的革兰氏阴性菌。该菌株不能降解纤维素和淀粉,也不能液化明胶。16SRNA分析表明其与Agarivorans sp.的同源性在99%以上,故命名为Agarivorans sp.SM0527。同时,本文对SM0527发酵产琼胶酶的条件进行了优化:人工海水配制,含琼脂0.5%,酵母粉0.5%,发酵起始pH 7.0,500 ml三角瓶中装培养基100ml,接种量为5%,培养时间为60小时。在上述条件下,发酵液中琼胶酶的酶活可达到3300 IU/ml。发酵的粗酶液通过离心、硫酸铵沉淀和阴离子交换层析,得到一个分子量大约90 kD的琼胶酶。该酶最适反应pH值为8.5,最适反应温度为45℃,最适反应时间为10 min。
本文使用SM0527所产的琼胶酶及本试验室保存的拟康氏木霉Trichodermapseudokoningii K9301所产的纤维素酶试验了多种红藻原生质体的制备。结果表明含琼胶酶10%-15%,纤维素酶1%-2%的混合酶对于条斑紫菜、江蓠、三叉仙菜、海头红细胞壁都有良好的酶解效果。在使用葡萄糖作为渗透稳定剂的条件下,条斑紫菜、江蓠、海头红原生质体产量分别达到了9.4×10~5、1.6×10~6、2.3×10~6个原生质体/克鲜藻。
Viable single cells and protoplasts of algae can be used in physiological, biochemical and genetic studies,biotechnology and mariculture.In addition,they can be employed to obtain economically valuable compounds.Single cells and protoplasts can be isolated by enzyme treatment of seaweed thalli.Complex enzyme systems including cellulases,pectinases,xylanases and agarases are necessary for obtaining protoplasts.
Red algae are a very heterogeneous group with respect to the chemical composition of the cell walls and intercellular matrix,which can change depending on the life-cycle stage,physiological status and cultural conditions.Therefore,finding new sources of enzymes degrading red algal polysaccharides and selecting enzyme complexes for thalli maceration are beneficial to the development of red seaweed cell technologies.Agarases are specific enzymes catalyzing the hydrolysis of agar,which is the major component of the cell wall matrix in red algae.
The Agarase-Producing bacteria was isolated from red algae collected in Yantai. The strain is Gram-negative,couldn't hydrolyze the cellulose and starch,and couldn't fluidify glutin.16S rDNA sequence analysis of the strain revealed high similarity with the Agarivorans sp.:therefor it was named Agarivorans sp.SM0527.
At the same time,the liquid state fermentation media for agarase production by SM0527 was optimized.The optimized medium contained agarose 0.5%;yeast extract 0.5%;each 500 ml gular flask containing 100 ml of medium,inoculum size 5%and a harvest time of 60h.Under these optimized conditions,agarase activity of 3300 IU/ml in the crude culture filtrate was obtained.
By centrifugation、ammonium sulfate fractionation and anion- exchange,a homogenousβ-agarase was purified from the crude culture filtrate of SM0527 with a gain rate of 10.2%and a purification of 8.2 folds.The molecular weight of the purified agarase was approximately 90 kDa.The optimal pH for the agarase activity was at pH 8.5,and agarase was optimally active at 45℃.Furthermore,the optimal reaction time of the enzyme assay was l0 min.
The agarase from SM0527 and the cellulases excreted by Trichoderma pseudokoningii K9301(a strain preserved in our lab)were employed to isolate protoplasts from some species of red algae.Experiments revealed that this complex enzyme system which contained the agarase of 10%to 15%and the cellulases of 1% to 2%had good effects on red algae including Porphyra yezoensis,Gracilaria verrucosa,Ceramium koudoi and Plocamium telfariae.With glucose of 2 M,an effective and economical penetrate in preparing protoplasts,yields of these protoplasts reached about 9.4×10~5 To 2.3×10~6 cells per gram of fresh weight algae.
红藻门包括多种形态、性质各异的海藻,其细胞壁成分随着生长时期、生理状态、培养条件的不同而处于动态变化中。因此找到一种能有效降解红藻细胞壁的酶以大量制备原生质体,就成为开展红藻相关研究的关键。红藻细胞壁共有的主要成分是琼胶——包括琼脂糖和琼脂胶,而琼胶酶就是专一降解这种多糖的水解酶。
本文从在烟台沿海采集的多种红藻表面分离到一株高产琼胶酶的革兰氏阴性菌。该菌株不能降解纤维素和淀粉,也不能液化明胶。16SRNA分析表明其与Agarivorans sp.的同源性在99%以上,故命名为Agarivorans sp.SM0527。同时,本文对SM0527发酵产琼胶酶的条件进行了优化:人工海水配制,含琼脂0.5%,酵母粉0.5%,发酵起始pH 7.0,500 ml三角瓶中装培养基100ml,接种量为5%,培养时间为60小时。在上述条件下,发酵液中琼胶酶的酶活可达到3300 IU/ml。发酵的粗酶液通过离心、硫酸铵沉淀和阴离子交换层析,得到一个分子量大约90 kD的琼胶酶。该酶最适反应pH值为8.5,最适反应温度为45℃,最适反应时间为10 min。
本文使用SM0527所产的琼胶酶及本试验室保存的拟康氏木霉Trichodermapseudokoningii K9301所产的纤维素酶试验了多种红藻原生质体的制备。结果表明含琼胶酶10%-15%,纤维素酶1%-2%的混合酶对于条斑紫菜、江蓠、三叉仙菜、海头红细胞壁都有良好的酶解效果。在使用葡萄糖作为渗透稳定剂的条件下,条斑紫菜、江蓠、海头红原生质体产量分别达到了9.4×10~5、1.6×10~6、2.3×10~6个原生质体/克鲜藻。
Viable single cells and protoplasts of algae can be used in physiological, biochemical and genetic studies,biotechnology and mariculture.In addition,they can be employed to obtain economically valuable compounds.Single cells and protoplasts can be isolated by enzyme treatment of seaweed thalli.Complex enzyme systems including cellulases,pectinases,xylanases and agarases are necessary for obtaining protoplasts.
Red algae are a very heterogeneous group with respect to the chemical composition of the cell walls and intercellular matrix,which can change depending on the life-cycle stage,physiological status and cultural conditions.Therefore,finding new sources of enzymes degrading red algal polysaccharides and selecting enzyme complexes for thalli maceration are beneficial to the development of red seaweed cell technologies.Agarases are specific enzymes catalyzing the hydrolysis of agar,which is the major component of the cell wall matrix in red algae.
The Agarase-Producing bacteria was isolated from red algae collected in Yantai. The strain is Gram-negative,couldn't hydrolyze the cellulose and starch,and couldn't fluidify glutin.16S rDNA sequence analysis of the strain revealed high similarity with the Agarivorans sp.:therefor it was named Agarivorans sp.SM0527.
At the same time,the liquid state fermentation media for agarase production by SM0527 was optimized.The optimized medium contained agarose 0.5%;yeast extract 0.5%;each 500 ml gular flask containing 100 ml of medium,inoculum size 5%and a harvest time of 60h.Under these optimized conditions,agarase activity of 3300 IU/ml in the crude culture filtrate was obtained.
By centrifugation、ammonium sulfate fractionation and anion- exchange,a homogenousβ-agarase was purified from the crude culture filtrate of SM0527 with a gain rate of 10.2%and a purification of 8.2 folds.The molecular weight of the purified agarase was approximately 90 kDa.The optimal pH for the agarase activity was at pH 8.5,and agarase was optimally active at 45℃.Furthermore,the optimal reaction time of the enzyme assay was l0 min.
The agarase from SM0527 and the cellulases excreted by Trichoderma pseudokoningii K9301(a strain preserved in our lab)were employed to isolate protoplasts from some species of red algae.Experiments revealed that this complex enzyme system which contained the agarase of 10%to 15%and the cellulases of 1% to 2%had good effects on red algae including Porphyra yezoensis,Gracilaria verrucosa,Ceramium koudoi and Plocamium telfariae.With glucose of 2 M,an effective and economical penetrate in preparing protoplasts,yields of these protoplasts reached about 9.4×10~5 To 2.3×10~6 cells per gram of fresh weight algae.
引文
1.陈昌生 坛紫菜和条斑紫菜的原生质体电融合 生物工程学报1992年01期
2.陈海敏 严小军 郑立 张伟伟 林伟 琼胶的降解及其产物的分析 郑州工程学院学报2003年03期
3.陈惠源 蔡俊鹏 刘江涛 海洋细菌产琼胶酶的条件优化 现代食品科技2005年03期
4.陈菊琦 活体生物酶分离石花菜原生质体的研究 厦门水产学院学报1999年07期
5.陈菊琦 坛紫菜 不同部位营养细胞分离培养的比较研究 厦门水产学院学报1989年01期
6.褚燕 于文功 韩峰 琼胶酶高产海洋假单胞菌CY-24的筛选及培养条件的优化 中国海洋药物2003年05期
7.蔡俊鹏 刘江涛 产琼胶酶菌株的筛选及其胞内外酶活的测定 天然药物研究与开发2005年06期
8.邓元告 左正宏 陈奕欣 郑微云 坛紫菜叶绿体DNA的快速提取 厦门大学学报(自然科学版)2003年06期
9.杜宗军 王祥红 李筠 陈吉祥 琼胶酶研究进展 微生物学通报2003年01期
10.杜宗军 王鹏 李筠等 两株琼胶酶高产细菌的筛选和鉴定 海洋科学2002年03期
11.付万冬 韩宝芹 王常红 刘万顺 琼胶降解菌F-6筛选、培养条件及对条斑紫菜细胞解离作用的研究 海洋与湖沼2007年04期
12.郭宝太 毕玉平 单雷 李广存 条斑紫菜高纯度总DNA及其质粒状DNA的提取 海洋学报2000年02期
13.郭琪 王静雪 海洋微生物酶的研究概况 水产科学2005年12期
14.古静燕 韩文君 李天东 王君明 阮期平Paenibacillus sp1MY03菌株产胞外琼胶酶的条件优化 绵阳师范学院学报2007年05期
15.胡晓珂 江晓路 管华诗 海藻多糖降解酶的性质和作用机理 微生物学报2001年06期
16.林学政 沈继红 李广友 海藻原生质体融合及杂交技术的研究进展2000年 08期
17.初建松 刘万顺 张朝阳 江蓠原生质体分离和培养的初步研究 海洋通报1998年06期
18.刘江涛 蔡俊鹏 吴冰 两株琼胶酶高产细菌的筛选和鉴定 现代食品科技2005年01期
19.刘江涛 蔡俊鹏 吴冰 琼胶酶及其综合应用的研究概况 现代食品科技2005年01期
20.李能章 邱荣蓉 彭远义 琼胶酶的研究进展 生命科学研究2006年02期
21.缪伏荣 李忠荣 琼胶的降解及其产物的开发应用 现代农业科技2007年02期
22.马凌波 王素娟等 细菌酶酶解条斑紫菜体细胞及市内人工育苗的研究 水产学报1998年10期
23.欧昌荣 汤海青 管斌 琼胶酶生产菌的筛选、鉴定及其酶学性质的初步研究食品科学2005年06期
24.汤海青 管斌 王雪鹏 陈丽红 欧昌荣 胡文浪琼胶降解菌AT-22的筛选和产酶性质 中国水产科学2005年02期
25.王广慧 张明 琼脂糖酶的研究进展 海洋通报 2006年03期
26.王明兹 庄惠如 陈必链 施巧琴 吴松刚 紫球藻原生质体分离研究 药物生物技术2001年08期
27.王静雪 江晓路 胡晓珂 细菌降解琼胶的研究进展 中国水产科学2001年03期
28.王静雪 江晓路 牟海津 管华诗 海洋弧菌QJH-12发酵产琼胶酶条件的优化海洋科学2007年07期
29.徐丽 刘江涛 蔡俊鹏 一株琼胶酶高产菌株的筛选鉴定及产酶条件的优化现代食品科技2006年04期
30.许星鸿 王萍 赵素珍 制备条斑紫菜原生质体的工具酶研究 淮海工学院学报(自然科学版)2007年03期
31.谢恩义 马家海 礁膜原生质体的分离及培养 水产学报2004年01期
32.严兴洪 紫菜种内原生质体的融合和融合体再生 上海水产大学学报2001年 02期
33.严兴洪 田中次郎 有贺佑胜 条斑紫菜色彩突变体的诱导、分离和特性分析水产学报2000年03期
34.杨震 唐学玺 戴继勋 沈颂东 贾鹏举 酶解条件对条斑紫菜单细胞成活率影响的比较研究 青岛海洋大学学报2001年03期
35.余长缨 沈颂东 戴世勋 韩宝琴 从石鳖和帽贝中提取海藻解壁酶的初步研究 海洋通报2000年05期
36.张学成 程晓洁 施定基 江蓠属藻胆蛋白的研究 海洋学报1999年01期
37.张学成 程晓杰 隋正红 魏东 刘晨临 徐年军 江蓠属藻胆体的研究青岛海洋大学学报1999年02期
38.张学成 程晓杰 隋正红 魏东 刘晨临 徐年军江蓠属藻胆体的研究-藻胆体的荧光光谱特性 青岛海洋大学学报1999年03期
39.张亚萍 于文功 戴继勋 管华诗植物激素在条斑紫菜原生质体固体培养中的作用 海洋湖沼通报2000年04期
40.Aguirre-Lipperheide M.,Estrada-Rodriguez F.J.and Evans L.V.Facts,problems,and needs in seaweed tissue culture:an appraisal.J.Phycol.(1995)31:677-688.
41.Allouch,J.,M.Jam,W.Helbert,T.Barbeyron,B.Kloareg,B.Henrissat,and M.Czjzek.The three-dimensional structures of two beta-agarases.J.Biol.Chem.(2003)278:47171-47180.
42.Aoki T,Araki T,Kitamikado M Purification and characterization of a novel β-agarase from Vibrio sp.AP-2.Eur J Biochem(1990)187:461-465.
43.Benet H,Ar Gall E,Asensi A,Kloareg B Protoplast regeneration from gametophytes and sporophytes of some species in the order Laminariales (Phaeophyceae).Protoplasma(1997)199:39-48.
44.Chen Y.C.Development of protoplasts from holdfasts and vegetative thalli of Monostroma latissimum(Chlorophyta,Monostromatacae)for algal seed stock.J.Phycol.(2004)34:1075-1081.
45.C.R.K.Reddy,Shikh Dipakkore,G.Rajakrishna Kumar,Bhavanath Jha,Donald P.Cheney,Yuji Fujita An improved enzyme preparation for rapid mass production of protoplasts as seed stock for aquaculture of macrophytic marine green algae. Aquaculture 260 (2006) 290-297.
46. Didier Flament, Tristan Barbeyron, Murielle Jam, Philippe Potin, Mirjam Czjzek, Bernard Kloareg and Gurvan Michel Alpha-Agarases Define a New Family of Glycoside Hydrolases, Distinct from Beta-Agarase Families. Apply and environmental microbiology (2007), 17, 4691-4694.
47. E.V.Nikolaeva, A.I.Usov, A.P. Sinitsyn and A.H. Tambiev Degradation of agarophytic red algal cell wall components by new crude enzyme preparations. Journal of Applied Phycology 11: 385-389,1999.
48. Fujita, Y., and Migita, S. Isolation and culture of protoplasts from some seaweeds. Bull Fac Fish Nagasaki Univ (1985) 57:39-45.
49. Hongquan Liu, Wengong Yu, Jixun Dai, Qianhong Gong, Kunfeng Yang, Xinzhi Lu Cryopreservation of protoplasts of the alga Porphyra yezoensis by vitrification. Plant Science 166 (2004) 97-102.
50. HU Xiaoke, JIANG Xiaolu, GUAN Huashi Isolation of Protoplasts from Undada pinnatifida by Alginate Lyase Digestion. Journal of Ocean University of Oingdao. 2003 Vol.2 No.1 58—61.
51. Liu CL, Huang XH,Wang XL, Zhang XC, Li GY. Phylogenetic studies on two strains of Antarctic ice algae based on morphological and molecular characteristics. Phycologia, (2006) 45(2): 190-198.
52. Lyudmila A. Romanenko, Natalia V. Zhukova, Manfred Rohde, Anatoly M. Lysenko, Valery V. Mikhailov and Erko Stackebrandt Pseudoalteromonas agarivorans sp. nov., a novel marine agarolytic bacterium International. Journal of Systematic and Evolutionary Microbiology (2003), 53, 125-131.
53. Jingbao Li, Feng Han, Xinzhi Lu, Xiaoyan Fu, Cuiping Ma, Yan Chu and Wengong Yu A simple method of preparing diverse neoagaro-oligosaccharides with b-agarase. Carbohydrate Research 342 (2007) 1030-1033.
54. Isabelle Mussio and Anne-Marie Rusig Isolation of protoplasts from Fucus serratus and F. vesiculosus (Fucales,Phaeophyceae): factors affecting protoplast yield. Journal of Applied Phycology (2006) 18: 733-740.
55. Kajiwara, T., Hatanaka, A., Fujimura, T., Kawai, T., and Irie, M.. Isolation of protoplasts from marine brown algae Dictyotaceae plants. Nippon Suisan Gakkaishi (1988)54:1255.
56. Kirimura K, Masuda N, Iwasaki Y, Nakagawa H, Kobayashi R, Usami S Purification and characterization of a novel P-agarase from an alkalophilic bacterium, Alteromonas sp. E-1. J Biosci Bioeng (1999)87:436-441.
57. K.KEVEKORDES, J.BEARDALL and M.N.CLAYTON. A Novel Method for Extracting Protoplasts from Large Brown Algae. Journal of Experimental Botany Vol.44, No.10 1587-1593.
58. Lee DG Park GT, Kim NY, Lee EJ, Jang MIC, Shin YG, Park GS, Kim TM, Lee JH, Lee JH, Kim SJ, Lee SH Cloning, expression, and characterization of a glycoside hydrolase family 50 p-agarase from a marine Agarivorans isolate. Biotechnol Lett (2006)28:1925-1932.
59. M.C.Macia, W. Ludwig, K.H.Schleifer, M.J.Pujalte, and E.Garay Vibrio agarivorans sp. nov., a novel agarolytic marine bacterium. International Journal of Systematic and Evolutionary Microbiology (2001), 51, 2031-2036.
60. Midori Kurahashi and Akira Yokota Agarivorans albus gen. nov., sp. nov., a proteobacterium isolated from marine animals. International Journal of Systematic and Evolutionary Microbiology (2004). 54, 693-697.
61. M.Heidecker, L.H.Wegner, U.Zimmermann A Patch-Clamp Study of Ion Channels in Protoplasts Prepared from the Marine Alga Valonia utricularis. J. Membrane Biol. 172, 235-247 (1999).
62. Mollet JC, Verdus MC, Kling R, Morvan H Improved protoplast yield and cell wall regeneration in Gracilaria verrucosa (Huds.) Papenfuss (Gracilariales, Rhodophyta). J. Exp. Bot.(1995) 46:239-247.
63. Murielle JAM, Didier FLAMENT, Julie ALLOUCH The endo-p-agarases AgaA and AgaB from the marine bacterium Zobellia galactanivorans: two paralogue enzymes with different molecular organizations and catalytic behaviours. Biochem. J. (2005)385,703-713
64. Ohta Y, Hatada Y, Ito S, Horikoshi K High-level expression of a neoagarobiose-producing beta-agarase gene from Agarivorans sp. JAMB-A11 in Bacillus subtilis and enzymic properties of the recombinant enzyme. Biotechnol Appl Biochem (2005)41:183-191.
65. Ohta Y, Hatada Y, Miyazaki M, Nogi Y, Ito S, Horikoshi K Purification and characterization of a novel α-agarase from a Thalassomonas sp. Curr Microbiol (2005) 50:212-216.
66. Ohta Y, Hatada Y, Nogi Y, Miyazaki M, Li Z, Akita M, Hidaka Y, Goda S, Ito S, Horikoshi K Enzymatic properties and nucleotide and amino acid sequences of a thermostable P-agarase from a novel species of deep-sea Microbulbifer. Appl Microbiol Biotechnol (2004)64:505-514.
67. Polne-Fuller M, Gibor A Calluses, cells and protoplasts in studies towards genetic improvement of seaweeds. Aquaculture (1986)57: 117-123.
Polne-Fuller M, Saga N, Gibor A Algal cell, callus and tissue cultures and selection of algal strains. Beih. Nova Hedwigia (1986)83:30-36.
68. Q. Wang, R. Gafny, N. Sahar, I. Sela, M. Mawassi, E. Tanne, A. Perl, Cryopreservation of grapevine (Vitis vinifera L.) embryogenic cell suspensions by encapsulation-dehydration and subsequent plant regeneration, Plant Sci. 162 (2002) 551-558.
69. Srinivasa Rao, Uppalapati, and Yuji Fujita A simple method for mass isolation of protoplasts from species of Monostroma, Enteromorpha and Ulva (Chlorophyta. Ulvales). Journal of Applied Phycology 14: 165-168, 2002.
70. R. Langis, P.L. Steponkus, Cryopreservtion of rye protoplasts by vitrification, Plant Physiol. 92 (1990) 666-671.
71. Rusig A.-M. and Cosson J. Plant regeneration from protoplasts of Enteromorpha intestinalis (Chlorophyta, Ulvophyceae) as seedstock for macroagal culture. J. appl. Phycol. (2001)13: 103-108.
72. Shenghao Liu, Chenlin Liu, Xiaohang Huang, Yingmei Chai, Bailin Cong Optimization of parameters for isolation of protoplasts from the Antarctic sea ice alga Chlamydomonas sp. ICE-L. J Appl Phycol (2006) 18:783-786.
73. Tambiev AH, Sinitsyn AP, Nikolaeva EV, Anikina MI The effect of the land fungi exoenzymes on the cell wall of red seaweeds. Biotechnologia (1997)2: 10-14.
74. T. Matsumoto, C. Takahashi, A. Sakai, Y. Nako, Cryopreservation of in vitro-grown apical meristems of hybrid statice by three different procedures, Sci. Hor. 76(1998)105-114.
75. Tomoaki Wakabayashi, Toru Kuboi, Takuya Tuboi, Masayuki Kaji, and Masakazu Hara Preparation of High Yields of Algal Protoplasts Using Buccal Juice of Sea Hare and Commercial Cellulase. Mar. Biotechnol. 1, 407-410, 1999.
76. Vera, J., Alvarez, R., Murano, E., Slebe, J. C. and Leon, O. Identification of a marine agarolytic pseudoalteromonas isolate and characterization of its extracellular agarase. Appl. Environ. Microbiol. (1998) 64,4378-4383.
77. Wang J, Mou H, Jiang X, Guan H Characterization of a novel β -agarase from marine Alteromonas sp. SY37-12 and its degrading products. Appl Microbiol Biotechnol (2006) 71:833-839.
78. Wei-wei Zhang and Li Sun Cloning, Characterization, and Molecular Application of a Beta-Agarase Gene from Vibrio sp. Strain V134. Apply and environmental microbiology (2007), 9, 2825-2831.
79. Xiao Ting Fu, Hong Lin and Sang Moo Kim Purification and characterization of a novel β -agarase, AgaA34, from Agarivorans albus YKW-34. Appl Microbiol Biotechnol. Accepted: 25 November 2007.
80. Yuzuru Mizukami, Masanori Okauchi, Hitoshi Kito, Shin-ichi Ishimoto, Toshiro Ishida, Mituru Fuseya Culture and development of electrically fused protoplasts from red marine algae, Porphyra yezoensis and P. suborbiculata. Aquaculture 132 (1995)361-367.
2.陈海敏 严小军 郑立 张伟伟 林伟 琼胶的降解及其产物的分析 郑州工程学院学报2003年03期
3.陈惠源 蔡俊鹏 刘江涛 海洋细菌产琼胶酶的条件优化 现代食品科技2005年03期
4.陈菊琦 活体生物酶分离石花菜原生质体的研究 厦门水产学院学报1999年07期
5.陈菊琦 坛紫菜 不同部位营养细胞分离培养的比较研究 厦门水产学院学报1989年01期
6.褚燕 于文功 韩峰 琼胶酶高产海洋假单胞菌CY-24的筛选及培养条件的优化 中国海洋药物2003年05期
7.蔡俊鹏 刘江涛 产琼胶酶菌株的筛选及其胞内外酶活的测定 天然药物研究与开发2005年06期
8.邓元告 左正宏 陈奕欣 郑微云 坛紫菜叶绿体DNA的快速提取 厦门大学学报(自然科学版)2003年06期
9.杜宗军 王祥红 李筠 陈吉祥 琼胶酶研究进展 微生物学通报2003年01期
10.杜宗军 王鹏 李筠等 两株琼胶酶高产细菌的筛选和鉴定 海洋科学2002年03期
11.付万冬 韩宝芹 王常红 刘万顺 琼胶降解菌F-6筛选、培养条件及对条斑紫菜细胞解离作用的研究 海洋与湖沼2007年04期
12.郭宝太 毕玉平 单雷 李广存 条斑紫菜高纯度总DNA及其质粒状DNA的提取 海洋学报2000年02期
13.郭琪 王静雪 海洋微生物酶的研究概况 水产科学2005年12期
14.古静燕 韩文君 李天东 王君明 阮期平Paenibacillus sp1MY03菌株产胞外琼胶酶的条件优化 绵阳师范学院学报2007年05期
15.胡晓珂 江晓路 管华诗 海藻多糖降解酶的性质和作用机理 微生物学报2001年06期
16.林学政 沈继红 李广友 海藻原生质体融合及杂交技术的研究进展2000年 08期
17.初建松 刘万顺 张朝阳 江蓠原生质体分离和培养的初步研究 海洋通报1998年06期
18.刘江涛 蔡俊鹏 吴冰 两株琼胶酶高产细菌的筛选和鉴定 现代食品科技2005年01期
19.刘江涛 蔡俊鹏 吴冰 琼胶酶及其综合应用的研究概况 现代食品科技2005年01期
20.李能章 邱荣蓉 彭远义 琼胶酶的研究进展 生命科学研究2006年02期
21.缪伏荣 李忠荣 琼胶的降解及其产物的开发应用 现代农业科技2007年02期
22.马凌波 王素娟等 细菌酶酶解条斑紫菜体细胞及市内人工育苗的研究 水产学报1998年10期
23.欧昌荣 汤海青 管斌 琼胶酶生产菌的筛选、鉴定及其酶学性质的初步研究食品科学2005年06期
24.汤海青 管斌 王雪鹏 陈丽红 欧昌荣 胡文浪琼胶降解菌AT-22的筛选和产酶性质 中国水产科学2005年02期
25.王广慧 张明 琼脂糖酶的研究进展 海洋通报 2006年03期
26.王明兹 庄惠如 陈必链 施巧琴 吴松刚 紫球藻原生质体分离研究 药物生物技术2001年08期
27.王静雪 江晓路 胡晓珂 细菌降解琼胶的研究进展 中国水产科学2001年03期
28.王静雪 江晓路 牟海津 管华诗 海洋弧菌QJH-12发酵产琼胶酶条件的优化海洋科学2007年07期
29.徐丽 刘江涛 蔡俊鹏 一株琼胶酶高产菌株的筛选鉴定及产酶条件的优化现代食品科技2006年04期
30.许星鸿 王萍 赵素珍 制备条斑紫菜原生质体的工具酶研究 淮海工学院学报(自然科学版)2007年03期
31.谢恩义 马家海 礁膜原生质体的分离及培养 水产学报2004年01期
32.严兴洪 紫菜种内原生质体的融合和融合体再生 上海水产大学学报2001年 02期
33.严兴洪 田中次郎 有贺佑胜 条斑紫菜色彩突变体的诱导、分离和特性分析水产学报2000年03期
34.杨震 唐学玺 戴继勋 沈颂东 贾鹏举 酶解条件对条斑紫菜单细胞成活率影响的比较研究 青岛海洋大学学报2001年03期
35.余长缨 沈颂东 戴世勋 韩宝琴 从石鳖和帽贝中提取海藻解壁酶的初步研究 海洋通报2000年05期
36.张学成 程晓洁 施定基 江蓠属藻胆蛋白的研究 海洋学报1999年01期
37.张学成 程晓杰 隋正红 魏东 刘晨临 徐年军 江蓠属藻胆体的研究青岛海洋大学学报1999年02期
38.张学成 程晓杰 隋正红 魏东 刘晨临 徐年军江蓠属藻胆体的研究-藻胆体的荧光光谱特性 青岛海洋大学学报1999年03期
39.张亚萍 于文功 戴继勋 管华诗植物激素在条斑紫菜原生质体固体培养中的作用 海洋湖沼通报2000年04期
40.Aguirre-Lipperheide M.,Estrada-Rodriguez F.J.and Evans L.V.Facts,problems,and needs in seaweed tissue culture:an appraisal.J.Phycol.(1995)31:677-688.
41.Allouch,J.,M.Jam,W.Helbert,T.Barbeyron,B.Kloareg,B.Henrissat,and M.Czjzek.The three-dimensional structures of two beta-agarases.J.Biol.Chem.(2003)278:47171-47180.
42.Aoki T,Araki T,Kitamikado M Purification and characterization of a novel β-agarase from Vibrio sp.AP-2.Eur J Biochem(1990)187:461-465.
43.Benet H,Ar Gall E,Asensi A,Kloareg B Protoplast regeneration from gametophytes and sporophytes of some species in the order Laminariales (Phaeophyceae).Protoplasma(1997)199:39-48.
44.Chen Y.C.Development of protoplasts from holdfasts and vegetative thalli of Monostroma latissimum(Chlorophyta,Monostromatacae)for algal seed stock.J.Phycol.(2004)34:1075-1081.
45.C.R.K.Reddy,Shikh Dipakkore,G.Rajakrishna Kumar,Bhavanath Jha,Donald P.Cheney,Yuji Fujita An improved enzyme preparation for rapid mass production of protoplasts as seed stock for aquaculture of macrophytic marine green algae. Aquaculture 260 (2006) 290-297.
46. Didier Flament, Tristan Barbeyron, Murielle Jam, Philippe Potin, Mirjam Czjzek, Bernard Kloareg and Gurvan Michel Alpha-Agarases Define a New Family of Glycoside Hydrolases, Distinct from Beta-Agarase Families. Apply and environmental microbiology (2007), 17, 4691-4694.
47. E.V.Nikolaeva, A.I.Usov, A.P. Sinitsyn and A.H. Tambiev Degradation of agarophytic red algal cell wall components by new crude enzyme preparations. Journal of Applied Phycology 11: 385-389,1999.
48. Fujita, Y., and Migita, S. Isolation and culture of protoplasts from some seaweeds. Bull Fac Fish Nagasaki Univ (1985) 57:39-45.
49. Hongquan Liu, Wengong Yu, Jixun Dai, Qianhong Gong, Kunfeng Yang, Xinzhi Lu Cryopreservation of protoplasts of the alga Porphyra yezoensis by vitrification. Plant Science 166 (2004) 97-102.
50. HU Xiaoke, JIANG Xiaolu, GUAN Huashi Isolation of Protoplasts from Undada pinnatifida by Alginate Lyase Digestion. Journal of Ocean University of Oingdao. 2003 Vol.2 No.1 58—61.
51. Liu CL, Huang XH,Wang XL, Zhang XC, Li GY. Phylogenetic studies on two strains of Antarctic ice algae based on morphological and molecular characteristics. Phycologia, (2006) 45(2): 190-198.
52. Lyudmila A. Romanenko, Natalia V. Zhukova, Manfred Rohde, Anatoly M. Lysenko, Valery V. Mikhailov and Erko Stackebrandt Pseudoalteromonas agarivorans sp. nov., a novel marine agarolytic bacterium International. Journal of Systematic and Evolutionary Microbiology (2003), 53, 125-131.
53. Jingbao Li, Feng Han, Xinzhi Lu, Xiaoyan Fu, Cuiping Ma, Yan Chu and Wengong Yu A simple method of preparing diverse neoagaro-oligosaccharides with b-agarase. Carbohydrate Research 342 (2007) 1030-1033.
54. Isabelle Mussio and Anne-Marie Rusig Isolation of protoplasts from Fucus serratus and F. vesiculosus (Fucales,Phaeophyceae): factors affecting protoplast yield. Journal of Applied Phycology (2006) 18: 733-740.
55. Kajiwara, T., Hatanaka, A., Fujimura, T., Kawai, T., and Irie, M.. Isolation of protoplasts from marine brown algae Dictyotaceae plants. Nippon Suisan Gakkaishi (1988)54:1255.
56. Kirimura K, Masuda N, Iwasaki Y, Nakagawa H, Kobayashi R, Usami S Purification and characterization of a novel P-agarase from an alkalophilic bacterium, Alteromonas sp. E-1. J Biosci Bioeng (1999)87:436-441.
57. K.KEVEKORDES, J.BEARDALL and M.N.CLAYTON. A Novel Method for Extracting Protoplasts from Large Brown Algae. Journal of Experimental Botany Vol.44, No.10 1587-1593.
58. Lee DG Park GT, Kim NY, Lee EJ, Jang MIC, Shin YG, Park GS, Kim TM, Lee JH, Lee JH, Kim SJ, Lee SH Cloning, expression, and characterization of a glycoside hydrolase family 50 p-agarase from a marine Agarivorans isolate. Biotechnol Lett (2006)28:1925-1932.
59. M.C.Macia, W. Ludwig, K.H.Schleifer, M.J.Pujalte, and E.Garay Vibrio agarivorans sp. nov., a novel agarolytic marine bacterium. International Journal of Systematic and Evolutionary Microbiology (2001), 51, 2031-2036.
60. Midori Kurahashi and Akira Yokota Agarivorans albus gen. nov., sp. nov., a proteobacterium isolated from marine animals. International Journal of Systematic and Evolutionary Microbiology (2004). 54, 693-697.
61. M.Heidecker, L.H.Wegner, U.Zimmermann A Patch-Clamp Study of Ion Channels in Protoplasts Prepared from the Marine Alga Valonia utricularis. J. Membrane Biol. 172, 235-247 (1999).
62. Mollet JC, Verdus MC, Kling R, Morvan H Improved protoplast yield and cell wall regeneration in Gracilaria verrucosa (Huds.) Papenfuss (Gracilariales, Rhodophyta). J. Exp. Bot.(1995) 46:239-247.
63. Murielle JAM, Didier FLAMENT, Julie ALLOUCH The endo-p-agarases AgaA and AgaB from the marine bacterium Zobellia galactanivorans: two paralogue enzymes with different molecular organizations and catalytic behaviours. Biochem. J. (2005)385,703-713
64. Ohta Y, Hatada Y, Ito S, Horikoshi K High-level expression of a neoagarobiose-producing beta-agarase gene from Agarivorans sp. JAMB-A11 in Bacillus subtilis and enzymic properties of the recombinant enzyme. Biotechnol Appl Biochem (2005)41:183-191.
65. Ohta Y, Hatada Y, Miyazaki M, Nogi Y, Ito S, Horikoshi K Purification and characterization of a novel α-agarase from a Thalassomonas sp. Curr Microbiol (2005) 50:212-216.
66. Ohta Y, Hatada Y, Nogi Y, Miyazaki M, Li Z, Akita M, Hidaka Y, Goda S, Ito S, Horikoshi K Enzymatic properties and nucleotide and amino acid sequences of a thermostable P-agarase from a novel species of deep-sea Microbulbifer. Appl Microbiol Biotechnol (2004)64:505-514.
67. Polne-Fuller M, Gibor A Calluses, cells and protoplasts in studies towards genetic improvement of seaweeds. Aquaculture (1986)57: 117-123.
Polne-Fuller M, Saga N, Gibor A Algal cell, callus and tissue cultures and selection of algal strains. Beih. Nova Hedwigia (1986)83:30-36.
68. Q. Wang, R. Gafny, N. Sahar, I. Sela, M. Mawassi, E. Tanne, A. Perl, Cryopreservation of grapevine (Vitis vinifera L.) embryogenic cell suspensions by encapsulation-dehydration and subsequent plant regeneration, Plant Sci. 162 (2002) 551-558.
69. Srinivasa Rao, Uppalapati, and Yuji Fujita A simple method for mass isolation of protoplasts from species of Monostroma, Enteromorpha and Ulva (Chlorophyta. Ulvales). Journal of Applied Phycology 14: 165-168, 2002.
70. R. Langis, P.L. Steponkus, Cryopreservtion of rye protoplasts by vitrification, Plant Physiol. 92 (1990) 666-671.
71. Rusig A.-M. and Cosson J. Plant regeneration from protoplasts of Enteromorpha intestinalis (Chlorophyta, Ulvophyceae) as seedstock for macroagal culture. J. appl. Phycol. (2001)13: 103-108.
72. Shenghao Liu, Chenlin Liu, Xiaohang Huang, Yingmei Chai, Bailin Cong Optimization of parameters for isolation of protoplasts from the Antarctic sea ice alga Chlamydomonas sp. ICE-L. J Appl Phycol (2006) 18:783-786.
73. Tambiev AH, Sinitsyn AP, Nikolaeva EV, Anikina MI The effect of the land fungi exoenzymes on the cell wall of red seaweeds. Biotechnologia (1997)2: 10-14.
74. T. Matsumoto, C. Takahashi, A. Sakai, Y. Nako, Cryopreservation of in vitro-grown apical meristems of hybrid statice by three different procedures, Sci. Hor. 76(1998)105-114.
75. Tomoaki Wakabayashi, Toru Kuboi, Takuya Tuboi, Masayuki Kaji, and Masakazu Hara Preparation of High Yields of Algal Protoplasts Using Buccal Juice of Sea Hare and Commercial Cellulase. Mar. Biotechnol. 1, 407-410, 1999.
76. Vera, J., Alvarez, R., Murano, E., Slebe, J. C. and Leon, O. Identification of a marine agarolytic pseudoalteromonas isolate and characterization of its extracellular agarase. Appl. Environ. Microbiol. (1998) 64,4378-4383.
77. Wang J, Mou H, Jiang X, Guan H Characterization of a novel β -agarase from marine Alteromonas sp. SY37-12 and its degrading products. Appl Microbiol Biotechnol (2006) 71:833-839.
78. Wei-wei Zhang and Li Sun Cloning, Characterization, and Molecular Application of a Beta-Agarase Gene from Vibrio sp. Strain V134. Apply and environmental microbiology (2007), 9, 2825-2831.
79. Xiao Ting Fu, Hong Lin and Sang Moo Kim Purification and characterization of a novel β -agarase, AgaA34, from Agarivorans albus YKW-34. Appl Microbiol Biotechnol. Accepted: 25 November 2007.
80. Yuzuru Mizukami, Masanori Okauchi, Hitoshi Kito, Shin-ichi Ishimoto, Toshiro Ishida, Mituru Fuseya Culture and development of electrically fused protoplasts from red marine algae, Porphyra yezoensis and P. suborbiculata. Aquaculture 132 (1995)361-367.