【作者】 张起；

【导师】 陈秀兰；

【作者基本信息】 山东大学 ， 微生物学， 2008， 硕士

【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。更多还原

【Abstract】 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.更多还原