【作者】 马悦欣；

【导师】 董双林； 江晓路；

【作者基本信息】 中国海洋大学 ， 水产养殖， 2008， 博士

【摘要】 本论文目的是从海洋环境筛选高活性的κ-卡拉胶酶产生菌株,对筛选菌株的产酶培养条件和培养基进行优化,在此基础上对该菌株进行诱变以提高其酶活力,纯化其κ-卡拉胶酶,并研究该酶的酶学性质,利用此酶制备κ-卡拉胶寡糖,并对其酶解产物的组成和结构进行分析,为κ-卡拉胶酶酶制剂和κ-卡拉胶寡糖的工业化生产提供理论和技术支撑。通过富集培养技术、初筛和复筛方法从多种海藻和刺参肠道筛选出33株具有κ-卡拉胶降解活性的菌株,其中从刺参肠道筛选出的AJ5菌株产酶活力最高,为1.274 U/mL。依据形态学和生理学特征及16S rRNA基因序列分析,将该菌株鉴定为假交替单胞菌属（Pseudoalteromonas）,通过比较发现该菌株与已报道该属中的惟一一种产κ-卡拉胶酶菌P. carrageenovora有多种生理生化特征不同。通过单因素试验和正交试验对Pseudoalteromonas sp.AJ5菌株产胞外κ-卡拉胶酶的培养条件进行了优化。实验确定Pseudoalteromonas sp. AJ5菌株的最佳培养条件为:250 mL三角瓶装入75 mL发酵培养基、摇床转速150 r/min、接种量7%、pH8.0、培养温度28℃;最佳培养基组成为:κ-卡拉胶1 g/L、牛肉膏2 g/L、NaCl20 g/L、K₂HPO₄·3H₂O 1 g/L、MgSO₄·7H₂O 0.5 g/L、MnCl2·4H₂O 0.2 g/L、FePO₄·4H₂O 0.01 g/L。以Pseudoalteromonas sp.AJ5菌株为出发菌株,经紫外线、甲基磺酸乙酯复合诱变和自然选育,获得一株酶活力显著提高的突变株Pseudoalteromonas sp.AJ5-913,发酵产酶活力达6.788 U/mL,比优化前提高了2.9倍。对该突变株进行液体发酵制备出κ-卡拉胶酶酶液,通过30%-80%硫酸铵分级沉淀、Sephadex G-200凝胶过滤层析和CM-纤维素52阳离子交换层析技术对κ-卡拉胶酶进行纯化和精制,获得电泳纯度的酶蛋白组份,SDS-PAGE确定此κ-卡拉胶酶的分子量为35 kDa。采用Edman降解法测得该酶的N端氨基酸序列为NPTCHIAKPGETTILQECRS,通过与已报道细菌κ-卡拉胶酶的N-末端氨基酸序列比较,没有发现与该酶同样的N-末端氨基酸序列,初步确定为一新κ-卡拉胶酶。采用等电聚焦电泳测得该酶的等电点pI为8.5。通过对该酶酶学性质研究,确定该酶反应的最适pH范围为8,且酶活力在pH6.6-8.6范围内比较稳定,最适温度为55℃,酶活力在28℃下稳定,但经50℃–75℃处理30 min,95%的酶活力丧失,最适NaCl浓度50 mmol/L,金属离子Zn²⁺, Co²⁺和Cu²⁺几乎完全抑制酶的活性。动力学参数测定结果表明,AJ5-913菌株的κ-卡拉胶酶水解κ-卡拉胶符合米氏动力学方程,采用双倒数法作图法求得其米氏常数Km值为9.8±0.2 mg/mL。AJ5-913菌株的κ-卡拉胶酶专门水解κ-卡拉胶,对τ-和λ-卡拉胶和琼脂糖没有水解作用。利用AJ5-913菌株所产的κ-卡拉胶酶对κ-卡拉胶进行酶解制备κ-卡拉胶寡糖,通过电喷雾离子化飞行时间质谱（ESI-TOF-MS）和13C-NMR分析该酶的水解产物,确定该κ-卡拉胶酶专门水解κ-卡拉胶3,6-内醚-D-半乳糖残基和4-硫酸基-D-半乳糖之间的β-1,4糖苷键,产生3,6-内醚-D-半乳糖作为非还原端,D-半乳糖作为还原端的κ-新卡拉寡糖,主要产物是κ-新卡拉二糖硫酸盐、κ-新卡拉四糖硫酸盐、κ-新卡拉六糖硫酸盐、κ-新卡拉八糖硫酸盐和κ-新卡拉十糖硫酸盐,与已报道细菌的κ-卡拉胶酶的酶解产物有所不同。寡糖的抗病毒活性实验显示,3.12μg/mL-200μg/mL的κ-新卡拉寡糖对单纯疱疹病毒1型（HSV-1）的吸附有抑制作用,并且呈现明显的量效关系,表明κ-新卡拉寡糖可干扰HSV-1毒株向Vero细胞的吸附。更多还原

【Abstract】 This paper aims at screening aκ-carrageenase-producing bacterium with high enzyme activity, optimizing its culture conditions and medium components, mutagenizing the bacterium to obtain the mutant with higher activity, purifying theκ-carrageenase from the mutant cultural supernatant, studying the enzymatic properties, preparingκ-carrageenan-derived oligosaccharides fromκ-carrageenan using the enzyme, analyzing the composition and structure of the enzyme hydrolyzed products, providing the theoretical and technological supports for commercial production ofκ-carrageenase andκ-carrageenan oligosaccharides. By enrichment culture technique aκ-carrageenan-degrading bacterium AJ5, capable of utilizingκ-carrageenan as sole source of carbon and energy, was isolated from the intestine of holothurian Apostichopus japonicus. The strain was identified as the genus Pseudoalteromonas sp. according to its morphological and physiological characterization and 16S rRNA gene analysis. It was found that the strain had different physiological characteristics compared with the only one bacterium, P. carrageenovora, in this genus. The culture conditions and medium components for the bacterium have been standardized for the maximal productivity of the extracellularκ-carrageenase using the single factor and orthogonal tests. The optimal culture conditions was found as following:75 mL medium in 250 mL Erlenmeyer flask, shaking speed of 150 r/min, inoculum volume 7%, pH8.0 and temperature 28°C. The optimal medium components were observed as the following:κ-carrageenan 1 g/L, beef extract 2 g/L, NaCl20 g/L, K₂HPO₄·3H₂O 1 g/L, MgSO₄·7H₂O 0.5 g/L, MnCl₂·4H₂O 0.2 g/L, FePO₄·4H₂O 0.01 g/L. By using the complex mutagenesis of UV irradiation and Ethyl Methanesulphonate （EMS） treatment, the mutant Pseudoalteromonas sp.AJ5-913 withκ-carrageenase activity of 6.788 U/mL was obtained from Pseudoalteromonas sp.AJ5, which was 2.9-fold higher than that of the parent culture. An extracellularκ-carrageenase was purified from Pseudoalteromonas sp.AJ5-913 cultural supernatant by ammonium sulfate fractionation, gel filtration chromatography （Sephadex G-200） and cation-exchange chromatography （CM-cellulose 52）. The purified enzyme yielded a single band on SDS-PAGE with the molecular mass of 35 kDa. The sequence of the 20 amino acids at the N-terminal of the enzyme was N-P-T-C-H-I-A-K-P-G-E-T-T-I-L-Q-E-C-R-S. Compared with known N-terminal amino acid residues ofκ-carrageenases, noκ-carrageenase with the same corresponding N-terminal amino acid sequence was observed, indicating that this protein might be a novelκ-carrageenase. The pI ofκ-carrageenase was 8.5 on isoelectric focusing. The optimum pH for the enzyme was 8.0 and its activity was stable in the pH range of 6.6–8.6. The optimum temperature was 55°C and the enzyme was stable at 28°C, but 95% of the activity was lost at 50°C-75°C for 30 min. The activity of the enzyme was optimum at the presence of 50 mmol/L NaCl. The enzyme activity was almost completely inhibited by Co²⁺, Cu²⁺ and Zn²⁺ at 1 mmol/L. Thisκ-carrageenase showed Michaelis-type kinetics when hydrolyzing κ-carrageenan, as calculated from Lineweaver plot, the apparent Km value was 9.8±0.2 mg/mL. The enzyme specifically hydrolyzedκ-carrageenan.τ- andλ-carrageenans and agarose were not hydrolyzed by thisκ-carrageenase. The composition and structure of main enzyme hydrolyzed products examined by ESI-TOF-MS and 13C-NMR wereκ-neocarrabiose, -tetraose, -hexaose, -octaose, and -decaose sulfates with 3-linkedβ-D-galactopyranose 4-sulfate as the reducing end, indicating theκ-carrageenase from Pseudoalteromonas sp. AJ5-913 specifically hydrolyzed theβ-1, 4 glycosidic linkage between 3,6-anhydro-D-galactose and D-galactose. The main enzyme hydrolyzed products of this enzyme were different from those ofκ-carrageenases from other bacterial strains. The anti-HSV-1 activities ofκ-neocarraoligosaccharides were determined.κ-Neocarraoligosaccharides （3.12-200μg/mL） could interfere absorption of HSV-1 to Vero cells and there was obvious relationship between the concentrations and the effects ofκ-neocarraoligosaccharides.更多还原