【作者】 赵雪淞；

【导师】 周义发；

【作者基本信息】 东北师范大学 ， 生物化学与分子生物学， 2009， 博士

【摘要】 人参（Panax ginseng C.A Maye）为五加科人参属植物,是我国的传统名贵中药。人参皂甙是人参的有效成分,到目前为止,已经从人参中发现40多种人参皂甙。各种人参皂甙的含量不同,结构多样,药理活性也有很大的差别。研究发现某些在人参中含量很低的稀有人参皂甙具有很高的药用价值和应用前景。但是,稀有人参皂甙含量太低,很难从人参中直接提取,依靠目前的化学合成技术也难以工业生产。制备这些稀有人参皂甙唯一可行的方法是改变某些高含量人参皂甙的化学结构来完成。化学转化法通常选择性较差,产率较低,同时容易造成环境污染。生物转化则具备条件温和、选择性较好、副产物少、得率高、无污染和容易工业化生产等优点,被认为是制备稀有人参皂甙最具有潜力的方法。本论文通过筛选发现,番茄褐孢霉产生的高选择性糖苷水解酶能将人参中含量最高的人参皂甙Rb₁水解为唯一产物——稀有人参皂甙Rd。本文探索了这种β-葡萄糖苷酶的分离纯化条件,研究了它的分子特征和性质,为工业应用奠定了基础。本论文的主要结果如下:1、从40种植物病原真菌中筛选出七种能够不同程度地转化人参皂甙的真菌,各种真菌的转化底物和产物不同。其中番茄褐孢霉能够特异性转化人参皂甙Rb₁成为Rd。最佳转化条件为:加底物时间,24 h;底物浓度,0.25 mg/ml;培养液pH值,pH5-6;培养时间,8 d;培养温度,37℃。番茄褐孢霉产生转化人参皂甙Rb₁成为Rd的糖苷酶的最佳产酶条件为:V8汁培养基,液体培养84小时。2、培养84 h的番茄褐孢霉V8汁培养液经过过滤离心、DEAE-cellulose阴离子交换柱层析、硫酸铵沉淀、Sepharose CL-6B凝胶过滤柱层析、Phenyl Sepharose CL-4B疏水柱层析以及Mono Q阴离子交换柱层析等多步分离技术结合,成功分离纯化出一种电泳纯的β-葡萄糖苷酶G-I（人参皂甙Rb₁转化酶G-I）。G-I的纯化工艺具有很高的稳定性,能够保证其稳定制备。每50升番茄褐孢霉培养液约可获得0.5 mg活性蛋白,回收率为9%,比活力达17563 U/mg。3、Superose 6 10/300 GL凝胶过滤柱层析分析（HPLC）显示G-I的洗脱曲线为对称峰,分子量为80 KDa;经SDS聚丙烯酰胺凝胶电泳（SDS-PAGE）和等电聚焦聚丙烯酰胺凝胶电泳（IEF-PAGE）检测,图谱均显示为一条蛋白带,分子量为80 KDa,等电点（pI）为4.2,表明G-I已达至电泳纯,且为一种单链蛋白质,相对分子量为80 KDa。经胰蛋白酶水解、电喷雾四极杆飞行时间串联质谱（Q-TOF2）分析,获取了G-I中3个多肽链的氨基酸序列: 1. LVAHEENVR , 2.VGKDEGFAKAGGLSR ,3.LPLEAGESGTATFNVR。利用BLAST工具在NCBI非冗余蛋白数据库和欧洲生物信息学研究所（EBI）统一蛋白知识库中进行查询和比对,没有发现氨基酸完全相同的多肽段,表明此酶为一种新蛋白。将此肽段的氨基酸序列上传到统一蛋白知识数据库（UniProtKnowledgebase）,得到了其授予的序列接受号P85516。G-I的氨基酸序列与β-葡萄糖苷酶家族3的几种真菌β-葡萄糖苷酶有高度的同源性,可能属于此家族的成员之一。4、酶反应动力学实验证实,G-I具有较好的pH稳定性和热稳定性,在pH4.0～11.0范围内和40℃以下表现出良好的β-葡萄糖苷酶活性,最适反应pH值为pH6.0,最适反应温度为45℃。Cu²⁺,Zn²⁺在50mM时对酶活性有较强的抑制作用,相反,Na⁺、K⁺、Ca²⁺、Mn²⁺和Mg²⁺对酶活性有轻微的刺激作用。0.25 M的SDS没有影响G-I的酶活性,0.25 mM的EDTA只是轻微抑制了G-I的活性。G-I的Km值为0.18 mM,最大反应初速度Vmax为5.52 mM/min。底物专一性分析发现,G-I能高特异性水解人工合成的底物pNPG,还能水解β-葡萄糖苷键连接的二糖如纤维二糖、龙胆二糖和槐二糖,再次表明此酶为一种β-葡萄糖苷酶。G-I对人参皂甙的底物专一性研究表明,此酶对人参皂甙Rb₁表现了很强的水解活性,可达到pNPG的22.1%,而不水解人参皂甙Rb₂、Rc和Rd,说明此酶对人参皂甙Rb₁中C-20位的β（1→6）糖苷键更具有特异性,不水解人参皂甙其它糖苷键,G-I对人参皂甙的这种高选择性水解为Rd的工业制备奠定了基础。更多还原

【Abstract】 Panax ginseng C. A. Meyer has been used as a medicine in China over 2000 years. Ginsenosides are the major active components of ginseng. More than 40 ginsenosides have been isolated and identified. It has been found that the minor ginsenosides such as Rg3, CK and Rd have very good bioactivities. It is difficult to prepare minor ginsenosides by extraction from ginseng because of their low concentration. Based on current technologies, it is impossible to prepare the minor ginsenosides by chemical synthesis. At present, a possible pathway for preparation of the minor ginsenosides is transformation from structurally related major ginsenosides.The amount of the major ginsenoside Rb₁ is high in ginseng and it has the same aglycone （protopanaxadiol） as the minor ginsenosides. Rb₁ has one or more sugar residues at the C-20 position than the minor ginsenosides. Theoretically, the minor ginsenosides can be obtained by hydrolysis of Rb₁ to remove one or more glucose residue at position C-20. Chemical transformation usually has poor selectivity and generates more environmental pollution. Biotransformation is thought to have more potential for conversion because of its high specificity and environmental compatibility. Some efforts have been made to look for suitable enzymes that can convert Rb₁ into the minor ginsenosides. However, most lack specificity which results in a low yield of Rd.This paper reports that a novel ginsenoside Rb₁-hydrolyzingβ-D-glucosidase （G-I） secreted by phytopathogenic fungus C.fulvum which convert ginsenoside Rb₁ to Rd with high specificity （Rd as sole product） was purified and characterized. This enzyme by Cladosporium fulvum (（syn. Fulvia fulva） would be very useful for the preparation of the minor ginsenoside Rd in industry.The main results obtained from this work are as follows:1. Forty phytopathogenic fungi were tested for their ability to transform the major ginsenosides to the active minor ginsenosides, and seven fungi were identified to have this ability among them by TLC and HPLC. C. fulvum, a tomato pathogen, was found to transform major ginsenoside Rb₁ to Rd as the sole product. The following optimum conditions for transforming Rd by C. fulvum were determined: the time of substrate addition, 24 h; substrate concentration, 0.25 mg ml^-1; temperature, 37°C; pH, 5.0; and biotransformation period, 8 days. At these optimum conditions, the maximum yield was 86% （molar ratio）. The optimum conditions of producting ginsenoside Rb₁-hydrolyzingβ-D-glucosidase by C. fulvum were evaluated to be following, the medium: V8 juice medium; and the time of cultivation: 84 h. This fungus is very potential to be applied on the preparation for Rd in pharmaceutical industry.2. A novel ginsenoside Rb₁-hydrolyzingβ-glucosidase （G-I） secreted by phytopathogenic fungus C. fulvum was purified to homogeneity using a six-step purification procedure: ion-exchange chromatography on DEAE-cellulose, 30-80% （NH4）2SO4 precipitation, gel filtration chromatography on Sepharose CL-6B, hydrophobic interaction chromatography on Phenyl Sepharose CL-4B, ion-exchange chromatography on Mono Q HR 5/5 and hydroxyapatite chromatography on Bio-Scale CHT20-1. The yield was 9%, the specific activity and purification fold was 17563 U/mg protein and 399 fold, respectively.3、The purified G-Ιwas a monomer with native molecular weight of approximately 80 KDa and pI value of 4.2. The oligopeptide fragment obtained after enzymatic digestion of G-I was sequenced by nanoESI-MS/MS （Q-TOF2）. Three peptide sequences （1. LVAHEENVR, 2.VGKDEGFAKAGGLSR, 3.LPLEAGESGTATFNVR） were obtained and subjected to the UniProt Knowledgebase （European Bioinformatics Institute） using the WU-BLAST2 network service in a search for proteins that matched the amino acid sequences of G-I, but no sequences were retrieved. G-I may therefore be a novel glycosidase. The protein sequence data reported in this paper will appear in the UniProt Knowledgebase under the accession number P85516. The amino acid sequence homology analysis showed that G-I possessed high homologous with the family 3β-glucosidases.4、For G-I, the optimal pH was 6.0 and the optimal temperature was 45°C. G-I was highly stable within pH 4.0-11.0 and below 40°C. The Km and Vmax values of G-I against pNPG were 0.18 mM and 5.52 mM/min, respectively. G-I was inhibited by Cu²⁺ and Zn²⁺ ions but not inhibited by 0.25 M SDS. G-I was slightly activated by Na⁺, K⁺, Ca²⁺, Mn²⁺ and Mg²⁺. Of the substrates tested, G-I specifically hydrolyzed theβ-（1→6）-glucosidic linkage at the C-20 site of gensinoside Rb₁ to form ginsenoside Rd, without hydrolyzing otherβ-D-glucosidic linkages of Rb₁. Besides, G-I can hydrolyze pNPG andβ-linked disaccharides such as cellobiose, sophorose and gentiobiose, but exhibited very low activity against other aryl-glycosides and methyl-α-glycosides.更多还原