一种番茄褐孢霉产生的高选择性转化人参皂甙Rb_1为Rd的β-葡萄糖苷酶
|
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Rb_1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Rb_1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Rb_1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Rb_1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℃。Cu~(2+),Zn~(2+)在50mM时对酶活性有较强的抑制作用,相反,Na~+、K~+、Ca~(2+)、Mn~(2+)和Mg~(2+)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Rb_1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Rb_2、Rc和Rd,说明此酶对人参皂甙Rb_1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。
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 Rb_1 is high in ginseng and it has the same aglycone (protopanaxadiol) as the minor ginsenosides. Rb_1 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 Rb_1 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 Rb_1 into the minor ginsenosides. However, most lack specificity which results in a low yield of Rd.
This paper reports that a novel ginsenoside Rb_1-hydrolyzingβ-D-glucosidase (G-I) secreted by phytopathogenic fungus C.fulvum which convert ginsenoside Rb_1 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 Rb_1 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 Rb_1-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 Rb_1-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 Cu~(2+) and Zn~(2+) ions but not inhibited by 0.25 M SDS. G-I was slightly activated by Na~+, K~+, Ca~(2+), Mn~(2+) and Mg~(2+). Of the substrates tested, G-I specifically hydrolyzed theβ-(1→6)-glucosidic linkage at the C-20 site of gensinoside Rb_1 to form ginsenoside Rd, without hydrolyzing otherβ-D-glucosidic linkages of Rb_1. 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.
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Rb_1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Rb_1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Rb_1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℃。Cu~(2+),Zn~(2+)在50mM时对酶活性有较强的抑制作用,相反,Na~+、K~+、Ca~(2+)、Mn~(2+)和Mg~(2+)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Rb_1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Rb_2、Rc和Rd,说明此酶对人参皂甙Rb_1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。
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 Rb_1 is high in ginseng and it has the same aglycone (protopanaxadiol) as the minor ginsenosides. Rb_1 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 Rb_1 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 Rb_1 into the minor ginsenosides. However, most lack specificity which results in a low yield of Rd.
This paper reports that a novel ginsenoside Rb_1-hydrolyzingβ-D-glucosidase (G-I) secreted by phytopathogenic fungus C.fulvum which convert ginsenoside Rb_1 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 Rb_1 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 Rb_1-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 Rb_1-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 Cu~(2+) and Zn~(2+) ions but not inhibited by 0.25 M SDS. G-I was slightly activated by Na~+, K~+, Ca~(2+), Mn~(2+) and Mg~(2+). Of the substrates tested, G-I specifically hydrolyzed theβ-(1→6)-glucosidic linkage at the C-20 site of gensinoside Rb_1 to form ginsenoside Rd, without hydrolyzing otherβ-D-glucosidic linkages of Rb_1. 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.
引文
[1]王铁生.中国人参[M].天津:天津科学技术出版社,2001,87-125.
[2] Fuzzati N, Analysis methods of ginsenosides[J]. Journal of Chromatography B, 2004, 812: 119-133.
[3] Park JD, Rhee DK, Lee YH. Biological activities and chemistry of saponins from Panax ginseng C. A. Meyer[J]. Phytochemistry Reviews, 2005, 4: 159–175.
[4]张丹.酶法改变人参皂甙Rg3糖基以获得高抗肿瘤活活物质-人参皂甙Rh2的研究[D].大连:大连轻工业学院, 2000.
[5] Chang YS, Seo EK, Gyllenhaal C, et al. Panax ginseng: A Role in Cancer Therapy? [J]. INTEGRATIVE CANCER THERAPIES, 2003, 2(1): 13-33.
[6] Attele AS, Wu JN, Yuan CS. Ginseng Pharmacology: MULTIPLE CONSTITUENTS AND MULTIPLE ACTIONS [J]. Biochemical Pharmacology, 1999, 58: 1685–1693.
[7]窦得强,靳玲,陈英杰.人参的化学成分及药理活性的研究进展与展望[J].沈阳药科大学学报, 1999, 16(2):151一156.
[8] Tsang D, Yeung HW, Tso WW,et al. Ginseng saponins: Influence on neuro-transmitter uptake in rat brain synaptosomes [J]. Planta Med, 1985, (3):221–224.
[9] Benishin CG. Actions of ginsenoside Rb1 on choline uptake in central cholinergic nerve endings [J]. Neurochem Int, 1992, (21):1–5.
[10]马天才,于庆海.人参对学习记忆的药理影响[J].中草药,1990, 21(7):38-40.
[11] Yamaguchi Y, Higashi M, Kobayashi H. Effects of ginsenosides on maze performance and brain choline acetyltransferase activity in scoplamine-treated young rats and aged rats[J]. Eur J Pharmacol, 1997, 329(1):37-41.
[12]王爱民,曹颖林,王玉坤,等.中国人参根、茎叶皂甙对大鼠的学习记忆及脑内单胺类递质含量的影响[J].中国中药杂志,1995,20(8):493～495.
[13] Gillis CN. Panax ginseng Pharmarcology: a nitric oxide link [J]. Biochem Pharmacol, 1997, 54(1):1-8.
[14] Lim JH, Wen TC, Matsuda S, et al. Protection of ischemic hippocampal neurons by ginsenoside Rb1, a main ingredient of ginseng root [J]. Neurosci Res, 1997, 28:191–200.
[15] Wen TC,Yoshimura H, Matsuda S, et al. Ginseng root prevents learning disability and neuronal loss in gerbils with 5-minute forebrain ischemia[J]. Acta Neuropathol, 1996, 91: 15–22.
[16]刘正湘,刘晓春.人参皂甙Rb1与Re对大鼠缺血再灌注心肌细胞凋亡的影响[J].中国组织化学与细胞化学杂志,2002 ,11(4) :374.
[17]李志刚,蓝荣芳.人参皂甙Rg1抗OxLDL诱导内皮细胞凋亡及分子机制[J] .中国组织化学与细胞化学杂志,2002 ,11(3) :248-251.
[18]阮秋容,宋建欣.人参皂甙对血管内皮细胞纤溶酶原激活物抑制和核因子-KB的作用[J] .中华心血管病杂志,2004 ,32 (4) :230-234.
[19]孙晓霞,夏映红,钟国赣.人参皂甙单体Rb1对大鼠在体心脏收缩性能的影响[J] .长春中医学院学报,2003,19(1) :43-49.
[20] Ma LY, Xiao PG. Effects of saponins of Panax notoginseng on intracellular free Ca2+ concentration in dissociated neurons [J]. Chinese Pharmaceutical Journal, 1998, 33(8):467-/ 469.
[21] Ma LY, Xiao PG, Liang FQ, et al. Protective effects of Panax notoginseng saponins on primary cortical cultures of rat [J]. Chinese Pharmaceutical Journal, 1998, 33(3):143-145.
[22] Xu Qing Fang, Fang Xiao Ling, Chen Dao Feng. Pharmacokinetics and bioavailability of ginsenoside Rb1 and Rg1 from Panax notoginseng in rats [J]. Journal of Ethnopharmacology, 2003, 84:187-/192.
[23]张仲苗,江波,章荣华,等.人参皂甙Rg3对小鼠免疫功能的影响[J].中药药理与临床,2004,20 (6):4-6.
[24]胡松华,林锋强.人参皂甙Rb1的免疫佐剂作用[J].中国兽医学报, 2003, 23(5):480- 482.
[25] Kenarova B, Neychev H, Hadjiivanova C, et al. Immunomodulating activity of ginsenoside Rg1 from Panax ginseng [J]. Jpn J Pharmacol, 1990, 54:447–454.
[26] Yang Z, Chen A, Sun HX, et al. Ginsenoside Rd elicits Th1 and Th2 immune responses to ovalbumin in mice [J]. Vaccine, 2007, 25:161–169.
[27]杨玉琪,李玛琳.人参皂甙的抗肿瘤作用及其机制[J].药学进展,2003,27(5):287-290.
[28]任莉莉,魏影非,杜惠兰等.人参抗肿瘤作用研究进展[J]. Chinese Traditional Patent Medicine, 2005, 27(8):947-950.
[29]夏丽娟,韩锐.人参皂甙Rh2体外对小鼠黑色素瘤细胞的分化诱导作用[J].药学学报,1996,31(10):742—745.
[30] Nakata H, Kikuchi Y, Tode T, et a1. Inhibitory effects ofginsenoside Rh2 on tumor growth in nude mice bearing human ovarian cancer cells [J]. Jpn J Cancer Ras, 1998, 89(7):733—740.
[31]李丽艳.人参皂甙的最新研究进展[J].四川省卫生管理干部学院学报,2004,23(4):293-295
[32]崔魏,赵洪艳,王燕嬉.人参皂甙抗衰老研究进展[J].中老年学杂志,2006,11:1578-1581.
[33] Chang MS, Lee SG, Rho HM. Transcriptional activation of Cu/Zn superoxide dismutase and catalasegenes by panaxadiol ginsenosides extracted from Panax ginseng [J]. Phytoyther Res, 1999, 13(8):641-644.
[34] Kim YH, Park KH, Rho HM. Transcriptional activation of the Cu, Zn superoxide dismutase gene through the AP2 site by ginsenoside Rb2 extracted from a medicinal plant,Panax ginseng [J]. J Biol Chem, 1996, 271(40):24539-24543.
[35] L Min, Juntian Z. Effects of ginsenoside Rb1 and Rg1 on synaptosomal free calcium level, ATPase, and calmodulin in rat hippocampus [J]. Chin Med J (Engl), 1995, (108):544–547.
[36] Cheng.Y, Shen LH, Zhang JT. Anti-amnestic and anti-aging effects of ginsenoside Rg1 and Rb1 and its mechanism of action [J]. Acta Pharmacol Sin, 2005, 26:143–149.
[37]胡莹,杨凌,杨胜利.糖苷酶序列分类法和作用机理的研究进展[J].药物生物技术, 2006, 13(1):066～070.
[38] Henrissat B. A classification of glycosyl hydrolases based on amino acid sequence similarities [J]. Biochem J, 1991, 280:309-312.
[39] Henrissat B, Bairoch A. Updating the sequence-based classification of glycosyl hydrolases [J]. Biochem J, 1996, 316:695-697.
[40] Carl SR, Stephen GW. Glycosidase mechanisms [J]. Current Opinionin Chemical Biology, 2000, 4:5732-5801.
[41] Davies G, Henrissat B. Structures and mechanisms of glycosyl hydrolases [J]. Structure, 1995, 3 :8532-8591.
[42] Mc CJ, Withers SG. Mechanisms of enzymatic glycoside hydrolysis [J]. Curr Opin Struct Biol, 1994, 4:8852-8921
[43]冯世江,李春,曹竹安.糖苷酶及其在糖基化合物改性中的研究[J].生物加工过程,2006,4(3):16-21.
[44] Rye CS and Withers SG. Glycosidase mechanisms [J]. Current Opinion in Chemical Biology, 2000, 4:573–580.
[45] Khaled M, Mohamed. Phenylpropanoid glucosides from Chrozophora oblique [J]. Phytochemistry, 2001, 58 :615-618.
[46] Ghufran Ahmad, Prem PY, Rakesh Maurya. Furanoflavonoid glycosides from Pongamia pinnata fruits [J]. Phytochemistry, 2004, 65:921-924.
[47] Wei L, Kazuo Koike, Yoshihisa Asada, et al. Biotransformation of umbelliferone by Panax ginseng root cultures [J]. Tetrahedron Letters, 2002, 43 :56332-56351.
[48] Sergio Riva. Biocatalytic modification of natural products [J]. Current Opinion in Chemical Biology, 2001, 5:1062-1111.
[49] Yu HS, Max Q, Guo Y, et al. Purification characterization of ginsenosideβ-glucosidase [J]. J Ginseng Res, 1999, 23 :50-54.
[50] Yosioka I, Sugawara T, Imai K, et al. Soil bacterial hydrolysis leading to genuine aglycone V on ginsenosides Rb1, Rb2 and Rc of the ginseng root saponins [J]. Chem Pharm Bull, 1972, 20 (11) :2418-2421.
[51] Yang L, He KJ, Yang Y, et al. Preparation of 20-β-D-glucopyranosyl-protopanaxadiol by enzyme degradation [P]. China Patent, ZL01133410. X. 2003205214
[52] Kida H, Akao T, Meselhy MR, et al. Enzymes responsible for the metabolism of saikosaponins from Eubacterium sp. A244, a human intestinal anaerobe [J]. Biol Pharm Bull, 1997, 20 (12) :1274-1278.
[53]吴少杰,杨志娟,朱丽华等.甘草皂甙生物转化的研究[J].中草药,2003, 36(6) :516-518.
[54] Choi YB, Kim KS, Rhee JS. Hydrolysis of soybean isoflavone glucosides by lactic acid bacteria [J]. Biotechnology Letters, 2002, 24(24) :2113-2116.
[55] Akao T, Kawabata K, Yanagisawa E. Balicalin, the predo-minant flavone glucuronide of scutellatiae radix, is absorbed from the rat gastrointestinal tract as the aglycone and restored to its original form. J Pharm Pharmacol [J], 2000, 52 (12) :1563-1568.
[56] Bokkenheuser VD, Shackleton CH, Winter J. Hydrolysis of dietary flavonoid glycosides by strains of intestinal bacteroides from humans [J]. Biochem J, 1987, 248 (3) :953-956.
[57] Zhang C, Yu H, Jin F et al. Purification and characterization of ginsenoside-α- arabinofuranase hydrolyzing ginsenoside Rc into Rd from the fresh root of Panax ginseng [J]. Process Biochemistry, 2002, 37:793–798.
[58] Hu Y, Luan H, Yang L et al. Purification and characterization of a novel ginsenoside-hydrolyzingβ-D-glucosidase from the China white jade snail (Achatina fulica) [J]. Enzyme and Microbial Technology, 2007, 40:1358–1366.
[59] Luan H, Liu X, Yang L et al. Purification and characterization of a novel stable ginsenoside Rb1-hydrolyzingβ-D-glucosidase from China white jade snail [J]. Process Biochemistry, 2006, 41:1974–1980.
[60] Kim DS, Cue YS, Jin FX et al. Ginsenoside Rh2 prepared from enzyme reaction [J].大连轻工业学院学报,2001,20(2):34-37.
[61] Shin HY, Lee JH, Lee JY et al. Purification and Characterization of Ginsenoside Ra-Hydrolyzingβ-D-Xylosidase from Bifidobacterium breve K-110, a Human Intestinal Anaerobic Bacterium [J]. Biol. Pharm. Bull, 2003, 26(8):1170-1173.
[62] Shin HY, Park SY, Sung JH et al. Purification and Characterization ofα-L- Arabinopyranosidase andα-L-Arabinofuranosidase from Bifidobacterium breve K-110, a Human Intestinal Anaerobic Bacterium Metabolizing Ginsenoside Rb2 and Rc[J]. Applied and Environmental Microbiology, 2003, 57:7116-7123.
[63] Park SY, Bae EA, Sung JH et al. Purification and Characterization of Ginsenoside Rb1-Metabolizingβ-Glucosidase from Fusobacterium K-60, a Human Intestinal Anaerobic Bacterium [J]. Biosci. Biotechnol.Biochem, 2001, 65(5):1163—1169.
[64] Chen LQ, Kim MK, Lee JW, et al. Conversion of major ginsenoside Rb1 to ginsenoside F2 by Caulobacter leidyia[J]. Biotechnol Lett, 2006, 28:1121–1127.
[65] Dong A, Ye M, Guo H, et al. Microbial transformation of ginsenoside Rb1 by Rhizopus stolonifer and Curvularia lunata [J]. Biotechnol Lett, 2003, 25:339–344.
[66] Cheng LQ, Na JR, Bang MH, et al. Conversion of major ginsenoside Rb1 to 20(S)- ginsenoside Rg3 by Microbacterium sp. GS514 [J]. Phytochemistry, 2008, 69:218–224.
[67] Sung RK, Yukio S, Kei S et al. Marked Production of Ginsenosides Rd, F2, Rg3, and Compound K by Enzymatic Method [J]. Chem Pharm Bull, 2007, 55(10):1522—1527.
[68]马娜,宋宏新.人参糖苷酶提取纯化及性质研究[J].陕西中医,2003,24(12):236-239.
[69]赵立亚,鱼红闪,金凤燮.酶法生产稀有人参皂甙及其产物成分的分析[J].大连轻工业学院学报,2002,21(2):176-180.
[70]王娟.番茄叶霉病原菌转化人参皂甙的研究[D].东北师范大学,2008.
[71] Su JH, Xu JH, Lu WY, et al. Enzymatic transformation of ginsenoside Rg3 to Rh2 using newly isolated Fusarium proliferatum ECU2042 [J]. Journal of Molecular Catalysis B: Enzymatic, 2006, 38:113–118.
[72] Sandrock, RW and VanEtten, HD. Fungal sensitivity to and enzymatic degradation of the phytoanticipin a-tomatine [J]. Phytopathology, 1998, 88:137-143.
[73] Yokozawa T and Dong E. Role of ginsenoside Rd in cisplatin induced renal injury: special reference to DNA fragmentation [J]. Nephron, 2001, 89:433-438.
[74] Yokozawa, T and Owada S. Effects of ginsenoside Rd cephaloride induced renal disorder [J]. Nephron, 1999, 81:200-207.
[75] Yokozawa, T and Liu ZW. The role of ginsenoside Rd in cisplatin-induced acute renal failure [J]. Ren. Fail, 2000, 22:115-127.
[76] Yokozawa, TA, Satoh and Cho EJ. Ginsenoside-Rd attenuates oxidative damage related to aging in senescence-accelerated mice [J]. J Pharm Pharmacol, 2004, 56:107-113.
[77] Shi QQ, Hao J, Bouissac Y, et al. Ginsenoside Rd from Panax notoginseng enhances astrocyte differentiation from neural stem cells [J]. Life Sci, 2005, 76:983-995.
[78] Yokozawa, T, Liu ZW and Dong E. A study of ginsenoside-Rd in a renal ischemia-reperfusion model[J]. Nephron, 1998, 78:201-206.
[79] Ji QC, Harkey MR, Henderson GL, et al. Quantitative determination of ginsenosides by high-performance liquid chromatography- tandem mass spectrometry [J]. Phytochem Anal, 2001, 12:320–326.
[80]高聆.褐孢霉产生的人参皂甙Rb1水解酶的分离纯化及其性质[D].东北师范大学,2008.
[81] Bisaria, VS and Mishra, S. Regulatory aspects of cellulase biosynthesis and secretion [J]. CRC Crit Rev Biotechnol, 1989, 9: 61-103.
[82] Kubicek, CP, Messner, R, Gruber, F, et al. The Trichoderma cellulase regulatory puzzle: from the interior life of a secretory fungus [J]. Enz Microb Technol, 1993, 15: 90-99.
[83] Rosi I, Vinel, M and Domezio, M. Characterization ofβ-glucosidase activity in yeasts of enological origin [J]. J Appl Bact, 1994, 77: 519-527.
[84] Esen, A.β-glucosidases: overview inβ-Glucosidases: Biochemistry and Molecular Biology [M], Esen, A, Ed, American Chemical Society, Washington, DC, 1993, 1-14.
[85] Tull D, Wither SG, Gikes NR, et al. Glumatic Acid 274 is the Nucleophile in the Active Site of a“Retaining”Exoglucanase from Cellulononas firi [J]. The Journal of Biological Chemistry, l991, 266:15621-15625.
[86] Grabnitz F, Seiss M, Rucknagel Karl P. Structure of theβ-glucosidase gene bal A of clostridium thermocellum sequence analysis reveals a super family of cellulose andβ-glucosidase including human lactase/ phcoriz in hydrolase [J]. Eur J Biochem, 1991, 200:301-309.
[87] Withers, SG and Street, IP. Identification of a covalent a-D-glucopyranosyl enzyme intermediate formed on ap-glucosidase [J]. J. Am. Chem. Soc, 1988, 110:8551-8553.
[88] Rudick MJ, Elbein AD. Glycoprotein enzymes secreted by Aspergillus fumigatus. Purification and properties ofβ-glucosidase [J]. J Biol Chem, 1973, 248:6506-6513.
[89] Meyer HP, Canevascini G. Separation and Some Properties of Two Intracellularβ-Glucosidases of Sporotrichum (Chrysosporium) thermophile [J]. Appl Environ Microbiol, 1981, 41:924–931.
[90] Umezurike GM. The subunit structure ofβ-glucosidase from Botryodiplodia theobromae Pat [J]. Biochem J, 1975, 145:361-368.
[91] Filho EXF. Purification and characterization of aβ-glucosidase from solid-state cultures of Humicola grisea var. thermoidea [J]. Can J Microbiol, 1996, 42:1–5.
[92] Coughlan MP. The properties of fungal and bacterial cellulases with comments on their production and application [J]. Biotechnol Genet Eng Rev, 1985, 3: 39–109.
[93] Zhang C, Li D, Yu H, et al. Purification and characterization of piceid-β-d-glucosidase from Aspergillus oryzae [J]. Process Biochem, 2007, 42: 83-88.
[94] Chirico WJ, Brown RD. Purification and characterization of aβ-glucosidase from Trichoderma reese i[J]. Eur J Biochem, 1987, 165:333?341.
[95] Issam SM, Mohamed G, Farid L, et al. Production, Purification, and Biochemical Characterization of Twoβ-Glucosidases from Sclerotinia sclerotiorum [J]. Appl Biochem Biotechnol, 2003, 111:29-39.
[96] Kempton, JB, Withers, SG. Mechanism of Agrobacteriumβ-glucosidase: Kinetic studies [J]. Biochemistry, 1992, 31:9961-9969.
[97] Li Y-K, Chu S-H, Sung Y-H. Purification, Characterization and Mechanistic Study ofβ-Glucosidase from Flavobacterium meningosepticum (ATCC 13253) [J]. Journal of the Chinese Chemical Society (Taipei), 1998, 45(5): 603-610.
[98] Clarke AJ, Bray MR, Strating H.β-glucosidases, (β-glucanases and xylanases) [M]. In A. Esen ed, O-Glucosidases: Biochemistry and Molecular Biology, ACS Symposium Series 533. American Chemical Society, Washington DC, 1993, 27-41.
[99] Cheng LQ, Kim MK, Lee JW, et al. Conversion of major ginsenoside Rb1 to ginsenoside F2 by Caulobacter leidyia [J]. Biotechnol Lett, 2006, 28:1121-1127.
[100] Dong A, Ye M, Guo H, et al. Microbial transformation of ginsenoside Rb1 by Rhizopus stolonifer and Curvularia lunata [J]. Biotechnol Lett, 2003, 25:339-344.
[101] Lin MC, Wang KC, Lee SS. Transformation of ginsenosides Rg and Rb , and crude Sanchi saponins by human intestinal microflora 1 1[J]. J Chin Chem Soc, 2001, 48:113-120.
[2] Fuzzati N, Analysis methods of ginsenosides[J]. Journal of Chromatography B, 2004, 812: 119-133.
[3] Park JD, Rhee DK, Lee YH. Biological activities and chemistry of saponins from Panax ginseng C. A. Meyer[J]. Phytochemistry Reviews, 2005, 4: 159–175.
[4]张丹.酶法改变人参皂甙Rg3糖基以获得高抗肿瘤活活物质-人参皂甙Rh2的研究[D].大连:大连轻工业学院, 2000.
[5] Chang YS, Seo EK, Gyllenhaal C, et al. Panax ginseng: A Role in Cancer Therapy? [J]. INTEGRATIVE CANCER THERAPIES, 2003, 2(1): 13-33.
[6] Attele AS, Wu JN, Yuan CS. Ginseng Pharmacology: MULTIPLE CONSTITUENTS AND MULTIPLE ACTIONS [J]. Biochemical Pharmacology, 1999, 58: 1685–1693.
[7]窦得强,靳玲,陈英杰.人参的化学成分及药理活性的研究进展与展望[J].沈阳药科大学学报, 1999, 16(2):151一156.
[8] Tsang D, Yeung HW, Tso WW,et al. Ginseng saponins: Influence on neuro-transmitter uptake in rat brain synaptosomes [J]. Planta Med, 1985, (3):221–224.
[9] Benishin CG. Actions of ginsenoside Rb1 on choline uptake in central cholinergic nerve endings [J]. Neurochem Int, 1992, (21):1–5.
[10]马天才,于庆海.人参对学习记忆的药理影响[J].中草药,1990, 21(7):38-40.
[11] Yamaguchi Y, Higashi M, Kobayashi H. Effects of ginsenosides on maze performance and brain choline acetyltransferase activity in scoplamine-treated young rats and aged rats[J]. Eur J Pharmacol, 1997, 329(1):37-41.
[12]王爱民,曹颖林,王玉坤,等.中国人参根、茎叶皂甙对大鼠的学习记忆及脑内单胺类递质含量的影响[J].中国中药杂志,1995,20(8):493～495.
[13] Gillis CN. Panax ginseng Pharmarcology: a nitric oxide link [J]. Biochem Pharmacol, 1997, 54(1):1-8.
[14] Lim JH, Wen TC, Matsuda S, et al. Protection of ischemic hippocampal neurons by ginsenoside Rb1, a main ingredient of ginseng root [J]. Neurosci Res, 1997, 28:191–200.
[15] Wen TC,Yoshimura H, Matsuda S, et al. Ginseng root prevents learning disability and neuronal loss in gerbils with 5-minute forebrain ischemia[J]. Acta Neuropathol, 1996, 91: 15–22.
[16]刘正湘,刘晓春.人参皂甙Rb1与Re对大鼠缺血再灌注心肌细胞凋亡的影响[J].中国组织化学与细胞化学杂志,2002 ,11(4) :374.
[17]李志刚,蓝荣芳.人参皂甙Rg1抗OxLDL诱导内皮细胞凋亡及分子机制[J] .中国组织化学与细胞化学杂志,2002 ,11(3) :248-251.
[18]阮秋容,宋建欣.人参皂甙对血管内皮细胞纤溶酶原激活物抑制和核因子-KB的作用[J] .中华心血管病杂志,2004 ,32 (4) :230-234.
[19]孙晓霞,夏映红,钟国赣.人参皂甙单体Rb1对大鼠在体心脏收缩性能的影响[J] .长春中医学院学报,2003,19(1) :43-49.
[20] Ma LY, Xiao PG. Effects of saponins of Panax notoginseng on intracellular free Ca2+ concentration in dissociated neurons [J]. Chinese Pharmaceutical Journal, 1998, 33(8):467-/ 469.
[21] Ma LY, Xiao PG, Liang FQ, et al. Protective effects of Panax notoginseng saponins on primary cortical cultures of rat [J]. Chinese Pharmaceutical Journal, 1998, 33(3):143-145.
[22] Xu Qing Fang, Fang Xiao Ling, Chen Dao Feng. Pharmacokinetics and bioavailability of ginsenoside Rb1 and Rg1 from Panax notoginseng in rats [J]. Journal of Ethnopharmacology, 2003, 84:187-/192.
[23]张仲苗,江波,章荣华,等.人参皂甙Rg3对小鼠免疫功能的影响[J].中药药理与临床,2004,20 (6):4-6.
[24]胡松华,林锋强.人参皂甙Rb1的免疫佐剂作用[J].中国兽医学报, 2003, 23(5):480- 482.
[25] Kenarova B, Neychev H, Hadjiivanova C, et al. Immunomodulating activity of ginsenoside Rg1 from Panax ginseng [J]. Jpn J Pharmacol, 1990, 54:447–454.
[26] Yang Z, Chen A, Sun HX, et al. Ginsenoside Rd elicits Th1 and Th2 immune responses to ovalbumin in mice [J]. Vaccine, 2007, 25:161–169.
[27]杨玉琪,李玛琳.人参皂甙的抗肿瘤作用及其机制[J].药学进展,2003,27(5):287-290.
[28]任莉莉,魏影非,杜惠兰等.人参抗肿瘤作用研究进展[J]. Chinese Traditional Patent Medicine, 2005, 27(8):947-950.
[29]夏丽娟,韩锐.人参皂甙Rh2体外对小鼠黑色素瘤细胞的分化诱导作用[J].药学学报,1996,31(10):742—745.
[30] Nakata H, Kikuchi Y, Tode T, et a1. Inhibitory effects ofginsenoside Rh2 on tumor growth in nude mice bearing human ovarian cancer cells [J]. Jpn J Cancer Ras, 1998, 89(7):733—740.
[31]李丽艳.人参皂甙的最新研究进展[J].四川省卫生管理干部学院学报,2004,23(4):293-295
[32]崔魏,赵洪艳,王燕嬉.人参皂甙抗衰老研究进展[J].中老年学杂志,2006,11:1578-1581.
[33] Chang MS, Lee SG, Rho HM. Transcriptional activation of Cu/Zn superoxide dismutase and catalasegenes by panaxadiol ginsenosides extracted from Panax ginseng [J]. Phytoyther Res, 1999, 13(8):641-644.
[34] Kim YH, Park KH, Rho HM. Transcriptional activation of the Cu, Zn superoxide dismutase gene through the AP2 site by ginsenoside Rb2 extracted from a medicinal plant,Panax ginseng [J]. J Biol Chem, 1996, 271(40):24539-24543.
[35] L Min, Juntian Z. Effects of ginsenoside Rb1 and Rg1 on synaptosomal free calcium level, ATPase, and calmodulin in rat hippocampus [J]. Chin Med J (Engl), 1995, (108):544–547.
[36] Cheng.Y, Shen LH, Zhang JT. Anti-amnestic and anti-aging effects of ginsenoside Rg1 and Rb1 and its mechanism of action [J]. Acta Pharmacol Sin, 2005, 26:143–149.
[37]胡莹,杨凌,杨胜利.糖苷酶序列分类法和作用机理的研究进展[J].药物生物技术, 2006, 13(1):066～070.
[38] Henrissat B. A classification of glycosyl hydrolases based on amino acid sequence similarities [J]. Biochem J, 1991, 280:309-312.
[39] Henrissat B, Bairoch A. Updating the sequence-based classification of glycosyl hydrolases [J]. Biochem J, 1996, 316:695-697.
[40] Carl SR, Stephen GW. Glycosidase mechanisms [J]. Current Opinionin Chemical Biology, 2000, 4:5732-5801.
[41] Davies G, Henrissat B. Structures and mechanisms of glycosyl hydrolases [J]. Structure, 1995, 3 :8532-8591.
[42] Mc CJ, Withers SG. Mechanisms of enzymatic glycoside hydrolysis [J]. Curr Opin Struct Biol, 1994, 4:8852-8921
[43]冯世江,李春,曹竹安.糖苷酶及其在糖基化合物改性中的研究[J].生物加工过程,2006,4(3):16-21.
[44] Rye CS and Withers SG. Glycosidase mechanisms [J]. Current Opinion in Chemical Biology, 2000, 4:573–580.
[45] Khaled M, Mohamed. Phenylpropanoid glucosides from Chrozophora oblique [J]. Phytochemistry, 2001, 58 :615-618.
[46] Ghufran Ahmad, Prem PY, Rakesh Maurya. Furanoflavonoid glycosides from Pongamia pinnata fruits [J]. Phytochemistry, 2004, 65:921-924.
[47] Wei L, Kazuo Koike, Yoshihisa Asada, et al. Biotransformation of umbelliferone by Panax ginseng root cultures [J]. Tetrahedron Letters, 2002, 43 :56332-56351.
[48] Sergio Riva. Biocatalytic modification of natural products [J]. Current Opinion in Chemical Biology, 2001, 5:1062-1111.
[49] Yu HS, Max Q, Guo Y, et al. Purification characterization of ginsenosideβ-glucosidase [J]. J Ginseng Res, 1999, 23 :50-54.
[50] Yosioka I, Sugawara T, Imai K, et al. Soil bacterial hydrolysis leading to genuine aglycone V on ginsenosides Rb1, Rb2 and Rc of the ginseng root saponins [J]. Chem Pharm Bull, 1972, 20 (11) :2418-2421.
[51] Yang L, He KJ, Yang Y, et al. Preparation of 20-β-D-glucopyranosyl-protopanaxadiol by enzyme degradation [P]. China Patent, ZL01133410. X. 2003205214
[52] Kida H, Akao T, Meselhy MR, et al. Enzymes responsible for the metabolism of saikosaponins from Eubacterium sp. A244, a human intestinal anaerobe [J]. Biol Pharm Bull, 1997, 20 (12) :1274-1278.
[53]吴少杰,杨志娟,朱丽华等.甘草皂甙生物转化的研究[J].中草药,2003, 36(6) :516-518.
[54] Choi YB, Kim KS, Rhee JS. Hydrolysis of soybean isoflavone glucosides by lactic acid bacteria [J]. Biotechnology Letters, 2002, 24(24) :2113-2116.
[55] Akao T, Kawabata K, Yanagisawa E. Balicalin, the predo-minant flavone glucuronide of scutellatiae radix, is absorbed from the rat gastrointestinal tract as the aglycone and restored to its original form. J Pharm Pharmacol [J], 2000, 52 (12) :1563-1568.
[56] Bokkenheuser VD, Shackleton CH, Winter J. Hydrolysis of dietary flavonoid glycosides by strains of intestinal bacteroides from humans [J]. Biochem J, 1987, 248 (3) :953-956.
[57] Zhang C, Yu H, Jin F et al. Purification and characterization of ginsenoside-α- arabinofuranase hydrolyzing ginsenoside Rc into Rd from the fresh root of Panax ginseng [J]. Process Biochemistry, 2002, 37:793–798.
[58] Hu Y, Luan H, Yang L et al. Purification and characterization of a novel ginsenoside-hydrolyzingβ-D-glucosidase from the China white jade snail (Achatina fulica) [J]. Enzyme and Microbial Technology, 2007, 40:1358–1366.
[59] Luan H, Liu X, Yang L et al. Purification and characterization of a novel stable ginsenoside Rb1-hydrolyzingβ-D-glucosidase from China white jade snail [J]. Process Biochemistry, 2006, 41:1974–1980.
[60] Kim DS, Cue YS, Jin FX et al. Ginsenoside Rh2 prepared from enzyme reaction [J].大连轻工业学院学报,2001,20(2):34-37.
[61] Shin HY, Lee JH, Lee JY et al. Purification and Characterization of Ginsenoside Ra-Hydrolyzingβ-D-Xylosidase from Bifidobacterium breve K-110, a Human Intestinal Anaerobic Bacterium [J]. Biol. Pharm. Bull, 2003, 26(8):1170-1173.
[62] Shin HY, Park SY, Sung JH et al. Purification and Characterization ofα-L- Arabinopyranosidase andα-L-Arabinofuranosidase from Bifidobacterium breve K-110, a Human Intestinal Anaerobic Bacterium Metabolizing Ginsenoside Rb2 and Rc[J]. Applied and Environmental Microbiology, 2003, 57:7116-7123.
[63] Park SY, Bae EA, Sung JH et al. Purification and Characterization of Ginsenoside Rb1-Metabolizingβ-Glucosidase from Fusobacterium K-60, a Human Intestinal Anaerobic Bacterium [J]. Biosci. Biotechnol.Biochem, 2001, 65(5):1163—1169.
[64] Chen LQ, Kim MK, Lee JW, et al. Conversion of major ginsenoside Rb1 to ginsenoside F2 by Caulobacter leidyia[J]. Biotechnol Lett, 2006, 28:1121–1127.
[65] Dong A, Ye M, Guo H, et al. Microbial transformation of ginsenoside Rb1 by Rhizopus stolonifer and Curvularia lunata [J]. Biotechnol Lett, 2003, 25:339–344.
[66] Cheng LQ, Na JR, Bang MH, et al. Conversion of major ginsenoside Rb1 to 20(S)- ginsenoside Rg3 by Microbacterium sp. GS514 [J]. Phytochemistry, 2008, 69:218–224.
[67] Sung RK, Yukio S, Kei S et al. Marked Production of Ginsenosides Rd, F2, Rg3, and Compound K by Enzymatic Method [J]. Chem Pharm Bull, 2007, 55(10):1522—1527.
[68]马娜,宋宏新.人参糖苷酶提取纯化及性质研究[J].陕西中医,2003,24(12):236-239.
[69]赵立亚,鱼红闪,金凤燮.酶法生产稀有人参皂甙及其产物成分的分析[J].大连轻工业学院学报,2002,21(2):176-180.
[70]王娟.番茄叶霉病原菌转化人参皂甙的研究[D].东北师范大学,2008.
[71] Su JH, Xu JH, Lu WY, et al. Enzymatic transformation of ginsenoside Rg3 to Rh2 using newly isolated Fusarium proliferatum ECU2042 [J]. Journal of Molecular Catalysis B: Enzymatic, 2006, 38:113–118.
[72] Sandrock, RW and VanEtten, HD. Fungal sensitivity to and enzymatic degradation of the phytoanticipin a-tomatine [J]. Phytopathology, 1998, 88:137-143.
[73] Yokozawa T and Dong E. Role of ginsenoside Rd in cisplatin induced renal injury: special reference to DNA fragmentation [J]. Nephron, 2001, 89:433-438.
[74] Yokozawa, T and Owada S. Effects of ginsenoside Rd cephaloride induced renal disorder [J]. Nephron, 1999, 81:200-207.
[75] Yokozawa, T and Liu ZW. The role of ginsenoside Rd in cisplatin-induced acute renal failure [J]. Ren. Fail, 2000, 22:115-127.
[76] Yokozawa, TA, Satoh and Cho EJ. Ginsenoside-Rd attenuates oxidative damage related to aging in senescence-accelerated mice [J]. J Pharm Pharmacol, 2004, 56:107-113.
[77] Shi QQ, Hao J, Bouissac Y, et al. Ginsenoside Rd from Panax notoginseng enhances astrocyte differentiation from neural stem cells [J]. Life Sci, 2005, 76:983-995.
[78] Yokozawa, T, Liu ZW and Dong E. A study of ginsenoside-Rd in a renal ischemia-reperfusion model[J]. Nephron, 1998, 78:201-206.
[79] Ji QC, Harkey MR, Henderson GL, et al. Quantitative determination of ginsenosides by high-performance liquid chromatography- tandem mass spectrometry [J]. Phytochem Anal, 2001, 12:320–326.
[80]高聆.褐孢霉产生的人参皂甙Rb1水解酶的分离纯化及其性质[D].东北师范大学,2008.
[81] Bisaria, VS and Mishra, S. Regulatory aspects of cellulase biosynthesis and secretion [J]. CRC Crit Rev Biotechnol, 1989, 9: 61-103.
[82] Kubicek, CP, Messner, R, Gruber, F, et al. The Trichoderma cellulase regulatory puzzle: from the interior life of a secretory fungus [J]. Enz Microb Technol, 1993, 15: 90-99.
[83] Rosi I, Vinel, M and Domezio, M. Characterization ofβ-glucosidase activity in yeasts of enological origin [J]. J Appl Bact, 1994, 77: 519-527.
[84] Esen, A.β-glucosidases: overview inβ-Glucosidases: Biochemistry and Molecular Biology [M], Esen, A, Ed, American Chemical Society, Washington, DC, 1993, 1-14.
[85] Tull D, Wither SG, Gikes NR, et al. Glumatic Acid 274 is the Nucleophile in the Active Site of a“Retaining”Exoglucanase from Cellulononas firi [J]. The Journal of Biological Chemistry, l991, 266:15621-15625.
[86] Grabnitz F, Seiss M, Rucknagel Karl P. Structure of theβ-glucosidase gene bal A of clostridium thermocellum sequence analysis reveals a super family of cellulose andβ-glucosidase including human lactase/ phcoriz in hydrolase [J]. Eur J Biochem, 1991, 200:301-309.
[87] Withers, SG and Street, IP. Identification of a covalent a-D-glucopyranosyl enzyme intermediate formed on ap-glucosidase [J]. J. Am. Chem. Soc, 1988, 110:8551-8553.
[88] Rudick MJ, Elbein AD. Glycoprotein enzymes secreted by Aspergillus fumigatus. Purification and properties ofβ-glucosidase [J]. J Biol Chem, 1973, 248:6506-6513.
[89] Meyer HP, Canevascini G. Separation and Some Properties of Two Intracellularβ-Glucosidases of Sporotrichum (Chrysosporium) thermophile [J]. Appl Environ Microbiol, 1981, 41:924–931.
[90] Umezurike GM. The subunit structure ofβ-glucosidase from Botryodiplodia theobromae Pat [J]. Biochem J, 1975, 145:361-368.
[91] Filho EXF. Purification and characterization of aβ-glucosidase from solid-state cultures of Humicola grisea var. thermoidea [J]. Can J Microbiol, 1996, 42:1–5.
[92] Coughlan MP. The properties of fungal and bacterial cellulases with comments on their production and application [J]. Biotechnol Genet Eng Rev, 1985, 3: 39–109.
[93] Zhang C, Li D, Yu H, et al. Purification and characterization of piceid-β-d-glucosidase from Aspergillus oryzae [J]. Process Biochem, 2007, 42: 83-88.
[94] Chirico WJ, Brown RD. Purification and characterization of aβ-glucosidase from Trichoderma reese i[J]. Eur J Biochem, 1987, 165:333?341.
[95] Issam SM, Mohamed G, Farid L, et al. Production, Purification, and Biochemical Characterization of Twoβ-Glucosidases from Sclerotinia sclerotiorum [J]. Appl Biochem Biotechnol, 2003, 111:29-39.
[96] Kempton, JB, Withers, SG. Mechanism of Agrobacteriumβ-glucosidase: Kinetic studies [J]. Biochemistry, 1992, 31:9961-9969.
[97] Li Y-K, Chu S-H, Sung Y-H. Purification, Characterization and Mechanistic Study ofβ-Glucosidase from Flavobacterium meningosepticum (ATCC 13253) [J]. Journal of the Chinese Chemical Society (Taipei), 1998, 45(5): 603-610.
[98] Clarke AJ, Bray MR, Strating H.β-glucosidases, (β-glucanases and xylanases) [M]. In A. Esen ed, O-Glucosidases: Biochemistry and Molecular Biology, ACS Symposium Series 533. American Chemical Society, Washington DC, 1993, 27-41.
[99] Cheng LQ, Kim MK, Lee JW, et al. Conversion of major ginsenoside Rb1 to ginsenoside F2 by Caulobacter leidyia [J]. Biotechnol Lett, 2006, 28:1121-1127.
[100] Dong A, Ye M, Guo H, et al. Microbial transformation of ginsenoside Rb1 by Rhizopus stolonifer and Curvularia lunata [J]. Biotechnol Lett, 2003, 25:339-344.
[101] Lin MC, Wang KC, Lee SS. Transformation of ginsenosides Rg and Rb , and crude Sanchi saponins by human intestinal microflora 1 1[J]. J Chin Chem Soc, 2001, 48:113-120.