A Highly Selective β-D-glucosidase Hydrolyzing Ginsenoside Rb₁ To Rd From Cladosporium Fulvum

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.