| Timosaponin BII, a furostanol saponin purified from Anemarrhena asphodeloides Bunge, can significantly improve the learning and memory abilities, up-regulate the number of nAChRs and increase the rCBF (regional cerebral blood flow) of rat in our previous studies. Recently Timosaponin BII has been studying as a new candidate for anti-vascular dementia and anti-alzheimer's disease drug. In order to find new target compounds that having high activity and low toxicity with similar structure to timosaponin BII for structure-activity relationship, we have been looking for an effective method to modify structural of this compound. Biotransformation is a biochemical reaction to modify the structure of the xenobiotics by vegetal celluar or organ, animal cellular, microorganism and its cellular, and isolated enzyme, which is mainly enzyme-catalyzed reaction. Advantages often associated with biotransformation are pronounced exquisite chemoselectivity, regioselectivity, stereoselectivity, less by-product, and easy to operate under mild conditions. Biotransformation is a technology which has potentially application and market value for widening diversity of natural product, searching for lead compounds, enhancing the sustainability of the rare species of natural resources, improving preparation efficiency and reducing the costs. However, there are not many reports on biotransformation of steroidal saponin and its relevant research, especially on the hydrolysis and glycosylation at C3-sugar chain.In this thesis, timosaponin BII as substrate, over 40 enzymes and a hundred microorganisms were screened, and an enzyme (Toruzyme 3.0L) and two microorganisms (Aspergillus niger AS 3.0739, Pseudomonas fluorescens) were found to be able to transform BII into its corresponding derivatives.Toruzyme 3.0L, a commercial CGTase from Thermoanaerobacter sp., was found that it could synthesize glucosylation derivatives for timosaponin BII. Nine products (Product 1～9) with different degrees of glucosylation were purified and their structures were elucidated on the basis of 13C-NMR, HR-ESI-MS and FAB-MS spectra data. In this work, we found that Toruzyme 3.0L was able to use non-activated sugar donor to synthesize glucosylation derivatives, and showed a high thermal tolerance with the most favorable enzymatic activity at 100℃.Aspergillus niger AS 3.0739, a common strain producing glucoamylase, was found to had the ability of hydrolyzing C-22 hydroxyl of timosaponin BII in whole-cell culture to generate timosaponin B (Product B1). While its crude enzymes could selectively hydrolyze the glycosyl groups of C-3 sugar chain at pH 8.0 to generate two de-glycosyl furostanol saponins (Product H1, H2), and then hydrolyze the glucosyl group at C-26 position of timosaponin BII to generate one de-glycosyl spirostanol saponin (timosaponin AIII, Product H4). The structure of Product H1 was determined to be (25S)-26-O-β-D-glucopyranosyl-22, 3-hydroxy-5β-furostside. The structure of Product H2 was determined to be (25S)-26-O-β-D-glucopyranosyl-22-hydroxy-5β-furost-3β, 26-diol-3-O-β-D-galactopyranoside;A Pseudomonas fluorescens (idenfied by China Pharmacy Microbial Culture Collection, CPCC), isolated from a unknown microorganism, incubated with BII to produce product with bigger polarity than that of BII product (Product H3). The big polarity product was separated and identified as (25S)-26-O-succinate-(1→6)-β- D-glucopyranosyl-22-hydroxy-5β-furostanol-3β,26-diol-3-O-β-D-glucopyranosyl-(1→2)-β-D-galactopyranoside based on the data of spectra.Whereas the specificity of toruzyme 3.0L with high efficiency and thermostability for glucosylation in timosaponin BII and understanding its protein structure, the specific enzyme was isolated to electrophoretic homogeneity by Gel filtration (S-200 HR), Anion-exchange (Q-HP) and Cation-exchange chromatography (SP-HP). After SDS-PAGE analysis, the protein was detected as a single band. The purified protein band on SDS-PAGE separation was submitted to the MS Laboratory, Center for Research of Proteome (Beijing, China), for protein sequencing. After the protein was digested with trypsin and the peptides were separated by the AB 4800 Plus MALDI-TOF/TOF? Proteomics Analyzer and Q-TOF2 (Waters Micromass, USA). These protein sequences were compared by the Basic Local Alignment Search Tool of National Center for Biotechnology Information (NCBI) and displayed the highest similarity to the Cyclodextrin-glycosyltransferase (CGTase, EC 2.4.1.19) from Thermoanaerobacterium thermosulfurigenes. The molecular mass of the protein was 78.4 kDa. The characteristics of purified enzyme was systematically investigated, and its optimal pH value was 8.0, and it had a very broad pH activity range; the optimum temperature for maximal enzyme activity was detected at 100°C. The enzyme showed a high thermal stability. After 6 h boiling, it still retained more than 60% of maximum activity.It is the found that theα-amylases and CGTase, ie. GH13 family enzyme could catalyze the glucosylation of steroidal saponin in one step. But, other kinds of amylases, such asγ-amylase (GH15 family), had no such the activity under the same reaction conditions. The mechanism of transglycosylation was hypothesized as follows: sugar-donor (dextrin) was first hydrolyzed to an activated glucose, and then the activated glucose was attached to the glucose-acceptor. We also first discovered that crude enzymes from Aspergillus. niger AS 3.0739 could selectively hydrolyze BII into its deglycosyl derivatives at pH 8.0. The whole-cell culture of Pseudomonas fluorescens could synthesize succinate derivatives in timosaponin BII. These studies not only enriched compound libraries of steroidal saponins with structural diversity, but also established a foundation for the research of structure-function relationship of timosaponin BII and guiding the transformation of steroidal saponins.
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