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A Highly Selective β-D-glucosidase Hydrolyzing Ginsenoside Rb1 to Rd from Cladosporium Fulvum

Author: ZhaoXueZuo
Tutor: ZhouYiFa
School: Northeast Normal University
Course: Biochemistry and Molecular Biology
Keywords: Ginsenoside Rb1 Ginsenoside Rd A tomato brown Neurospora (Cladosporium flulvum Cooke, synonyms Fulia fulva Ciferri) β - glucosidase Biotransformation
CLC: S567.51
Type: PhD thesis
Year: 2009
Downloads: 221
Quote: 2
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Abstract


Ginseng (Panax ginseng CA Maye) Araliaceae ginseng genus, is China's traditional precious Chinese medicine. Ginsenosides are the active ingredients of ginseng, so far, has found that more than 40 kinds of ginsenosides from ginseng. Ginsenosides content, structural diversity, very different pharmacological activity. The study found that some of the low levels of rare ginseng ginsenoside has high medicinal value and application prospect. The rare ginsenoside content is too low, it is difficult to directly from ginseng extract, rely on chemical synthesis technology is difficult to industrial production. The preparation of these rare ginsenoside only feasible way is to change some of the high levels of the chemical structure of ginsenosides. Chemical conversion process is generally poor selectivity and low yield, and likely to cause environmental pollution. Biotransformation have mild conditions, better selectivity, high yield byproducts, no pollution and industrial production, etc., is considered to be the most potential for the preparation of rare ginsenoside. The papers found through screening, the tomatoes brown Mortierella high selective glycoside hydrolase able 1 hydrolysis of a unique product - the rare ginsenoside Rd the highest content ginseng ginsenoside Rb. This article explores the conditions of the separation and purification of the β-glucosidase, and its molecular characteristics and properties, and laid the foundation for industrial applications. The main results of this paper are as follows: 1, from 40 plant pathogenic fungi screened seven kinds of fungi capable of transforming ginsenoside, the conversion of substrates and products of various fungi. The the tomatoes brown Neurospora is capable of specifically transformed ginseng saponin Rb of 1 become Rd. Optimal transformation conditions: plus substrate time 24 h; substrate concentration, 0.25 mg / mL; culture pH, pH5-6; culture time, 8 d; culture temperature of 37 ° C. Tomato brown Mortierella produce transformed ginsenoside Rb 1 Rd glycosidase best conditions for enzyme production: V8 juice medium liquid culture 84 hours. 2, cultured on V8 juice 84 h tomato brown Mortierella culture liquid by filtration centrifugation, DEAE-cellulose anion exchange column chromatography, ammonium sulfate precipitation and Sepharose CL-6B gel filtration column chromatography and phenyl Sepharose CL-4B hydrophobic interaction column layer The multi-step analysis and Mono Q anion exchange column chromatography separation technologies, successfully isolated and purified an electrophoretically pure beta-glucosidase GI (ginsenosides Rb 1 transforming enzymes GI). The purification process of the GI having a high stability, to be able to ensure its stable preparation. Every 50 liters of tomato Brown Mortierella culture medium to approximately 0.5 mg of active protein can be obtained, the recovery rate of 9%, up to 17563 U / mg specific activity. 3, Superose 6 10/300 GL gel filtration column chromatography analysis (HPLC) shows the elution curve of a GI symmetrical peak, and a molecular weight of 80 kDa; by SDS polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing polyacrylamide gel electrophoresis (IEF-PAGE) detection, Spectrum showed a protein band, the molecular weight of 80 KDa, an isoelectric point (pI) was 4.2, indicating that the GI has reached to the electrophoretically pure, and is a single-chain protein , relative molecular weight of 80 KDa. The series by trypsin hydrolysis, electrospray ionization quadrupole time of flight mass spectrometry (Q-TOF2) Get GI 3 the amino acid sequence of the polypeptide chain: 1. LVAHEENVR, 2.VGKDEGFAKAGGLSR, 3.LPLEAGESGTATFNVR. BLAST tool to query and match in the NCBI non-redundant protein database and the European Bioinformatics Institute (EBI) the unification protein Knowledge Base, found no amino acids identical polypeptide segment, indicating that this enzyme as a new protein. The amino acid sequence of this peptide is uploaded to the unified protein knowledge database (UniProtKnowledgebase), has been granted a sequence accession number P85516. GI of the amino acid sequence and the β-glucosidase of several fungi of the family 3 of β-glucosidase enzymes have a high degree of homology, may belong to one of the members of this family. 4, and enzyme reaction kinetics experiments confirmed, GI has good pH stability and thermal stability within the range of pH4.0 ~ 11.0 and 40 ° C or less showed good β-glucosidase activity, and the optimum reaction pH pH6.0, the optimum reaction temperature of 45 ° C. Cu 2 , Zn 2 in 50mM strong inhibitory effect on the enzyme activity, contrary Na , K the Ca 2 , Mn, and Mg of 2 slight stimulation of enzyme activity. 0.25 M SDS did not affect the activity of the GI, 0.25 mM of EDTA is only slightly inhibited the activity of the GI. GI of Km value of 0.18 mM, the beginning of the maximum reaction velocity Vmax of 5.52 mM / min. Substrate specificity analysis showed that a GI can specifically hydrolyzes a synthetic the substrate pNPG, also available in the hydrolysis of β-glucosidase key connection disaccharide such as cellobiose, gentiobiose, and Cassia disaccharide, again indicate that this The enzyme is a β-glucosidase. GI substrate specificity ginsenosides, The enzyme ginsenosides Rb 1 showed strong hydrolytic activity, reached 22.1% of the pNPG without hydrolysis ginsenosides Rb 2 , Rc, Rd, description of this enzyme the ginsenosides Rb C-20 β (1 → 6) glycosidic bond is more specific, not hydrolyzed ginsenosides The glycosidic bond GI ginsenosides this highly selective hydrolysis laid the foundation for the industrial preparation Rd.

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CLC: > Agricultural Sciences > Crop > Economic crops > Medicinal crops > Shade drugs > Ginseng
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