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Application and Glycoside hydrolases

Application and Glycoside hydrolases. General catalytic functions. Biological functions of glycosyl hydrolases. For mammalians Insemination (beta-galactosidase, alpha-L-fucosidase, and many others) Alzheimer's Disease (alpha-D-mannosidase, beta-D-hexosaminidase, and beta-D-galactosidase)

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Application and Glycoside hydrolases

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  1. Application and Glycoside hydrolases

  2. General catalytic functions

  3. Biological functions of glycosyl hydrolases For mammalians • Insemination (beta-galactosidase, alpha-L-fucosidase, and many others) • Alzheimer's Disease (alpha-D-mannosidase, beta-D-hexosaminidase, and beta-D-galactosidase) • Gaucher disease (beta-glucosidase) • Fucosidosis (alpha-L-fucosidase) • Diabetes control (Alpha-amylase) • Many others For plants and microbes • Protection, energy producing…… For virus • Bio-recognition and infection, such as H5N1.

  4. Several thousands of genes of glycohydrolases have been cloned. • CAZY-Carbohydrate-Active enZYmes http://afmb.cnrs-mrs.fr/CAZY/index.html Supported by French National Center for Scientific Research (CNRS)

  5. Current studies in our Lab • β-Glucosidase (family 1 and 3) • Chitinase (family 18) • Chitosanase (family 46 and 75) • α-L-Arabinofuranosidase (family 54) • α-L-Fucosidase I, II (family 29) • β-1,3-Glucosyltransferase (family 72) • β-1,6-Glucosyltransferase (family 17) • Laminaripentaose hydrolase (LPHase, β-1,3-glucanase, family 64) • Proteases (β-secretase, human signal peptide peptidase)

  6. Enzymatic Preparation of Oligosaccharides Goal: • 1. Large-scale • 2. With specific size

  7. Natural polysaccharides

  8. Natural polysaccharides .

  9. Cellulose $ ~0Glucose $10 /kgCellobiose $2,000/kgCellotriose $3,000/gDerivative of cellooligosaccharides $ ?

  10. Induction Screening Microbes Gene Cloning Fermentation Expressed Enzyme Enzyme Degradation of Polysaccharides Oligosaccharides Catalytic Mechanism Analysis Mutated Enzyme Cloned Gene Directed- Evolution

  11. Common Technology Fermentation Bioseparation Genetic Engineering DNA cloning Recombinant DNA Directed-evolution Transgenic tech. Expression system Phage display

  12. Screening of microbes with chitosanase Bacillus cereus Paecilomyces lilacinusAspergillus fumigatus

  13. Oligosaccharides Electro-spray Mass Analysis Cheng, C-Y., and Li, Y-K. (2000) Biotechnol. Appl. Biochem. 32, 197-203.

  14. Cloning process of AF chitosanase

  15. Two types of hydrolases Endo-type : broad product specificity Exo-type : high product specificity

  16. Bacilluschitosanase X-ray structure

  17. Endo-CNS / Exo-CNS conversion 73 GGSRDpclggTHPD 180 QG ATsrtckpGSDT

  18. Two types of hydrolases Endo-type : broad product specificity Exo-type : high product specificity

  19. Catalysis laminaripentaose hydrolase Naturally occurring enzymes can produce pentameric sugar!! Nakabayashi, M., Nishijima, T., Ehara, G., Nikaidou, N., Nishihashi, H., and Watanabe, T. (1998) J. Ferment. Bioeng. 85, 459-464.

  20. Chitobiose preparation by chitinase

  21. Techniques for the acceleration of evolution: • Mutagenesis based on protein structure • Direct evolution by DNA shuffling Are you ready to learn these modern biotechniques?

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