Application and Application and Glycoside Glycoside

hydrolaseshydrolases

General catalytic functions

O

OROH

HOHO

Y

Glycosidase

H2O

O

OHOH

HOHO

Y

+ ROH

O

OROH

HOHO

Y

Transglycosidase

R'OH

O

OR'OH

HOHO

Y

+ ROH

R'OH can be alcohols, carbohydrates

Biological functions of glycosyl hydrolases

For mammaliansFor 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 microbesFor plants and microbes

Protection, energy producing……

For virusFor virus

Bio-recognition and infection, such as H5N1.

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)

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)

Current studies in our LabCurrent studies in our Lab

Enzymatic Enzymatic Preparation of Preparation of OligosaccharidesOligosaccharides

Goal:Goal:

1. Large-scale1. Large-scale

2. With specific size2. With specific size

Natural polysaccharidesNatural polysaccharides

Natural polysaccharidesNatural polysaccharides

OOH

HO

O

HO

OO

HO

OHOHO HOHO

OOHO

OHOHO

HO

HO

HO

HO

O

HOO

HO

HO

HO

( )n

n)(H2N

OOH

HO

HOO

H2N

OHO

HOO

H2N

OHOHO

HO

)(

HO

HOHO

OO

HOO

OHO

HO

OHO

AcHNAcHNAcHNHO

HO

OOH

HO

O

HO

OHO

O

HO

OHOHO

HO

HOHO( )n

( n)

Cellulose

Chitin

Chitosan

Xylan

-1,3-glucan

n.

Cellulose $ ~0Glucose $10 /kgCellobiose $2,000/kgCellotriose $3,000/g

Derivative of cellooligosaccharides $ ?

FermentationFermentation

Degradation of Polysaccharide

s

Degradation of Polysaccharide

s

Oligosaccharides

MutatedEnzyme

MutatedEnzyme

Cloned GeneCloned Gene

CatalyticMechanism

ExpressedEnzyme

ExpressedEnzyme

Enzyme

Enzyme

Induction

Gene Cloning

ScreeningMicrobes

Directed-Evolution

Analysis

Common TechnologyCommon Technology

FermentationGenetic

Engineering Bioseparation

DNA cloning DNA cloning Recombinant DNA Recombinant DNA Directed-evolution Directed-evolution Transgenic tech. Transgenic tech. Expression systemExpression system

Phage displayPhage display

Bacillus cereus Paecilomyces lilacinus Aspergillus fumigatus

Screening of microbes with chitosanaseScreening of microbes with chitosanase

Oligosaccharides Electro-spray Mass AnalysisOligosaccharides Electro-spray Mass Analysis

Cheng, C-Y., and Li, Y-K. (2000) Biotechnol. Appl. Biochem. 32, 197-203.

Cloning process of AF Cloning process of AF chitosanasechitosanase

Two types of Two types of hydrolaseshydrolases

Endo-type : broad product specificity

Exo-type : high product specificity

BacillusBacillus chitosanase X-ray structure chitosanase X-ray structure

Endo-CNS / Exo-CNS Endo-CNS / Exo-CNS conversionconversion

7373

GGSRDpGGSRDpcclggTHPDlggTHPD

180180

QG ATsrtQG ATsrtcckpGSDTkpGSDT

Two types of Two types of hydrolaseshydrolases

Endo-type : broad product specificity

Exo-type : high product specificity

)n(

OOH

OO

HOHO

HO

HO

HO

HO

O

HOO

HO

HO

HO

n= 2

Catalysis laminaripentaose hydrolaseCatalysis laminaripentaose hydrolase

Naturally occurring enzymes can produce Naturally occurring enzymes can produce pentameric sugar!!pentameric sugar!!

Nakabayashi, M., Nishijima, T., Ehara, G., Nikaidou, N., Nishihashi, H., and Watanabe, T. (1998) J. Ferment. Bioeng. 85, 459-464.

Chitobiose preparation by chitinase

Micromass Q-Tof 1-DEC-2003chitobiose

50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000m/z0

100

%

MAHESH113003-03 30 (1.323) Cm (25:32) TOF MS ES+ 4.47e3870.95446.97

447.99

655.97

871.96

872.96

O

OH

OH

NH

CCH3

O

OHO

OH

OH

NH

CCH3

O

OHONa+

Techniques for the acceleration of evolution:

• Mutagenesis based on protein structure

• Direct evolution by DNA shuffling

Are you ready to learn these modern

biotechniques?