Application and Glycoside
hydrolases
General catalytic functions
Y
Y
O
HO
HO
Glycosidase
OR
OH
O
HO
HO
OH
+
ROH
OH
H2O
Y
HO
HO
Y
O
Transglycosidase
OR
OH
HO
HO
R'OH
R'OH can be alcohols, carbohydrates
O
OR'
OH
+
ROH
Biological functions of glycosyl hydrolases
For mammalians
 Insemination (beta-galactosidase, alpha-L-fucosidase, and many
others)
 Alzheimer's Disease (alpha-D-mannosidase, beta-Dhexosaminidase, 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.
 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)
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)
Enzymatic Preparation of
Oligosaccharides
Goal:
1. Large-scale
2. With specific size
Natural polysaccharides
Natural polysaccharides
HO
HO
HO
HO
HO
(
O
)
HO
HO
HO
.
HO
HO
O
( O
HO
H 2N
O
HO
Chitin
Ac H N
O
)O
n HO
H 2N
OH
Chitosan
H 2N
HO
O
HO
(O
OH
O
HO
HO
HO
HO
O
HO
HO
O
n HO
Ac H N
HO
HO
Xylan
O
)
HO
Ac H N
OH
O
O
O
(
Cellulose
HO
HO
HO
HO
HO
HO
HO
OH
HO
) O
n HO
( O
HO
O
n HO
O
O
HO
HO
O
O
O
HO
HO
) O
n
O
OH
HO
 -1,3-glucan
Cellulose
Glucose
Cellobiose
Cellotriose
$ ~0
$10 /kg
$2,000/kg
$3,000/g
Derivative of
cellooligosaccharides $ ?
Induction
Screening
Microbes
Gene Cloning
Fermentation
Enzym
e
Degradation of
Polysaccharides
Oligosaccharide
s
Analysis
Mutate
d
Enzym
e
Cloned Gene
DirectedEvolution
Expresse
d
Enzyme
Catalytic
Mechanism
Common Technology
Fermentation
Genetic
Engineering
DNA cloning
Recombinant DNA
Directed-evolution
Transgenic tech.
Expression system
Bioseparation
Phage display
Screening of microbes with chitosanase
Bacillus cereus
Paecilomyces lilacinus
Aspergillus fumigatus
Oligosaccharides Electro-spray Mass Analysis
Cheng, C-Y., and Li, Y-K. (2000) Biotechnol. Appl. Biochem. 32, 197-203.
Cloning process of AF chitosanase
Two types of hydrolases
Endo-type : broad product specificity
Exo-type : high product specificity
Bacillus chitosanase X-ray structure
Endo-CNS / Exo-CNS conversion
73
GGSRDpclggTHPD
180
QG ATsrtckpGSDT
Two types of hydrolases
Endo-type : broad product specificity
Exo-type : high product specificity
Catalysis laminaripentaose hydrolase
HO
HO
O
HO
HO
HO
HO
O
(O
HO
HO
HO
)O
n
O
OH
HO
n= 2
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.
Chitobiose preparation by chitinase
chitobiose
M
icrom
assQ
-Tof
1-DEC-2003
M
AHESH113003-0330(1.323)Cm(25:32)
870.95
446.97
100
OH
OH
O
HO
O
HO
HO
%
O
NH
CO
NH
C O
TO
FM
SES+
4.47e3
OH
+
Na
CH 3
CH 3
871.96
447.99
872.96
655.97
0
50
100
150
200
250
300
350
400
450
500
550
600
650
700
750
800
850
900
950
m
/z
1000
Techniques for the acceleration of
evolution:
• Mutagenesis based on protein structure
• Direct evolution by DNA shuffling
Are you ready to learn these
modern biotechniques?