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sea cucumber

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by

Sulhee Lee

on 4 November 2014

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Transcript of sea cucumber


Speaker: Sulhee Lee
Introduction
-1,3-glucanase
well-known class of enzymes widespread
Sea cucumber
Stichopus japonicus
In bacteria,
degradation of polysaccharides
that can be present
in their environment and
be used
as an energy source
In fungi,
development of cell wall architecture
in yeasts and filamentous fungi
In plant,
aspects of plants physiology and development
such as germination, growth, defense against pathogens, flowering, cellular and tissue development and differentiation
In the animal kingdom,
commonly found in
marine echinoderms
digestion of algal food
and also play some
important roles in
embryogenesis
structure of
-1,3-glucanase
economically
important echinoderms
widely cultured in China
utilize -1,3-glucans from
algae
as
main source of glucose
the abilities of marine species to
utilize nourishment
depend on the
activities of digestive enzymes
existing in their
digestive tract
However,
little information

about the digestive enzymes

from sea cucumbers

has been known up to now
Materials
Sea cucumber was collected from Yellow Sea
The gut was taken out and stored before use for experiment
DEAE Sepharose CL-6B : 10 - 10
4
6
Butyl-Sepharose 4 Fast Flow
Enzyme activity assay
One unit of -1,3-glucanase activity
the amount of enzyme that produces 1 ug of D-glucose per
minute under the standard assay conditions
Protein determination
the
method of Lowry
et al.
using
bovine serum albumin
as a standard
Enzyme activity stain
Ammonium sulfate precipitation
SDS-PAGE and Native PAGE
12% separating and 5% stacking gels
50 g
washed three times with ice cold D.W.
150 mL of ice-cold
phosphate sodium bfr.
centrifugation
80% saturation of ammonium sulfate
The final precipitate was crude enzyme which was dissolved in Tris-HCl buffer
concentrated by ultrafiltration
Enzyme purification
DEAE-Sepharose CL-6B (2.6 cm X 20 cm)
Buffer : 50 mM Tris-HCl (
pH 8.0
) buffer
Flow rate : 30 mL/h
Elution : a
linear gradient

of 0.2-1.5 M NaCl
Fractions (6 mL/fraction) containing
enzyme were pooled and
concentrated by UF
Butyl-Sepharose 4 Fast Flow (1.8 cm X 20 cm)
Buffer : 10 mM sodium phosphate (
pH 7.0
) buffer containing 0.2 M ammonium sulfate
Flow rate : 60 mL/h
Elution : a
step wise
method of 0.05, 0.02, 0.01 M ammonium sulfate
Enzyme purification
The enzyme-active fraction (2 mL)
gel elution after electrophoresis
concentrated by UF

used further experiments
as the finally purified enzyme
Determination of pH and temperature optimum and their stability
pH
Range :
pH 3.0 - 12.0
Stability : the residual activity
Temperature
Range :
10 - 90

Stability : the residual activity
Km and Vmax of enzyme
Kinetics : using
various concentrations of laminarin

A
Lineweaver-Burk
double reciprocal plot analysis

Michaelis-Menten equation : using substrate concentrations of 0.25, 0.5, 0.75, 1.0, 1.25 and 1.5 mg/mL laminarin
Protein identification by mass spectrometry
The
trypsin-digested protein
was subjected to Q-TOF mass spectrometry with
electrospray ion-source
(Q-TOF: quardrupole time-of-flight)
Result and Discussion
Enzyme purification
Enzyme purification
Optimum pH and
temperature
Effect by metal ions
Kinetics of
the purified enzyme
On the basis of a
Lineweaver-Burk
plot

Substrate :
Laminarin

Km value : 19.8 ug mL

Vmax value : 2,000 ug min mg
-1
-1
-1
Protein identification
by mass spectrometry
Peptide segment 1 : WCFKSCVMFVNLPHMK
Peptide segment 2 : CDFKFDVVQTTLLQNK
Peptide segment 3 : SSGGLYPDVLK

Searching the three-peptide segments of the purified glucanase from the protein BlastX

Do not show

any significant homology with other
echinoderm glucanase listed in the BLAST
similar to those from
Brachionus plicatilis, Bursaphelenchus xylophilus, Chaetomium indicum
, and
Trichoderma asperellum
similar to
Bacillus clausii
NM-1
lower than
B. circulans
IAM1165
similar to
B. circulans
IAM1165
-
lower
than
Pichia pastoris, Rhizoctonia soani,
and
Rhizopus microsporus
var
. microsporus
-
higher
than
Rhizoctonia soani
-
lower
than
Pichia pastoris
The searching range was extended to all animal -1,3-glucanases and all echinoderm proteins,

there were still negative result
Full transcript