Loading presentation...

Present Remotely

Send the link below via email or IM


Present to your audience

Start remote presentation

  • Invited audience members will follow you as you navigate and present
  • People invited to a presentation do not need a Prezi account
  • This link expires 10 minutes after you close the presentation
  • A maximum of 30 users can follow your presentation
  • Learn more about this feature in our knowledge base article

Do you really want to delete this prezi?

Neither you, nor the coeditors you shared it with will be able to recover it again.


sea cucumber

No description

Sulhee Lee

on 4 November 2014

Comments (0)

Please log in to add your comment.

Report abuse

Transcript of sea cucumber

Speaker: Sulhee Lee
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
structure of
important echinoderms
widely cultured in China
utilize -1,3-glucans from
main source of glucose
the abilities of marine species to
utilize nourishment
depend on the
activities of digestive enzymes
existing in their
digestive tract
little information

about the digestive enzymes

from sea cucumbers

has been known up to now
Sea cucumber was collected from Yellow Sea
The gut was taken out and stored before use for experiment
DEAE Sepharose CL-6B : 10 - 10
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
method of Lowry
et al.
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.
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
Range :
pH 3.0 - 12.0
Stability : the residual activity
Range :
10 - 90

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

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
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
Effect by metal ions
Kinetics of
the purified enzyme
On the basis of a

Substrate :

Km value : 19.8 ug mL

Vmax value : 2,000 ug min mg
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
lower than
B. circulans
similar to
B. circulans
Pichia pastoris, Rhizoctonia soani,
Rhizopus microsporus
. microsporus
Rhizoctonia soani
Pichia pastoris
The searching range was extended to all animal -1,3-glucanases and all echinoderm proteins,

there were still negative result
Full transcript