SYNTHESIS AND CHARACTERIZATION OF
CROSS-LINKED CELLULASE ENZYME
AGGREGATES (CLEAS) BY ETHANOL AND
ACETONE DESOLVATION TECHNIQUE
1
Presentation
by
Jagdish Singh
Department of Biotechnology
Mata Gujri College,
Fatehgarh Sahib, Punjab.
Introduction
Cellulase are most prominent group of hydrolytic
enzymes in industry.
• They catalyze the hydrolysis of
β-1, 4 linkages
present in cellulose to convert it into glucose.
• They are produced in nature by plants, fungi,
bacteria, and even some protozoa, molluscs, and
nematodes.
• They are multienzyme complexes of three major types
of enzymes:
1. Cellobiohydrolase
2. Endo β-glucanase
3. Β- glucosidase
•
2
 In
enzymatic wool treatment, the diffusion
of the enzyme inside the wool fibre causes
unacceptable losses of strength.
 It was thought that if the cellulase were
chemically modified in order to increase
their molecular weight, their attack would
be restricted only to the surface of the
fibres, thus removing the cuticle, which is
the main interest.
 In this paper we have tried to synthesize
the supramolecule structure, CLEA by
chemical cross linking techniques.

3
Objectives for research work
Optimization of process parameters for
synthesis of CLEA by ethanol and acetone
desolvation method.
Functional and structural characterization of
free enzyme and CLEA.
4
OPTIMIZATION
OF PROCESS PARAMETERS
FOR SYNTHESIS OF
CLEA
Response surface methodology (RSM) is an effective
statistical tool and widely used in process
optimization, which includes experimental design,
condition optimization, model fitting, and validation.
 Response surface methodology (RSM) involving a
central composite design (CCD) with thirty
experiments conducting and a second-order polynomial
equation was employed to identify the relationship
between four significant variables that influence
CLEA synthesis significantly.
 Cellulase used here was of fungal origin obtained from
Trichoderma viride purchased from Hi-Media5
laboratories Pvt Ltd (Mumbai) kept at 4˚C.

CCD Design for CLEA Synthesis
CCD Design (I) for CLEA synthesis
Factor
Name
Units Low
actual
Value
High actual
(0)
(+1)
(-1)
X1
pH
--
1
8
9.5
X2
Calcium
%
0.75
6
6.5
X3
Time
Hrs
0.5
6
6.5
X4
Incubation
Hrs
0
4
4
6
time
SYNTHESIS OF CLEA
• 1 ml of free cellulase solution (0.2%) was prepared in
phosphate buffer (pH=7)..
Step-1
Step-2
• 10 mM sodium chloride was added.
• Different pH values and calcium carbonate was adjusted
Step-3
Incubated for different time as per design
.
• Chilled ethanol/acetone (desolvating agent) was added
Step-4
until solution become turbid.
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SYNTHESIS OF CLEA
Step-5
• 0.25ml glutaraldehyde (6%) was added. Incubated for
different time as per design
Step-6
• Resulting solution was centrifuged (6000 rpm and 4⁰C) for
10 minutes
Step-7
• Pellet was dissolved in phosphate buffer (pH 7) and
supernatent was discarded.
• Cellulase Residual activity was determined in pellet by
standard procedure.
Step-8
•.
8
Process of CLEA Synthesis
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EFFECT OF DIFFERENT PARAMETERS ON
SYNTHESIS OF CLEA


THE
Residual activity of CLEA was less (40%) as
compared to free enzyme.
So to improve the residual activity, effect of
following parameters on the synthesis of CLEA
was observed:
Sonication
Metal ions
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FUNCTIONAL PROPERTIES OF CLEA
1. Effect of pH on the activity of CLEAs
2. Effect of temperature on the activity
3. Effect of substrate concentration
4. Operational stability of CLEA
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STRUCTURAL PROPERTIES OF CLEA
1. FTIR spectroscopy
2. Particle size analysis
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Results and Discussion
13
Synthesis of CLEA using Response surface
methodology (RSM)
Residual activity (% recovery) 40.34 was achieved
by acetone with optimum parameters i.e.; pH
(X1) 6.5, calcium mM (X2) 0.75, time (X3) 3.5 h,
incubation period (X4) 2 h .
But when ethanol was used as desolvation reagent
only 39.61% residual activity was achieved by
optimum parameters i.e; pH (X1) 6.5, calcium
(X2) 0.75mM, time (X3) 2.5 h, incubation time
(X4) 2h
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3D SURFACE PLOTS FOR THE EFFECT OF PARAMETERS
(a)
(b
)
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(c)
(d
)
EFFECT OF SONICATION ON CLEA ACTIVITY
80
% Residual activity
70
60
50
40
30
20
10
0
0
10
15
20
Time (min)
25
30
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EFFECT OF METAL IONS ON CLEA ACTIVITY
100
90
Residual activity (%)
80
70
60
50
40
30
20
10
0
control
calcium
carbonate
nickel
potassium mercuric magnesium
sulphate
chloride
chloride
sulphate
Concentrations (750 ppm)
sodium
nitrite
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EFFECT OF DIFFERENT CALCIUM IONS ON CLEA
ACTIVITY
100
90
% Residual activity
80
70
60
50
40
30
20
10
0
control
calcium dibasic
phosphate
calcium
calcium chloride calcium nitrate
orthophosphate
calcium
carbonate
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EFFECT OF carbonate conc. on CLEA synthesis
Residual activity(% )
120
100
80
60
40
20
0
150
350
550
750
950
Calcium Carbonate(ppm)
1000
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FUNCTIONAL PROPERTIES OF CLEA
1. Effect of pH on the activity of CLEA activity
2. Effect of temperature on the CLEA activity
3. Effect of substrate concentration on the CLEA activity
4. Operational stability of CLEA
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EFFECT OF PH ON CLEA ACTIVITY
0.12
Enzyme Activity IU/ml)
0.1
0.08
0.06
0.04
0.02
0
3
4
5
6
7
8
pH
Free Enzyme
CLEA-I
CLEA-II
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EFFECT OF TEMPERATURE ON CLEA ACTIVITY
Enzyme activity (IU/ml)
0.2
0.18
0.16
0.14
0.12
0.1
0.08
0.06
0.04
0.02
0
20
Free Enzyme
30
40
Temp. (oC)
CLEA-I
50
60
CLEA-II
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KINETICS CHARACTERIZATION OF
ENZYME AND CLEA
Parameter
FREE
Enzyme activity of free enzyme, CLEA
Free
CLEA- I
CLEA- II
Km (%)
2.25
2.30
4.25
Vmax (IU/ml)
2.00
2.15
3.5
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OPERATIONAL STABILITY OF CLEA
Residual Activity (IU/ml)
1.4
1.2
1
0.8
0.6
0.4
0.2
0
0
1
2
3
4
5
6
7
8
9
Storage Time (Days)
Free enzyme
CLEA-I
CLEA-II
10
11
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STRUCTURAL PROPERTIES OF CLEA
The size and structural properties of CLEA affect its
activity. So following structural investigation was
performed:
1. FTIR spectroscopy
2. Particle size analysis
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Fig: FTIR spectrum of (a) free cellulase, (b) CLEA-I (c) CLEA-II
(a)
(b)
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(c)
PARTICLE SIZE ANALYSIS OF (A)FREE ENZYME (B)CLEA-I AND (C CLEA-II)
Free enzyme:
Size: 778 d.nm
Size of CLEA-I
2573 d.nm
Size of CLEA-II
4876 d.nm
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Summary of Research finding
1.
2.
3.
4.
Residual activity (% recovery) 40.34 was
achieved by acetone with optimum parameters
i.e.; pH (X1) 6.5, calcium mM (X2) 0.75, time
(X3) 3.5 h, incubation period (X4) 2 h .
But when ethanol was used as desolvation
reagent only
39.61% residual activity was
achieved by optimum parameters i.e; pH (X1)
6.5, calcium (X2) 0.75mM, time (X3) 2.5 h,
incubation time (X4) 2h
Metal ions such as calcium carbonate,
potassium chloride effects on the binding of
microstructures and enhance the residual
activity upto 86.2 and 85.1 %.
As the concentration of calcium carbonate
increases the activity of CLEA also increases in
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order of concentrations.
1)
2)
3)
4)
CLEA showed shift in the optimum tempertaure
and pH optima with respect to free enzyme.
Compared to free enzyme, CLEA has no
significant activity losses within 11 days at 4⁰C
which leads to long term operational stability.
CL EA found to be between range of 1-100 µm
by particle size analyzer.
FTIR spectrum shows amines, alkenes, nitro
groups bound on the surface of CLEAs.
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