SYNTHESIS AND CHARACTERIZATION OF CROSS-

LINKED CELLULASE ENZYME AGGREGATES (CLEAS) BY ETHANOL

AND ACETONE DESOLVATION TECHNIQUE

Presentation

byJagdish Singh

Department of BiotechnologyMata Gujri College,

Fatehgarh Sahib, Punjab.1

• 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. Cellobiohydrolase2. Endo β-glucanase3. Β- glucosidase

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Introduction

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.

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Objectives for research work

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Optimization of process parameters for synthesis of CLEA by ethanol and acetone desolvation method.Functional and structural characterization of free enzyme and CLEA.

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-Media laboratories Pvt Ltd (Mumbai) kept at 4˚C.

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CCD Design for CLEA Synthesis

CCD Design (I) for CLEA synthesis

Factor Name Units Low

actual

(-1)

Value

(0)

High actual

(+1)

X1 pH -- 1 8 9.5

X2 Calcium % 0.75 6 6.5

X3 Time Hrs 0.5 6 6.5

X4 Incubation

time

Hrs 0 4 4

SYNTHESIS OF CLEA

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SYNTHESIS OF CLEA

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Process of CLEA Synthesis

EFFECT OF DIFFERENT PARAMETERS ON THE SYNTHESIS OF CLEA

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:

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FUNCTIONAL PROPERTIES OF CLEA

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STRUCTURAL PROPERTIES OF CLEA

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Results and Discussion

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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

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(a) (b)

(c)

(d)

EFFECT OF SONICATION ON CLEA ACTIVITY

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EFFECT OF METAL IONS ON CLEA ACTIVITY

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EFFECT OF DIFFERENT CALCIUM IONS ON CLEA ACTIVITY

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EFFECT OF carbonate conc. on CLEA synthesis

FUNCTIONAL PROPERTIES OF CLEA

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EFFECT OF PH ON CLEA ACTIVITY

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EFFECT OF TEMPERATURE ON CLEA ACTIVITY

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Parameter 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

KINETICS CHARACTERIZATION OF FREE ENZYME AND CLEA

OPERATIONAL STABILITY OF CLEA

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STRUCTURAL PROPERTIES OF CLEA

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The size and structural properties of CLEA affect its activity. So following structural investigation was performed:

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(a)

(b)

(c)

Fig: FTIR spectrum of (a) free cellulase, (b) CLEA-I (c) CLEA-II

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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

1. 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 .

2. 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

3. 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 %.

4. As the concentration of calcium carbonate increases the activity of CLEA also increases in order of concentrations.

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Summary of Research finding

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1) CLEA showed shift in the optimum tempertaure and pH optima with respect to free enzyme.

2) Compared to free enzyme, CLEA has no significant activity losses within 11 days at 4⁰C which leads to long term operational stability.

3) CL EA found to be between range of 1-100 µm by particle size analyzer.

4) FTIR spectrum shows amines, alkenes, nitro groups bound on the surface of CLEAs.

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