Partial Purification of Cellulase Enzyme by Hexane and ...

Partial Purification of Cellulase Enzyme by Hexane and ...

SYNTHESIS AND CHARACTERIZATION OF CROSSLINKED 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 -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 -glucanase 3. - 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-Media 5 laboratories Pvt Ltd (Mumbai) kept at 4C.

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 time 6 SYNTHESIS OF CLEA 7 SYNTHESIS OF CLEA 8

Process of CLEA Synthesis 9 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: 10 FUNCTIONAL PROPERTIES OF CLEA 11 STRUCTURAL PROPERTIES OF CLEA 12

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 14 3D SURFACE PLOTS FOR THE EFFECT OF PARAMETERS

(a) (b ) 15 (c ) (d )

EFFECT OF SONICATION ON CLEA ACTIVITY 16 EFFECT OF METAL IONS ON CLEA ACTIVITY 17 EFFECT OF DIFFERENT CALCIUM IONS ON CLEA ACTIVITY

18 EFFECT OF carbonate conc. on CLEA synthesis 19 FUNCTIONAL PROPERTIES OF CLEA 20 EFFECT OF PH ON CLEA ACTIVITY 21

EFFECT OF TEMPERATURE ON CLEA ACTIVITY 22 KINETICS CHARACTERIZATION OF FREE ENZYME AND CLEA 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 23 OPERATIONAL STABILITY OF CLEA 24

STRUCTURAL PROPERTIES OF CLEA The size and structural properties of CLEA affect its activity. So following structural investigation was performed: 25 Fig: FTIR spectrum of (a) free cellulase, (b) CLEA-I (c) CLEA-II (a) (b)

26 (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 27 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 28 increases the activity of CLEA also increases in 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 4C which leads to long term operational stability. CL EA found to be between range of 1-100 m by particle size analyzer. FTIR spectrum shows amines, alkenes, nitro groups bound on the surface of CLEAs. 29 30

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