1. S YNTHESIS AND CHARACTERIZATION OF C ROSS - LINKED CELLULASE ENZYME AGGREGATES (CLEA S ) BY ETHANOL AND ACETONE DESOLVATION TECHNIQUE Presentation by Jagdish Singh Department of Biotechnology Mata Gujri College, Fatehgarh Sahib, Punjab. 1

2. 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 Introduction

3. 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

4. Objectives for research work 4 Optimization of process parameters for synthesis of CLEA by ethanol and acetone desolvation method. Functional and structural characterization of free enzyme and CLEA.

5. O PTIMIZATION 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. 5

6. 6 CCD Design for CLEA Synthesis CCD Design (I) for CLEA synthesis Factor NameUnits 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 X4Incubation time Hrs 0 4 4

7. S YNTHESIS OF CLEA 7 Step-1 1 ml of free cellulase solution (0.2%) was prepared in phosphate buffer (pH=7).. Step-2 10 mM sodium chloride was added. Step-3 Different pH values and calcium carbonate was adjusted Incubated for different time as per design. Step-4 Chilled ethanol/acetone (desolvating agent) was added until solution become turbid.

8. S YNTHESIS OF CLEA 8 Step-5 0.25ml glutaraldehyde (6%) was added. Incubated for different time as per design Step-6 Resulting solution was centrifuged (6000 rpm and 4 ⁰ C) for 10 minutes Step-7 Pellet was dissolved in phosphate buffer (pH 7) and supernatent was discarded. Step-8 Cellulase Residual activity was determined in pellet by standard procedure..

9. 9 Process of CLEA Synthesis

10. E FFECT 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 SonicationMetal ions

11. F UNCTIONAL PROPERTIES OF CLEA 11 1. Effect of pH on the activity of CLEAs2. Effect of temperature on the activity3. Effect of substrate concentration4. Operational stability of CLEA

12. S TRUCTURAL PROPERTIES OF CLEA 12 1. FTIR spectroscopy 2. Particle size analysis

13. Results and Discussion 13

14. 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

15. 3D S URFACE PLOTS FOR THE EFFECT OF P ARAMETERS 15 (a) (b ) (c) (d )

16. E FFECT OF SONICATION ON CLEA ACTIVITY 16

17. E FFECT OF METAL IONS ON CLEA ACTIVITY 17

18. E FFECT OF DIFFERENT CALCIUM IONS ON CLEA ACTIVITY 18

19. 19 E FFECT OF carbonate conc. on CLEA synthesis

20. F UNCTIONAL PROPERTIES OF CLEA 20 1. Effect of pH on the activity of CLEA activity2. Effect of temperature on the CLEA activity3. Effect of substrate concentration on the CLEA activity 4. Operational stability of CLEA

21. E FFECT OF P H ON CLEA ACTIVITY 21

22. E FFECT OF TEMPERATURE ON CLEA ACTIVITY 22

23. 23 ParameterEnzyme activity of free enzyme, CLEA FreeCLEA- ICLEA- II Km (%)2.252.304.25 V max (IU/ml)2.002.153.5 K INETICS CHARACTERIZATION OF FREE ENZYME AND CLEA

24. O PERATIONAL STABILITY OF CLEA 24

25. S TRUCTURAL PROPERTIES OF CLEA 25 1. FTIR spectroscopy 2. Particle size analysis The size and structural properties of CLEA affect its activity. So following structural investigation was performed:

26. 26 (a) (b) (c) Fig: FTIR spectrum of (a) free cellulase, (b) CLEA-I (c) CLEA-II

27. 27 P ARTICLE SIZE ANALYSIS OF ( A )F REE 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

28. 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. 28 Summary of Research finding

29. 29 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 4 ⁰ C 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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