ASHIQ_PRE_PHD_PRESENTATION.characterization of cellulase for psychrophilic fungus truncatella angustata and their evaluation of ailanthus excelsapptx

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1. Pre- Ph.D Presentation By Ashiq Hussain Magrey Under Supervision of Dr. Sanjay Sahay Department of Botany Govt. Post Graduate College , Biaora, Rajgarh, M.P Dr. Ragini G othalwal Dean L ife Sciences & Head Department of Biotechnology Barkatullah University, Bhopal Characterization of cellulases from Psychrophilic fungus Truncatella angustata and their evaluation for cellulysis of Ailanthus excelsa (Maha Neem) wood

2. I ntroduction Cellulase is the enzyme of industrial interest, plays a crucial role in hydrolysis of cellulose, a prime component of plant cell wall (Chandra et al ., 2010 ), and commercially have huge global market (Yoon et al ., 2014 ). The demand of cellulases will be strongly picked by the commercial production of biofuels in future (Yoon et al ., 2014).

3. Enzymatic hydrolysis of biomas

4. Objectives Biochemical characterization of partially purified cellulases (CMCase, FPase and Cellobiase) from fungal isolate encompassing effect of temperature, pH, metal ions, substrate concentration on individual enzyme activity; Study on the effect of glucose on individual enzyme activity to assess whether a condition of product inhibition exists there. Isolation of cellulose from Ailanthus excelsa (Maha neem) tree applying dilute sulfuric acid pretreatment of dried wood pieces; Treatment of A. excelsa cellulose, saw dust, wheat straw and rice straw with Truncatella angustata cellulases to see its potential to hydrolyze the cellulose under selected temperature, pH and incubation period conditions.

5. Method Screening prospective psychrotrophc fungus as source of cellulases from laboratory stock Cellulases production under SmF Partial characterization of Cellulases Dilute acid pretreatment of lignocellulosic materials (wheat straw, rice straw and woods of Ailanthus excelsa) Hydrolysis of pretreated lignocellulosic biomass Analysis of sugar yields in case different biomass

6. Enzyme production * In Erlenmeyer flasks (250 mL ) with 50 mL medium ( Czapek-Dox ) containing 1% cellulose as carbon source; * Inoculated with fungal mycelium, the medium incubated at 20°C for 5 days on orbital incubator shaker ( 120 rpm ); * The broth filtered through cheese cloth, the filtrate then was centrifuged at 5000 rpm for 10 min at 4°C, clear supernatant used as crude enzyme and stored at -20°C until used.

7. Enzyme assays Filter paper ase ( Fpase ) * ( FPase) activity was determined as described earlier (Ghose (1987 ) . The assay mixture (total volume of 2 mL) contained 50 mg of Whatman No.1 strip (1 × 6 cm) in 1 mL of 50 mM citrate buffer (pH 4.8) and 0.5 mL of diluted crude enzyme and was incubated at 20°C for 30 min. Carboxy Methyl Cellulose ( CMCase ) * CMCase activity was determined as described by ( Mandels Sternberg 1975) . The assay mixture, in a total volume of 2 mL , contains 0.5 mL of 1 mM of carboxyl methyl cellulose (CMC) in 50 mM citrate buffer (pH 4.8) and 0.5 mL of diluted crude enzyme. The mixture was incubated at 20°C for 30 min.

8. Enzyme assays Cellobiase * Cellobiase activity was assayed by incubating 0.5mL of supernatant with 0.5mL of 2% cellobiaose in 0.05M sodium citrate buffer (pH 4.8) at 20 o C for 30min. ( Lloyd and Whelan 1969) Sugar estimation by DNS method Sugar released in each of the three above reaction estimated by DNS ( 30g K-Na tartrate dissolved in 50 ml of dd water, further supplemented with 1g DNS and 20 ml 2N NaOH and final volume was made to 100 ml )method ( Miller 1959 ) at 540 nm.

9. The activity was calculated by the following formula: CMCase/ FPase/ Cellobiase activity (IU/ml) = x(mg glucose produced)x5560x22x2/30 1 volume of enzyme sample in dilution Enzyme conc. = ------------------------------------------- total volume of dilution = 1/dilution = 1/20 = 0.05 CMCase & FPase (IU/ml)= 0.37/Enzyme concentration that released 2 mg glucose   Cellobiase = 0.0926/enzyme concentration to release 1.0 mg glucose Enzyme activity calculation

10. Lignocellulosic substrates pretreatment method Substrates Wheat straw Mahaneem ( Ailanthus excelsa ) Saw dust Rice straw Pretreatmenent method Dilute acid (sulphuric acid (0.7%) method used ( Sahay and Rana 2017) Pretreated lignocellulose of A. excelsa

11. Collection of Saw dust from Timber S hop Bhopal

12. Results Detection of cellulases

13. FPase ( i.e. Total or exo and endoglucanase ) activity Effect of Temperature ( FPase ) The enzyme activity at 60 o C: 725.9 IU/L

14. Effect of pH ( FPase ) Maximum activity is at pH 10

15. Effect of metal ions ( FPase ) The enzyme is metallozyme , since Ca 2+ has positive effect, the highest enzyme activation found in presence of Mn 2+ and Cu 2+ . Among metal ions tested only Zn 2+ shows inhibitory effect

16. Stability at different pH ( FPase ) The maximum stability at pH was found.

17. CMCase activity Effect of temperature ( CMCase ) The maximum CMCase (Endo-β-1-4, glucanase or Endoglucanase ) activity found at 40 o C, a residual activity of 60 % at 5 o C, 90% at 10 o C, 75% at 20 o C and 88% at 50 o C was observed. The activity at 40 o C was found to be 279.6 IU/L.

18. Effect of pH ( CMCase ) The enzyme was acidophilic one, maximum activity at pH 4.0. Though, considerable residual activity found at pH 3.0 (80%), pH 5.0 (50%), pH 6.0 (70%) and pH 10.0-11.0 (50%)

19. Effect of metal ions ( CMCase ) The enzyme is metallozyme , since Ca 2+ had positive effect, the highest enzyme activation was found in presence of Cu 2+ . Among metal ions tested, Mn 2+ , Na 2+ and Zn 2+ were inhibitory ions, Zn 2+ showing highest inhibitory effect.

20. Effect of metal ions on CMCase activity

21. Stability at different temperature ( CMCase ) The enzyme shows stability at 4 o C and 60 o C, to the extent of 100%. The enzyme is thus considered to be thermostable one .

22. Stability at different pH ( CMCase ) The enzyme is stable at pH 8.0-9.0.

23. Cellobiase (β-glucosidases) activity Effect of temperature ( β- glucosidases ) The enzyme shows maximum stability at 60 o C, at other temperatures more 60% stability was shown.

24. Effect of pH ( β- glucosidases ) The maximum activity at pH 6.0, though a second peak activity was found at pH 9.0. Very low residual activity was found at pH 3.0 (32%), pH 5.0 (50%), pH 7.0 (50%) and pH 10.0-11.0 (less than 40 %).

25. Effect of metal ions (β-glucosidases) The enzyme is not a metallozyme , since Ca 2+ does not have positive effect. All the metal ions tested were found to have inhibitory effect, Mg 2+ showing highest inhibitory effect.

26. Effect of metal ions on β- glucosidases activity

27. S tability at different temperatures (β- glucosidases ) The enzyme was found stable at all temperature s

28. Stability at different pH(β- glucosidases ) The enzyme is stable at pH 9.0 .

29. Glucose inhibition test (β- glucosidases ) Positive inhibition of the enzyme by glucose was seen, almost 85% inhibition was found at 0.01% (w/v) exogenous glucose concentration .

30. Sugars in the wash (hemicelluloses hydrolysate ) The wash of the pretreated biomass was analysed for the presence of sugars. Maximum sugars were obtained from wheat straw and least from rice straw as in table below . Biomass Pentoses (mg/g) Reducing sugars (mg/g ) A excelsa 16.65±44 23.54±56 Wheat straw 23.76±64 28.45±62 Saw dust 14.32±37 19.42±42 Rice straw 12.86±32 15.65±24 ----------------------------------------------------------------------------------------

31. Hydrolysis rate of selected biomasses by T. angustata cellulases An initial slow hydrolysis for first 4-5 days, sharply enhanced on 6-7 days and then followed zig-zag pattern. The rate of hydrolysis of A. excelsa and wheat straw were found faster. At the sixth day A. excelsa , saw dust, wheat straw and rice straw releases glucose 2540mg/L, 2000 mg/L, 3500 mg/L and 700 mg/L respectively.

32. Conclusion The FPases had maximum activity at 60 o C, a residual activity of 35 % at 4 o C, 55% at 10 o C,35% at 40 o C was observed. The enzyme activity at 60 o C was found to be 725.9 IU/L, while at 4 o C and 30 o C the activity was 259.26 and 296.3 IU/L respectively. Three distinct types of activities at 4-10 o C, around 40 o C and around 60 o C were found . The enzyme was alkaliphilic with optimum pH 10. Though, considerable residual activity was also found at pH 3.0 (60%), pH 4.0 (50%), pH 6.0 (60%) and pH 9.0 (53%). The enzyme was metallozyme, showing the highest enzyme activation in presence of Mn 2 + and Cu 2+ . Among metal ions tested only Zn 2+ showed inhibitory effect.

33. Contd.. The enzyme was acidophilic one, maximum activity is at pH 4.0 though, considerable residual activity was also found at pH 3.0 (80%), pH 5.0 (50%), pH 6.0 (70%) and pH 10.0-11.0 (50 %). The enzyme was metallozyme, the highest activation was found in presence of Cu 2 + . Among metal ions tested, Mn 2+ , Na 2+ and Zn 2+ showed inhibitory effect. The enzyme showed stability at 4 o C and 60 o C, to the extent of 100%. The stability was maximum at pH 8.0-9.0. The maximum Cellobiase activity was found at 60 o C, residual activities of 43.6% at 4 o C and 41% at 10 o C was observed. The enzyme yielded maximum activity of 170 IU/L at 60 o C and 80 IU/L at 4 o C.

34. References Bai , X., Yuan, X., Wen , A., Li, J., Bai , Y., & Shao , T. (2016 ). Cloning, expression and characterization of a cold-adapted endo-1, 4-β-glucanase from Citrobacter farmeri A1, a symbiotic bacterium of Reticulitermes labralis .   The Journal of Life and Environmental Sciences,  4 , e2679. Chandra, M ., Kalra , A., Sharma, P. K., Kumar, H., & Sangwan , R. S. (2010 ). Optimization of cellulases production by Trichoderma citrinoviride on marc of Artemisia annua and its application for bioconversion process.  Biomass and Bioenergy ,  34 (5), 805-811. Ghose, T. K. (1987). Measurement of cellulase activities.  Pure and applied Chemistry ,  59 (2), 257-268. Mandels , M., & Sternberg, D. (1976 ). Recent advances in cellulase technology.  Journel of Ferment. Technology ,  54 (4), 267-286. Sahay , S., & Rana , R. S. (2017). Hemicellulose hydrolysate from Ailanthus excelsa wood potentially fermentable to ethanol.  Journal of Tropical Forest Science , 172-178.

35. List of P ublications Ashiq Magrey 1 *, Sanjay Sahay 3 , Ragini Gothalwal 1 . All Climates Carboxymethyl Cellulase from Psychrophilic Fungus Truncatella angustata (2019) Research & Reviews: A Journal of Microbiology and Virology, 9 (1) 2349-4360.ISSN : 2230-9853. Ashiq Magrey 1 *, Sanjay Sahay 2 , Ragini Gothalwal1 Cellulases for biofuel: A review (2018). International Journal of Recent Trends in Science And Technology, 17-25ISSN :2249-8109.

36. Awards & Conferences Attended I have attended 2 international and 5 national conferences and presented 2 Papers. Awards: Best paper presentation award. Workshops: 1 in MPCOST Bhopal. M embership of British Ecological Society (2017-18) Membership number 1011153 .

37. Thank You