Rhamnosidase Enzyme Assay protocol1.pptx

Alpha l Rhamnosidase assay protocol

1. Presentation On PROGRESS REPORT “Studies on Microbial α-L-Rhamnosidases, Significant in Pharmaceutical Industries” Kunwar Vishal Research Scholar (PHD202200002634) Life Sciences & Biotechnology Chhatrapati Shahu Ji Maharaj University, Kanpur

2. EC3.2.1.40 belongs to glycoside hydrolase (GH) families (GH13, GH78, and GH106 families) in the carbohydrate-active enzymes (CAZy) database  Hydrolyzes the non-reducing end of α-L-rhamnose. Naringin, rutin, quercitrin, hesperidin, diosgene, terpenyl glycosides and many other natural glycosides containing terminal α- l -rhamnose α-L-Rhamnosidases

3. Naringin α -L-Rhamnosidase Debittering and clearance of citrus fruit juices, Enhancement of wine aromas Derhamnosylation of many natural products containing terminal α- l -rhamnose to compounds of pharmaceutical interest Applications α -L-Rhamnosidase Prunin Bitterness reduced to 1/3rd Naringenin β-D- glucosidase

4. Substrate specificity

5. WL BLS YS DOO Fig: Screening of α -L-Rhamnosidase producing microbes Discoloration around colonies (Halo zone formation) is showing rhamnosidic activity

6. Screening of Extracellular secretion of α-L-Rhamnosidases We had four fungal strains which have been screened for α-L-Rhamnosidases which were showing prominent halo zone formation and they are: White Lemon (WL) Dark Olive Orange (DOO) Yellow Soil (YS) Black Soil (BLS) Now the cultures were grown in Liquid culture medium (75mL) containing, L- asparagine 2 gm, MgSO 4 .7H 2 O 0.5 gm, KH 2 PO 4 3 gm, 0.5% inducer in 1 L MilliQ water and initial pH was adjusted to 4.5. The inducers are Naringin (4',5,7-Trihydroxyflavanone), Rutin hydrate (Quercetin 3-O-rutinoside hydrate ) & Hesperidin. The culture flasks were incubated at 25℃ under stationary condition. Aliquots of one mL were withdrawn at the regular intervals of 24 hours and stored in -20℃ refrigerator. The presence of α-L-Rhamnosidases activity using Davis method (1947) with some modifications is followed .

7. The reaction solution consisted of 900µL of 0.2M Sodium acetate/acetic acid buffer pH 4.5. Then added 50µL of 20Mm Naringin solution dissolved in methanol. 50µL of enzyme extract was added to above solution and incubated at 50℃ for 20 minutes, 100µL aliquot was withdrawn immediately and was added to 1.8ML 90% diethylene glycol (DEG) followed by the addition of 100µL 4N NaOH and mixed well. The samples were maintained at room temperature for 10-15 minutes and absorbance of the resultant solutions were measured spectrophotometrically at 420nm (Naringin and Naringenin show special transmittance curve near 420nm). The color intensity decreases which related to presence and activity of α-L-Rhamnosidases. To make α-L-Rhamnosidases preparation suitable for the conversion of Naringin to pruning, it must not contain β- glucosidase activity or it must be active in alkaline pH range where β- glucosidase has no activity. Reaction Reaction Stopper 200mM Sod. Acetate –acetic acid buffer 900 µL 50µL 20mM Naringin 50 µL enzyme extract 1.8mL Diethylene glycol Aliquot of 100 µL of reaction 100µL 4N NaOH For control experiment, enzyme extract were added to reaction after incubation and reaction stopped immediately as same manner.

8. BLS ASSAY DAYS OD@420 2 0.098 4 0.417 5 0.372 6 0.464 7 0.557 8 0.494 9 0.562 10 0.709 11 0.618 12 0.45 13 0.3 DAYS OD@420 R2 R2-Rn 2 2.799 2.799 3 2.215 2.799 0.584 4 1.738 2.799 1.061 5 1.692 2.799 1.107 6 1.699 2.799 1.1 8 1.528 2.799 1.271 9 1.443 2.799 1.356 10 1.489 2.799 1.31 11 1.515 2.799 1.284 1.743 0.8 0.5 1.572 WL ASSAY

9. Reaction R2-R 2.245 2.245 2.089 2.245 0.156 1.633 2.245 0.612 1.813 2.245 0.432 1.745 2.245 0.5 1.687 2.245 0.558 1.634 2.245 0.611 1.617 2.245 0.628 1.554 2.245 0.691 1.584 2.245 0.661 0.5 0.4 OD@420 Control E3 C-R 2.035 2.15 0.01 2.106 2.15 0.044 2.01 2.15 0.14 1.89 2.15 0.26 1.842 2.15 0.308 1.759 2.15 0.391 1.734 2.15 0.416 1.888 2.15 0.262 1.9 2.15 0.25 1.95 2.15 0.2 2 2.15 0.15 All these reactions were performed at 37°C for 15 min .

10. DAYS 2 0.41 3 0.468 4 0.5 5 0.719 6 0.757 7 0.845 8 0.849 9 0.679 10 0.722 11 0.734 12 0.55 13 0.35 All these reactions were performed at 50°C for 20 min .

11. HESPERIDIN CULTURE MEDIUM(HCM) C R C-R 1.403 1.404 -0.001 1.379 1.405 -0.026 1.384 1.391 -0.007 1.443 1.408 0.035 1.396 1.409 -0.013 1.404 1.395 0.009 1.412 1.381 0.031 1.422 1.376 0.046 1.402 1.373 0.029 1.423 1.378 0.045 DOO ASSAY HES 31/01/24 C R C-R 1.539 1.589 -0.05 1.459 1.462 -0.003 1.584 1.543 0.041 1.583 1.613 -0.03 1.576 1.568 0.008 1.579 1.593 -0.014 1.598 1.598 1.563 1.554 0.009 1.545 1.564 -0.019 1.61 1.571 0.039 YS ASSAY HES 28/01/24

12. C R C-R 1.414 1.421 -0.007 1.475 1.474 0.001 1.488 1.493 -0.005 1.496 1.485 0.011 1.471 1.51 -0.039 1.492 1.475 0.017 1.495 1.507 -0.012 1.485 1.486 -0.001 1.536 1.491 0.045 1.473 1.43 0.043 BLS ASSAY HES 25/01/24 CONTROL RXN C-R 1.397 1.381 0.016 1.491 1.478 0.013 1.483 1.496 -0.013 1.495 1.49 0.005 1.499 1.488 0.011 1.476 1.494 -0.018 1.467 1.482 -0.015 1.454 1.472 -0.018 1.462 1.467 -0.005 1.437 1.479 -0.042 WL ASSAY HES 24/01/24

13. RUTIN CULTURE MEDIUM(RCM) R C C-R 1.19 1.219 0.029 1.304 1.29 -0.014 1.312 1.286 -0.026 1.335 1.308 -0.027 1.321 1.32 -0.001 1.302 1.312 0.01 1.337 1.304 -0.033 1.339 1.308 -0.031 1.334 1.364 0.03 1.32 1.334 0.014 1.277 1.472 0.195 1.31 1.594 0.284 WL ASSAY RUT 05/02/24 YS ASSAY RUT 06/02/24 C R C-R 1.155 1.206 -0.051 1.325 1.294 0.031 1.381 1.306 0.075 1.362 1.353 0.009 1.341 1.351 -0.01 1.373 1.389 -0.016 1.338 1.354 -0.016 1.398 1.351 0.047 1.39 1.394 -0.004 1.349 1.363 -0.014 1.321 1.353 -0.032 1.385 1.363 0.022

14. C R C-R 1.36 1.384 -0.024 1.397 1.424 -0.027 1.384 1.366 0.018 1.399 1.42 -0.021 1.372 1.459 -0.087 1.434 1.434 1.443 1.444 0.001 1.428 1.423 0.005 1.528 1.433 0.095 1.557 1.421 0.136 1.459 1.431 0.028 1.464 1.437 0.027 DOO ASSAY RUT 07/02/24 BLS ASSAY RUT 01/02/24 C R C-R 1.314 1.308 0.006 1.377 1.391 -0.014 1.402 1.427 -0.025 1.379 1.349 0.03 1.416 1.437 -0.021 1.431 1.42 0.011 1.425 1.425 1.451 1.453 -0.002 1.403 1.44 -0.037 1.467 1.465 0.002 1.363 1.41 -0.047 1.424 1.396 0.028