PURIFICATION OF AMYLASE USING NANO ALUMINA

Under the guidance of

Dr. PUSHPA AGRAWAL Dean students affairs, RVCE

G VIJAY KUMAR Asst. prof, Dept of Biotechnology, RVCE

BY,
ARUN B R(1RV09BT010) NAVEEN J(1RV10BT404) VENKATESH M(1RV09BT054)

OBJECTIVE
Purification of amylase from the tinospora cordifolia. Characterization of the amylase obtained from tinospora cordifolia. Characterization of the nano material (Al2O3 and TiO2 ). Determination of adsorption isotherm.

MATERIALS AND INSTRUMENTS

Materials Tinospora cordifolia leaves Ethanol Distilled water Nano Powder Reagents for Lowrys Method Reagents for SDS PAGE Instruments Orbital shaker Cooling centrifuge Ion exchange chromatography kit (DEAECellulose) UV Spectrophotometer Colorimeter XRD Scanning Electron Microscope(SEM)

INTRODUCTION
Botanical Name(s): Tinospora Cordifolia Kingdom: Plantae Division: Magnoliophyta Class: Magnoliopsida Order: Ranunculales Family: Menispermaceae Genus: Tinospora

Tinospora cordifolia is a shrub that is native to India commonly known as Amrutha Balli. Its root, stems, and leaves are used in Ayurvedic medicine. It is used for the treatment of diabetes, high cholesterol, allergic rhinitis.

Alumina
Aluminium oxide is an amphoteric oxide with the chemical formula Al2O3. It is commonly referred to as alumina or corundum in its crystalline form. It has a widespread occurrence in nature and industry. Formula: Al2O3 Density: 3.95g/cm Melting point: 2,072C Molar mass: 101.961g/mol Boiling point: 2,977 C

Titanium dioxide
Titanium dioxide, also known as titanium(IV) oxide or titania, is the naturally occurring oxide of titanium It has a wide range of applications which include paint, sunscreen and food colouring.
Formula: TiO2 Density: 4.23 g/cm Molar mass: 79.866 g/mol Melting point: 1,843 C Boiling point: 2,972 C

Steps involved
Extraction of crude from Tinospora Cordifolia. Purification of amylase using Ion Exchange Chromatagraphy (Cationic exchanger). Purification of amylase using batch adsorption by ceramic material as a adsorbent at different pH. Purification of amylase using continuous adsorption at 6.8 pH. Analysis of amylase concentration using lowrys method Characterization of amylase by TLC method. Analysis of amylase activity using DNS method. Comparison of amylase using GC/MS with standard. Characterization of the alumina and Titanium oxide using SEM & XRD. Determination of mass transfer co-efficient.

Extraction of crude
Add 95 ml of ethanol in 5gm of sample. Kept in orbital shaker for 1 hr. Centrifuge at maximum speed for 15-20 min. Discard the supernatant and dry the pellets. 90 ml of water is added to the 5gm of pellet. Keep in shaker for 24 hrs. Filtrate the mixture.

Effect of pH on purification of amylase

10 ml of crude extract was added to 0.1 gm of alumina and kept in orbital shaker for 60 min of contact time and after adsorption the filtrate collected and analyzed for the concentration of amylase . The experiment was conducted at different pH ranging from 4 to 8. The optimum pH was found to be around 7

Breakthrough Curve
Break through curve
0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 0 10 20 30 40 50 60 70 80 90 Time (min) C/Co 10 mm 20 mm

Above Fig exhibits that the adsorbent is exhausted at a faster rate at a flow rate of 0.03 l/hr for bed height of 10 mm and at slower rate for the bed height 20mm, it was found that the saturation time was around 40 min and 60 min respectively. The experimental investigations inferred that adsorption could be effectively carried out at a higher bed height.

Breakthrough analysis
The effect of effluent flow rate on the experimental and predicted breakthrough curve at Q= .03 lit/hr is shown in figure. Figure show that the constant pattern wave approach obtained with a constant liquid phase driving force fits the experimental breakthrough curves. It was found that the half time was inversely proportional to the effluent flow rate and also directly proportional to the bed depth. Many correlations are available to explain Sherwood number as a function of Reynolds and Schmidt number. A review of the correlations suggests that the above equations give best predictions of the individual mass transfer coefficients.

OBSERVATIONS AND DISCUSSIONS

FIG. Scanning electron microscope images of magnification 50 m and 20 m Of alumina.

X RAY DIFFRACTION

Graph showing the X ray diffraction

THIN LAYER CHROMATOGRAPHY

Continuous adsorption column Characteristics

Experimental Condition Bed void fraction Bed void density kg/m3 Bed depth mm Flow rate l/hr Run 1 0.466 6.3 10 .03 Run 2 0.466 6.3 20 .03

Breakthrough analysis
The effect of effluent flow rate on the experimental and predicted breakthrough curve at Q= .03 lit/hr is shown in figure. Figure show that the constant pattern wave approach obtained with a constant liquid phase driving force fits the experimental breakthrough curves. It was found that the half time was inversely proportional to the effluent flow rate and also directly proportional to the bed depth. Many correlations are available to explain Sherwood number as a function of Reynolds and Schmidt number. A review of the correlations suggests that the above equations give best predictions of the individual mass transfer coefficients.

CONCLUSION
The amylase was extracted from tinospora cordifolia. The adsorption process where nano alumina was used as a adsorbent material purifies amylase to standards nearly equal to that obtained from ion exchange chromatography. Also the isotherms show that it is feasible to scale up the process. The conventional method of characterizing the purified product using SDS PAGE was not followed here, but instead the protein activity was studied and results were compared with those obtained when ion exchange chromatography was used to purify the amylase.

REFERENCES
Shilpa Tripathi, Vineet K Rathore, Jahnavi Gokhale and Parag Agrawal. Some study on alpha-amylase in Leather Industry International Journal of Applied Engineering and Technology 2012 Vol. 2 (2) April-June, pp.24-30. Gassenschmidt U, Janny KK, Tauscher B and Niebargell H (1995), Isolation and characterization of flocullating protein from tinospora cordifolia. BBA Biochemica et Biophysica Acta 1234: 477-484. K. A. Ghebremichael, K. R. Gunaratna and G. Dalhammar Single-step ion exchange purification of alpha-amylase from Tinospora cordifolia , Applied Microbiology and Biotechnology, Volume 70, Number 5 / May, 2006 ZHAO Wei YANG De-an SONG Jian-jing YANG Zi LIANG Chong, Influence of TAC on Dispersion of Aqueous Nano Alumina Suspension, Journal of Wuhan University of Technology, Volume 164, Issues 2-3, 30 May 2009, Pages 713-719 Roberta Brayner The toxicological impact of nanoparticles, Nanotoday, volume 3, Feb 2008 .

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