Agricultural Research Management Information System

Home
Research Summary
Search
About Us
- ARMIS
- Brochure
Contact Us
Report
User Request
Data Input
Help
- Operation Manual
  - PDF
  - Video
- Program Area & Commodity
We have reached 37600 number of research entries at this moment.

- Logout

Research Detail

Home
Research
Detail

Purification, Characterization and Effect of Physico-chemical Agents on the Stability of β-galactosidase from Betel Leaves

M S Shovon
Department of Biochemistry & Molecular Biology, University of Rajshahi, Rajshahi-6205, Bangladesh

S C D Sharma
Department of Biochemistry & Molecular Biology, University of Rajshahi, Rajshahi-6205, Bangladesh

N Roy
Department of Biochemistry & Molecular Biology, University of Rajshahi, Rajshahi-6205, Bangladesh

Abstract/ Executive Summary:

Context: β-galactosidase are present in a wide variety of organisms including plants, animals and microorganisms. Exploration of this enzyme from plant source will help to address the problems faced in the food and allied industries that look for enzymes with novel properties. Objectives: The aim of this study is to explore the purification, characterization and analysis of β-galactosidase from betel leaves. Materials and Methods: Aomal Bangla variety of betel leaf (Piper betle Linn.) was collected from betel vine. The column chromatographic method was done at 4°C using conventional method. The protein concentration was determined by UV-spectrophotometer at 280 nm. The activities of β-galactosidase were done by spectrophotometric method. All other reagents used in the study were of analytical grade. Unless specified, all the experimental conditions are maintained at 4°C. Results: After extraction of β-galactosidase from betel leaves, the crude enzyme was applied to DEAE-cellulose chromatography with sodium phosphate buffer (pH 7.0). The active fraction from DEAE-column chromatography was dialyzed with buffer and applied to CM-cellulose chromatography. The β-galactosidase activity from CM-cellulose chromatography was loaded to Sephadex G-75 Gel filtration chromatography. In this column, the enzyme β-galactosidase was eluted in a single peak. The homogeneity of purity was checked by disc gel electrophoresis and a single band was obtained. The optimum pH of β-GS-I, β-GS-II and β-GS-III were 3.5, 3.8 and 4.2, respectively. The optimum temperatures of the enzymes were 53, 51 and 56°C, respectively. Conclusion: The results obtained in this study suggest that three β-galactosidases namely β-GS-I, β-GS-II and β-GS-III were purified from betel leaves. This is the first report of purification and characterization of β-galactosidase from betel leaves.

Keywords: Betel leaves, β-galactosidase, MW, Characterisation, Stability.

Location: Department of Biochemistry & Molecular Biology, University of Rajshahi

Start Date:

End Date:

Program Area: Crop-Soil-Water Management

Commodity: Betel leaf

Objectives:

The aim of this study is to explore the purification, characterization and analysis of β-galactosidase from betel leaves.

Methodology:

Preparation of crude enzyme extract: The betel leaves (Aomal Bangla variety) were collected from Mohanpur upozila of Rajshahi. Fresh leaves (100 g in weigh) cut into small pieces and grinded in a mortar and pestle with 25 ml of cold 50 mM phosphate buffer, pH 7.4 and finally crushed into paste using homogenizer. The temperature was maintained at 4°C by putting ice in the outer chamber of homogenizer. The suspension was then filtered through double layers muslin cloth in the cool chamber. The filtrate collected was further clarified by centrifugation at 10000g for 15 min at 4°C. The clear supernatant was concentrated about 1/8th of the original volume by commercial sucrose at 4°C. Then it was dialyzed against 50 mM phosphate buffer, pH 7.4 for 24 h at 4°C. It was again centrifuged at 7000g for 15 min, to remove insoluble material and the clear supernatant was used as crude enzyme extract. Column chromatographies: The crude enzyme extract was loaded onto the DEAE-cellulose column (Sigma Chemical Co. USA), which was previously equilibrated with 50 mM phosphate buffer, pH 7.4 at 4°C. The protein was eluted from the column with the same buffer by stepwise elution containing increasing concentration of NaCl. Absorbance at 280 nm, protein concentration and β-galactosidase activity of each fraction were measured. The β-galactosidase containing fraction obtained from DEAE-cellulose was applied to CM-cellulose column (Sigma Chemical Co. USA) after dialysis against 0.1M sodium-acetate buffer, pH 5.2. The protein was eluted from the column with the same buffer by stepwise elution containing different concentration of NaCl. Absorbance at 280 nm, protein concentration and β-galactosidase activity of each fraction were measured. The impure enzyme active fraction obtained from CM-cellulose was applied on Sephadex G-75 column (Sigma Chemical Co. USA) after dialysis against 0.1 M Na-acetate buffer, pH 5.2. The protein was eluted from the column by the same buffer. Absorbance at 280 nm, protein concentration and enzyme activity of each fraction were measured. Polyacrylamide disc gel electrophoresis: The purity of the enzyme containing fractions was detected by polyacrylamide disc gel electrophoresis at room temperature on 7.5% gel, pH 8.3 as described by Ornstein (1964). Assay of β-galactosidase activity: β-galactosidase activity was assayed following the modified method as described by Lazan et al. (1993) using Methyl β-D-galactopyranoside as substrate. Amount of reducing sugar released was estimated by dinitrosalicyclic acid (Miller 1972). The β-galactosidase activity wasmeasured by estimating the amount of reducing sugar released from methyl-β-D-galactopyranoside. One unit of β-galactosidase activity was defined as the amount of enzyme that catalyzed the liberation of 1mg of galactose in 15 min at 37°C and the amount of galactose released was calculated from the standard curve prepared with galactose. The concentration of protein was determined following the method of Lowry et al. (1951) using BSA as standard. Determination of molecular weight: The molecular weight of β-galactosidase was determined by gel filtration on Sephadex G-150 as described by Andrews (1964). Catalase, β-galactosidase, carbonic anhydrase and lysozyme were used as marker proteins. The molecular weight of the subunits of β-galactosidases were determined by SDS-polyacrylamide gel electrophoresis as described by Weber and Osborn (1969). Estimation of total sugar, optimum pH and temperature: Total sugar contents of β-galactosidases were estimated by Phenol sulphuric acid method (Dubois 1956). The activities of β-galactosidases against Methyl-β-D-galactopyranoside as substrate was assayed using 0.1M buffers of different pH values (range, 2-10) at 37°C following the procedure as described above. The activities of the β-galactosidases were measured at different temperature (10-90°C) using sodium citrate buffer of pH, 3.6-4.2. At first the enzyme solutions were incubated at different temperature for 15 min and then the activities of the enzyme solutions were measured following the procedure as described above. Treatment with urea, acetic acid, calcium and various salts: To the β-galactosidases solution (in sodium citrate buffer of pH 3.6-4.2) of 0.5ml (0.25-0.30mg/ml), were added urea, calcium, acetic acid (all from BDH, England) and various salts at different concentrations and incubated for 10 min at 20°C. The mixtures were again incubated with the substrate for 15 min at 37°C and the enzyme activity was assayed.

Source of Information: J. bio-sci. 18: 108-115, 2010 ISSN 1023-8654

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

The results of the present study confirm that the activity of β-galactosidase in mature betel leaves is not due to a single β-galactosidase, but to three isomeric forms which can be purified by a combination of ion-exchange chromatography on DEAE- and CM-cellulose followed by gel filtration on Sephadex G-75. We also characterized and analyzed physico-chemical properties of these enzymes. As far we know, this is the first report of purification and characterization of β-galactosidase from betel leaves.

Knowledge Management: Journal