**2.5 Dialysis**

The ammonium sulphate precipitated enzyme of each isolate was dialyzed using acetylated dialysis tubings (Visking dialysis tubings, Sigma) [25] and a multiple dialyser (Pope Scientific Inc. Model 220, USA). The enzyme preparation was dialyzed using 0.2 M citrate phosphate buffer (pH 5.0) at 4°C for 24 hours under several changes of the buffer. The protein content of the dialyzed enzyme was afterwards determined using the Lowry *et al.* [22] method. Polygalacturonase activity of the dialyzed enzyme was also determined [23, 24].

#### **2.6 Enzyme assay**

## *2.6.1 Endo-polygalacturonase (PG)*

Reaction mixture consisted of 1 ml of 0.1% (w/v) pectin in 0.2 M citrate phosphate buffer (pH 5.0) as substrate and 0.5 ml enzyme. Controls consisted of only 1 ml of substrate with no enzyme added. The contents of both experimental and control tubes were incubated at 35°C for 1 hr. The reaction in each tube was terminated with 3 ml of dinitrosalicylic acid reagent (Appendix 1). Thereafter, 0.5 ml of enzyme was added to the controls. The contents of tubes were then boiled for 15 minutes. Optical density readings were taken at 540 nm using a colorimeter. The total reducing sugars released in the reaction mixtures was determined by the Dinitrosalicylate (DNSA) method [23, 24]. One unit of endo-polygalacturonase activity was defined as the amount enzyme in 1 ml of reaction mixture which released reducing sugars equivalent to 100 μg galacturonic acid per minute under specified assay conditions. Specific activity was expressed as enzyme units per mg protein.

### **2.7 Determination of protein concentration**

Using the Lowry *et al*. [22] method, protein concentration was determined at every stage of the investigation. Reaction was with copper in the presence of alkali (Appendix 1).

**165**

*Characterisation of Endo-Polygalacturonases Activities of Rice (*Oryza sativa*) Fungal Pathogens…*

Endo-Polygalacturonase from *Lasiodiplodia theobromae* and *Rhizoctonia solani* were subjected to further purification by gel filtration using a Sephadex G-100 column and ion-exchange chromatogeaphy using CM-Sephadex C-25 and CM-Sephadex C-50 columns. The Sephadex G-100 column was calibrated with

The effects of temperature, pH, substrate concentrations, certain cations and specific inhibitors on the activity of the purified endo-polygalacturonases from

The substrate used was 0.1% (w/v) pectin (Sigma-Aldrich, USA) in 0.2 M citrate phosphate buffer (pH 5.0). The reaction mixture consisted 1 ml of substrate and

The effect of heat (80°C) on the stability of the purified endo-polygalacturonase at different periods, 5, 10, 15, 20 and 25 minutes was carried out. The activity of the heated endo-polygalacturonase was determined by incubating 0.5 ml of enzyme plus 1 ml of 0.1% (w/v) pectin (Sigma-Aldrich, USA) in citrate phosphate buffer

The substrate used was 0.1% (w/v) pectin (Sigma-Aldrich, USA) in 0.2 M citrate phosphate buffer at different pH values ranging from pH 4.0–8.5. The reaction mixture consisted 1 ml of substrate and 0.5 ml of enzyme. Incubation was performed at

Concentrations which were 0.05–0.3% (w/v) pectin (Sigma-Aldrich, USA) constituted in 0.2 M citrate phosphate buffer (pH 5.0) were used as substrate in this investigation. The reaction mixture consisted 1 ml of substrate and 0.5 ml of

The cations NaCl and CaCl2 were used at different concentrations (5, 10, 15, 20 and 30 mM) for the activity of the purified endo-polygalacturonase. Each cation was constituted in 0.1% pectin in citrate phosphate buffer (pH 5.0. The reaction mixture was 1 ml of substrate and 0.5 ml of enzyme incubated at 35°C for 1 hr.

2,4-Dinitrophenol and ethylenediamine tetraacetic acid were the inhibitors used in this investigation. They were prepared at concentrations of 2, 4, 6, 8 and 10 mM

**2.8 Fractionation of enzyme using gel filtration and ion-exchange** 

**2.9 Characterisation of the purified endo-polygalacturonases**

*Lasiodiplodia theobromae* and *Rhizoctonia solani* were investigated.

0.5 ml of enzyme. Incubation was at a range of 5-70°C for 1 hr.

*DOI: http://dx.doi.org/10.5772/intechopen.94763*

proteins of known molecular weight [26, 27].

**chromatography**

**2.10 Effect of temperature**

**2.11 Heat stability test at 80°C**

(pH 5.0) substrate at 35°C for 1 hr.

enzyme, incubated at 35°C for 1 hr.

**2.15 Effects of certain inhibitors**

**2.14 Effects of cations**

**2.13 Effect of concentrations of substrate (pectin)**

**2.12 Effect of pH**

35°C for 1 hr.

*Characterisation of Endo-Polygalacturonases Activities of Rice (*Oryza sativa*) Fungal Pathogens… DOI: http://dx.doi.org/10.5772/intechopen.94763*

### **2.8 Fractionation of enzyme using gel filtration and ion-exchange chromatography**

Endo-Polygalacturonase from *Lasiodiplodia theobromae* and *Rhizoctonia solani* were subjected to further purification by gel filtration using a Sephadex G-100 column and ion-exchange chromatogeaphy using CM-Sephadex C-25 and CM-Sephadex C-50 columns. The Sephadex G-100 column was calibrated with proteins of known molecular weight [26, 27].

#### **2.9 Characterisation of the purified endo-polygalacturonases**

The effects of temperature, pH, substrate concentrations, certain cations and specific inhibitors on the activity of the purified endo-polygalacturonases from *Lasiodiplodia theobromae* and *Rhizoctonia solani* were investigated.

#### **2.10 Effect of temperature**

*Grain and Seed Proteins Functionality*

polygalacturonase activity [23, 24].

**2.4 Precipitation using ammonium sulphate**

Polygalacturonase activity was also determined [23, 24].

the dialyzed enzyme was also determined [23, 24].

**2.7 Determination of protein concentration**

**2.3 Enzyme extraction**

**2.5 Dialysis**

**2.6 Enzyme assay**

*2.6.1 Endo-polygalacturonase (PG)*

shaking at 30°C. Protein content was determined [22].

per ml of each isolate. These were the experimental flasks. Control flasks contained un-inoculated medium. Experimental and control flasks were incubated without

Contents of flasks were carefully filtered through glass fibre filter paper (Whatman GF/A) on the tenth day of inoculation of growth medium. Protein content of the filtrates was determined [22]. The filtrates were also assayed for

The crude enzymes of the isolates was treated with ammonium sulphate (analytical grade, Sigma) within 40–90% saturation. Precipitation was at 4°C for 24 hours. Centrifugation was done at 4000 rpm for 30 minutes at 4°C using a high speed cold centrifuge. The precipitate was re-constituted in 0.2 M citrate phosphate buffer (pH 5.0). Protein content of the precipitated enzyme was determined [22].

The ammonium sulphate precipitated enzyme of each isolate was dialyzed using acetylated dialysis tubings (Visking dialysis tubings, Sigma) [25] and a multiple dialyser (Pope Scientific Inc. Model 220, USA). The enzyme preparation was dialyzed using 0.2 M citrate phosphate buffer (pH 5.0) at 4°C for 24 hours under several changes of the buffer. The protein content of the dialyzed enzyme was afterwards determined using the Lowry *et al.* [22] method. Polygalacturonase activity of

Reaction mixture consisted of 1 ml of 0.1% (w/v) pectin in 0.2 M citrate phosphate buffer (pH 5.0) as substrate and 0.5 ml enzyme. Controls consisted of only 1 ml of substrate with no enzyme added. The contents of both experimental and control tubes were incubated at 35°C for 1 hr. The reaction in each tube was terminated with 3 ml of dinitrosalicylic acid reagent (Appendix 1). Thereafter, 0.5 ml of enzyme was added to the controls. The contents of tubes were then boiled for 15 minutes. Optical density readings were taken at 540 nm using a colorimeter. The total reducing sugars released in the reaction mixtures was determined by the Dinitrosalicylate (DNSA) method [23, 24]. One unit of endo-polygalacturonase activity was defined as the amount enzyme in 1 ml of reaction mixture which released reducing sugars equivalent to 100 μg galacturonic acid per minute under specified assay conditions. Specific activity was expressed as enzyme units per mg

Using the Lowry *et al*. [22] method, protein concentration was determined at every stage of the investigation. Reaction was with copper in the presence of alkali

**164**

protein.

(Appendix 1).

The substrate used was 0.1% (w/v) pectin (Sigma-Aldrich, USA) in 0.2 M citrate phosphate buffer (pH 5.0). The reaction mixture consisted 1 ml of substrate and 0.5 ml of enzyme. Incubation was at a range of 5-70°C for 1 hr.

#### **2.11 Heat stability test at 80°C**

The effect of heat (80°C) on the stability of the purified endo-polygalacturonase at different periods, 5, 10, 15, 20 and 25 minutes was carried out. The activity of the heated endo-polygalacturonase was determined by incubating 0.5 ml of enzyme plus 1 ml of 0.1% (w/v) pectin (Sigma-Aldrich, USA) in citrate phosphate buffer (pH 5.0) substrate at 35°C for 1 hr.

#### **2.12 Effect of pH**

The substrate used was 0.1% (w/v) pectin (Sigma-Aldrich, USA) in 0.2 M citrate phosphate buffer at different pH values ranging from pH 4.0–8.5. The reaction mixture consisted 1 ml of substrate and 0.5 ml of enzyme. Incubation was performed at 35°C for 1 hr.

#### **2.13 Effect of concentrations of substrate (pectin)**

Concentrations which were 0.05–0.3% (w/v) pectin (Sigma-Aldrich, USA) constituted in 0.2 M citrate phosphate buffer (pH 5.0) were used as substrate in this investigation. The reaction mixture consisted 1 ml of substrate and 0.5 ml of enzyme, incubated at 35°C for 1 hr.

#### **2.14 Effects of cations**

The cations NaCl and CaCl2 were used at different concentrations (5, 10, 15, 20 and 30 mM) for the activity of the purified endo-polygalacturonase. Each cation was constituted in 0.1% pectin in citrate phosphate buffer (pH 5.0. The reaction mixture was 1 ml of substrate and 0.5 ml of enzyme incubated at 35°C for 1 hr.

#### **2.15 Effects of certain inhibitors**

2,4-Dinitrophenol and ethylenediamine tetraacetic acid were the inhibitors used in this investigation. They were prepared at concentrations of 2, 4, 6, 8 and 10 mM

in 0.1% pectin (Sigma-Aldrich, USA) in citrate phosphate buffer at pH 5.0. These were the substrates.
