**2.2 Preparation of the sample for application in electrophoresis**

The preparation of proteic solutions for the application in electrophoresis is an important phase. It is important to highlight once again that most enzymes used industrially have microbial origin (fungi, yeasts or bacteria) and also that normally, their synthesis is followed by the elimination of a number of primary and secondary metabolites produced by the very microorganisms, as well as other compounds present in the cultivation medium, such as vitamins, salts, carbohydrates, amino acids and peptides.

In order to avoid the interference of such factors in the electrophoresis, especially when there is the application of enzymatic extracts without previous purification, it is necessary to pay close attention to the type of sample that is being prepared.

Below are some measures and precautions that must be adopted:


necessary, such as lyophilization, use of filtering membranes with defined molar mass, ammonium sulfate precipitation and even the use of solvents (ice acetone or ethanol). We must bear in mind that those two last processes need an additional dialysis for the removal of the ammonium sulfate or carbohydrates when the precipitation happens through the action of solvents.


### **2.3 Electrophoresis separation techniques**

100 Gel Electrophoresis – Advanced Techniques

The detection of enzymatic activities for industrial use, in electrophoresis gel, happens when PAGE (polyacrylamide gel electrophoresis) is employed, which is an electrophoresis performed in non-denaturing conditions. The Figure 1 illustrates some steps used to

In this kind of procedure, there is not preferably the addition of the sodium dodecyl sulfate – SDS detergent, β-mercaptoethanol or another reducing agent, such as dithiothreitol - DTT or urea. Also, the protein samples in its native form (not denaturated) are not boiled before their application in the gel because enzymes will lose its activities, if denaturated. The enzymatic activity may also be detected in gels of the SDS-PAGE type, if the samples were not boiled or added by any reducing agent that denatures the protein. Sodium dodecyl sulfate (SDS) is an anionic surfactant whose role is to bestow the proteins with uniform load density. SDS presents a high negative load and a hydrophobic tail that interacts with the polypeptidic chains in an approximated ratio of 1.4 g of SDS for each gram of protein, making them negatively loaded. In the lack of SDS, the proteins with equal mass may migrate differently in the pores of the gel due to the load differential of their tridimensional

PAGE may be performed in a pH 4.5 or 8.9, depending on the isoelectric point - pI, of the sample under study. In order to accomplished zymograms, it is performed SDS-PAGE; however, the samples generally correspond to a crude extract or a partial purified extract

The preparation of proteic solutions for the application in electrophoresis is an important phase. It is important to highlight once again that most enzymes used industrially have microbial origin (fungi, yeasts or bacteria) and also that normally, their synthesis is followed by the elimination of a number of primary and secondary metabolites produced by the very microorganisms, as well as other compounds present in the cultivation medium, such as

In order to avoid the interference of such factors in the electrophoresis, especially when there is the application of enzymatic extracts without previous purification, it is necessary to

i. Dialysis: This procedure aims at the removal of substances with smaller molar mass, such as salts, carbohydrates and amino acids, which may interfere in the electrophoresis

ii. Attention to the concentration of the sample applied in the electrophoresis. In general lines, around 10 µg of proteins is necessary for a good visualization in the electrophoresis gel after the dying phase. For the detection of the enzymatic activity, considerable enzymatic levels are necessary. If the protein solution presents a lower concentration, the application of any procedure for the concentration of proteins is

which are either not boiled or added by β-mercaptoethanol, DTT or urea.

**2.2 Preparation of the sample for application in electrophoresis** 

vitamins, salts, carbohydrates, amino acids and peptides.

pay close attention to the type of sample that is being prepared. Below are some measures and precautions that must be adopted:

**2.1 Preparation of polyacrylamide gel electrophoresis for activity enzymatic** 

**2. Some industrial enzymes and their applications** 

preparation of the electrophoresis gel.

structures.

quality;

#### **2.3.1 Electrophoresis in non-denaturing conditions (PAGE)**

Electrophoresis is going to be performed in pH (4.5 or 8.9), in a polyacrylamide gel that may range from 5 to 15%, depending on the size and the load of the protein under study. For proteins loaded negatively, the running buffer will consist of Tris-HCl and glycine, pH 8.9. For proteins loaded positively, there is going to be a buffer with β-alanine and glacial acetic acid, pH 4.5. Both procedures must be performed at 4ºC. Table 1 indicates necessary volumes to obtain PAGE gels with different concentrations.

#### **2.3.1.1 PAGE for acid proteins (-), (Davis, 1964)**


Gel Electrophoresis for Investigating Enzymes with Biotechnological Application 103

Add 50 µL of ammonium persulfate 10% and quickly put the mixture in the

 Wait until the gel solidifies and apply the samples (up to 30 μL) in different lanes; Connect the energy cables in the respective jacks of the energy source adjusted to 70

Turn on the source and wait for the samples to run throughout the gel before turning it off;

The electrophoresis must be performed in pH 8.9 and in the presence of SDS (sodium dodecyl sulfate), with the gel concentration ranging from 5 to 15%. Table 2 indicates

Adjust the pH with HCl concentrated for pH 8.9 and store it at 4-

Store at 4-6°C in a glass flask with Amberlite resin due to the

Dissolve with 17.5 mL of distilled H2O and adjust the pH to 6.75

Dissolve in an amount of distilled water sufficient for 1000mL,

degradation of acrylamide in acid and ammonia.

Tris-HCl 36.5 g TEMED 230 μL Distilled water 9 mL

Acrylamide 28 g Bis-acrylamide 0.74 g Distilled water 100 mL

SDS 0.21 g Distilled H2O 100 mL

with concentrated HCl and add: SDS 2 g Bromophenol blue 0.001 g Distilled water 100 mL Freeze aliquots for a further use.

Tris 3.025 g glycine 14.4 g SDS 1.0 g

Store at room temperature.

necessary volumes to obtain SDS-PAGE gels with different concentrations.

**Solution A** 

**Solution C** 

**Solution E** 

**Sample buffer** Tris-HCl 0.755 g Glycerol 1 mL

**Run Buffer** 

pH 8.9.

6°C.

electrophoresis bowl;

mAmps and 120 Volts;

Remove the gel carefully and process it.

**2.3.2 Electrophoresis gel SDS-PAGE, Laemmli (1970)** 

Place the comb;

Preparation of the samples and markers:

