**1.2 Some industrial enzymes and their applications**

Proteases, amylases, cellulases and lipases are used in the preparation of detergents and that is the greatest industrial market. Enzymes are added to increase wash efficiency. For so, they require lower usage temperatures, reduce wash periods and agitation costs. They act on both proteic and starchy residuals as well as on fats; they enhance clothes softness and restore color brightness. They require, in general, suitable thermostability and activity in alkaline pH.

Animal feed – the addition of cellulases, xylanases, proteases, lipases, ligninases and phytases in ruminants and monogastrics foods leads to the digestibility of grass and forage, reduces pollutants, decreases the release of carbon dioxide, soluble carbon hydrates and phosphorus. β-glucans increase food viscosity decreasing starch digestibility, but the addition of β-glucanases increases food assimilation, resulting in weight gain (Facchini et al., 2011a, 2011b).

Swine and poultry feed needs the addition of phytase, a phosphatase that acts on acid or alkaline media and dephosphorylates phytic acid releasing phosphorus to the environment (Haefner et al., 2005). That can damage the environments where the soil contains plenty of phosphate, as it is observed in Europe.

Xylanases and ligninases may also take part in the biobleaching of the cellulose pulp for the manufacturing of paper, whereas cellulases are used for the modification of the textile fiber properties, giving them the pre-wash effect. Those three enzymatic systems participate in the sugar cane hydrolysis for the bioethanol manufacturing. Such procedure has been widely adopted in many countries like Brazil, which has a number of flexpower vehicles (Betini et al., 2009; Michelin et al., 2009, 2011).

objective of any biological transformation is to obtain a short-term high conversion of substrate into product. Besides this characteristic, enzymes present a high specificity and selectivity, according to their metabolic role, acting in optimum conditions of pH and temperature. Also, they do not pose damage to the environment because they are

Applied biological catalysis had its origins in ancient China and Japan, in the manufacturing of foods and alcoholic beverages in which amylases and proteases of vegetal and microbial origins were employed. It dates back to the end of the XIX century with the introduction of standardized preparations of rennilases in the production of cheese. After this period, the implementation of new industrial applications of enzymes was slow, arising intensively in

The main reasons for the current importance of enzymes in the industrial scenario are due to the development of application processes of proteases in detergents, pectinases in juices and glucoamylase in the production of glucose from starch. Additionally, the employment of recombinant DNA techniques allowed the obtainment of high productivity and the most

Following, we present the application of enzymes in several technical sectors justifying the development of methods for the visualization of enzymatic activities in electrophoresis gels.

Proteases, amylases, cellulases and lipases are used in the preparation of detergents and that is the greatest industrial market. Enzymes are added to increase wash efficiency. For so, they require lower usage temperatures, reduce wash periods and agitation costs. They act on both proteic and starchy residuals as well as on fats; they enhance clothes softness and restore color brightness. They require, in general, suitable thermostability and activity in

Animal feed – the addition of cellulases, xylanases, proteases, lipases, ligninases and phytases in ruminants and monogastrics foods leads to the digestibility of grass and forage, reduces pollutants, decreases the release of carbon dioxide, soluble carbon hydrates and phosphorus. β-glucans increase food viscosity decreasing starch digestibility, but the addition of β-glucanases increases food assimilation, resulting in weight gain (Facchini et al.,

Swine and poultry feed needs the addition of phytase, a phosphatase that acts on acid or alkaline media and dephosphorylates phytic acid releasing phosphorus to the environment (Haefner et al., 2005). That can damage the environments where the soil contains plenty of

Xylanases and ligninases may also take part in the biobleaching of the cellulose pulp for the manufacturing of paper, whereas cellulases are used for the modification of the textile fiber properties, giving them the pre-wash effect. Those three enzymatic systems participate in the sugar cane hydrolysis for the bioethanol manufacturing. Such procedure has been widely adopted in many countries like Brazil, which has a number of flexpower vehicles

biodegradable.

the last 40 years.

alkaline pH.

2011a, 2011b).

phosphate, as it is observed in Europe.

(Betini et al., 2009; Michelin et al., 2009, 2011).

suitable design of enzymes.

**1.2 Some industrial enzymes and their applications** 

Proteases and lipases also have a role in the dairy industry, acting in the production of cheese (Gupta et al., 2002). Chemokine, extracted from the stomach of calves, acts in the Milk coagulation, leading to the formation of cheese. Lipases give the aroma and hot flavor in cheese (Hasan et al., 2006). Still in the food industry, pectinases and cellulases increase the extraction of oils through pressing (coconut, sunflower, soybean, olive, etc.).

One sector that has been widely economically explored is the application of amylases in processes of starch saccharification (Silva et al., 2009a, 2009b). For so, there is the need of several enzymes such as α-amylase, which forms maltooligosaccharides; glucoamylases and β-amylases, which hydrolyze starch to glucose and maltose and the glucosyltransferases with the production of cyclodextrins. With synergistic action of the amylolytic system, there is the production of maltose syrup used in breweries, as well as the glucose syrup which is preferably converted by glucose isomerase to fructose syrup, due to the high sweetening power. Such compounds may be used in the manufacturing of sauce, child feeding, gums, candies, ice-cream, pharmaceutical products, canned products etc. Amylases also act in the textile industry to remove starch added to cotton to increase resistance (Gupta et al., 2003).

In the pharmaceutical industry, the use of enzymes is increasingly growing, reflecting the *in vivo* catalysis potential. We can highlight the use of pancreatin, obtained from the swine pancreas, which is used as adjuvant in the digestive process of people who have genetic disorders leading to digestive problems or who, due to surgical removal of the pancreas or precocious aging, present digestive problems. Many enzymes are used as therapeutical agents, such as asparaginase and glutaminase, collagenase, hyaluronidase, ribonuclease, streptokinase, uricase and uroquinase (Prakashan, 2008).

Semi synthetic penicillins (ampicillin and amoxicillin) were launched in the market to replace penicillin, given the acquired resistance of some microorganisms. All kinds of penicillin have the same basic structure: 6-aminopenicillanic acid (6-APA), a thiazolidine ring bound to a beta lactam that takes a free amino group. In the synthesis of semi synthetic penicillin there is the enzymatic hydrolysis of G penicillin with the penicillin G acylase. After purified and concentrated, the 6-APA released in the hydrolysis is used as an intermediary in the synthesis of amoxicillin or ampicillin (Cabral et al., 2003).

The enzymes can be used in the cosmetics industry in creams against skin aging and acne, buffing cream, oral hygiene and hair dying.

Enzymes may be used in analytical applications, due to their high specificity, identifying substances in complex mixtures such as blood, urine and other biological fluids. They participate in tests for glucose (glucose oxidase), urea, amino acids, proteins, ethanol, etc (Godfrey & West, 1996).

In fine chemistry, the list of compounds produced by enzymatic biocatalysis is huge, and we highlight, as an example, vitamin C and several L-amino acids. Acrylamide is used as a monomer in the production of polyacrylamide, widely used as flocculating polymer. Acrylamide was initially produced chemically, but the technological disadvantages such as the formation of toxic residuals and the costs in the purification process made the enzymatic way a viable process.

Gel Electrophoresis for Investigating Enzymes with Biotechnological Application 101

iii. For the detection of activity in electrophoresis gel, the run must take place in low

iv. Attention must also be paid to the native load of proteins and the separation must depend only on its molar mass. For so, proteins may be mixed with SDS, becoming

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

> Tris-HCl 9.75 g HCl (1 M) 12 mL TEMED 0.05 mL Distilled water 25 mL

> Bis-acrylamide 0.32 g Distilled water 20 mL

Ammonium persulfate 0.007 g Distilled water 5 mL

through the action of solvents.

**2.3 Electrophoresis separation techniques** 

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

 **Dye**

temperatures, such as a refrigerator or a cold chamber.

negatively loaded, as it has already been described.

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

volumes to obtain PAGE gels with different concentrations.

**Solution A** 

**Solution C** 

**Solution G**

Glycerol

pH adjusted for pH 8.7

Acrylamide 9.6 g

pH adjusted for pH 8.9

Bromophenol blue

**Running buffer**  Tris-HCl 50mM glycine 36mM, pH 8.9

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
