**1. Introduction**

96 Gel Electrophoresis – Advanced Techniques

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Enzymes are vital biological catalysts with essential action in the metabolism of all living beings. Moreover, enzymes have a very significant role in various industrial sectors, including baking, brewery, detergents, textile, pharmaceutical, animal feed, cellulose pulp biobleaching, biofuels and others (Polizeli et al., 2005; 2009; 2011). Industrial enzymes are especially produced from microorganisms, such as bacteria, yeasts and filamentous fungi.

Here, we describe some basic knowledge about microbial enzymes with potential application in industry, their properties and some biochemical methods for the detection of amylase, pectinase, proteases, and xylanase activities on polyacrylamide gel electrophoresis. These methods are more qualitative procedures than quantitative, once they may be used to confirm the electrophoretic homogeneity of purified enzymes through chromatographic process. In addition, we described the principle of each method approached in this chapter, which grants a better understanding of each procedure.

### **1.1 Overall considerations about industrial enzymes**

Enzymes are optimum biological catalysts present in all living beings, and they, under adequate conditions, catalyze in their active sites the natural substrates from the metabolic route reactions. Quite often, the metabolic enzymes act in a sequence, and the product generated in a reaction becomes the substrate for the following phase, as diagramed:

$$\text{Substrate} \, 1 \xrightarrow{\text{enxyzme1}} \text{Product1} + \text{Enzyme} \, 1 \tag{1}$$

$$\text{Substrate} \newline 2 (= \text{Product1}) \xrightarrow{\text{enzyme 1}} \text{Product2} + \text{Enzyme 2} \tag{2}$$

Some properties of the enzymes make them excellent competitors against traditional chemical catalysts, due to their great catalytic efficacy (kcat), considering that the main

Gel Electrophoresis for Investigating Enzymes with Biotechnological Application 99

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,

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

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,

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

The enzymes can be used in the cosmetics industry in creams against skin aging and acne,

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

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

intermediary in the synthesis of amoxicillin or ampicillin (Cabral et al., 2003).

streptokinase, uricase and uroquinase (Prakashan, 2008).

buffing cream, oral hygiene and hair dying.

(Godfrey & West, 1996).

way a viable process.

olive, etc.).

al., 2003).

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 biodegradable.

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 last 40 years.

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 suitable design of enzymes.

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.
