Methods to Determine Enzymatic Activity

Pectinases constitute a group of enzymes widely used by the fruit and textile industries. Besides these industrial applications, these enzymes play an important biological role in protoplast fusion technology and plant pathology. Since the applications of pectinases in various fields are widening are becoming wider, it is important to understand the nature and properties of these enzymes for an efficient and effective application. For the past few years, intensive research has been carried out on isolation and characterization of pectinases. They are a group of enzymes that contribute to the degradation of pectin by various mechanisms and can be classified as esterases, eliminative depolymerases (lyases) and hydrolytic depolymerases (polygalacturonases). This chapter will describe the current state of knowledge of the main assays used to quantify pectinolytic enzymes and their activity.

Peroxidases are enzymes that use various peroxides as electron acceptors in oxidation, and are widespread in nature. Their activities are usually determined by spectrophotometric methods, which a condensation or oxidation product is formed in the presence of H2O2, or by titrimetric methods, using potassium permanganate or sodium thiosulfate, which measure free hydrogen peroxide in solution.

In this chapter methods for the selection of chitinolytic microorganisms is discussed, especially those based on the formation of clear hydrolytic zones due to colloidal chitin degradation. Also the main methods for the detection and measurement of chitinolytic activity are presented, including several assays for exochitinase and endochitinase, based on artificial substrates, as well as those based on reducing sugars from chitinous substrates, such as glycol-chitin or colloidal chitin. In addition, methods for the detection of chitinase in polyacrylamide gel electrophoresis are discussed.

Cellulase enzymes constitute a complex of glycoside hydrolases that are secreted by microorganisms, plants and some animals. These enzymes act on the hydrolysis of β-1,4–glucosidic bonds of the cellulose structure, the major polymer present on biomass. Recent attention has been given on cellulases use in the bioconversion process of lignocellulosic materials into bioethanol and biobased products, within the biorefinery concept. Besides that, cellulases have been largely applied at different industrial areas like textile, food, brewery and wine, animal feed, pulp and paper industries. This chapter will describe some current state of knowledge related to cellulase assays using soluble and insoluble substrates, focusing on the need to standardized methods in order to allow a comparison among different enzyme preparations.

Amylases are some of the most important industrial enzymes. Their family is comprised of enzymes with different specificities against a broad range of substrates. The group of endoamylases, which includes α-amylases, catalyzes the cleavage of internal α-1,4 linkages in amylose and amylopectin structures, releasing dextrins of various lengths. Exoamylases, on the other hand, act preferentially in external regions of the substrate, being represented by several enzymes which act towards molecules as small as maltose (e.g., maltase) until polysaccharides (e.g., glucoamylases). The third group comprises debranching amylases, including pullulanases and isoamylases, which act in α-1,6 bonds (branching linkages). Synergy between these groups of enzymes is crucial for the improvement of product release rate. Therefore, it is important to assess methods for the more specific detection as possible for each main group of amylolytic enzymes, in order to understand their synergy and evaluate potential microbial strains for their production. This chapter contains an overview of the mode of action of the main amylolytic enzymes and presents protocols for the quantification of the activity of the three major groups of amylases.

Xylanases are enzymes which catalyze the hydrolysis of 1,4-β-D-xylosidic linkages in xylan. In the present chapter several techniques concerning xylanase activity are discussed, specially those involving qualitative and quantitative assays, which are powerful tools used in screening, selection and enzyme production by xylanolytic microorganisms. This chapter also presents methodologies for enzyme characterization including optimum of pH and temperature, and detection of xylanases through the use of polyacrylamide gel electrophoresis.

Lipases hydrolyze triacylglycerols to diacylglycerols, monoacylglycerols, fatty acids, and glycerol in their natural environment. However, in non-aqueous media, these enzymes catalyze reverse reactions such as esterification and transesterification. The versatility of the molecular structure and catalytic properties of these enzymes allow them to be used in various industries such as: food, cosmetic, oleochemical, pharmaceutical and detergent. Since lipases have several applications and have been the focus of numerous research studies, this chapter contains the guidelines for screening of microorganisms that produce lipases and for meticulous enzymatic analyses, focusing on the need to standardize methods in order to compare the catalytic efficiency of lipases obtained by different sources in distinct laboratories.

Phenoloxidases are enzymes with broad substrate specificity tward a lot of phenols with several biotechnological applications. There are an expressive amount of methods in literature available for determining their activities. However, spectofotometric methods are much more practical and with very good evaluation by the users. Kinetic studies show that methods that measure the appearance of a product are much better over methods that measure substrate disappearance. Therefore, those kinds of methods are shown here.

Transglutaminases are enzymes capable of catalyzing acyl transfer reactions by introducing covalent cross-links between proteins, as well as peptides and various primary amines. These enzymes are very important due to their abilities to modify proteins and act as biological glues. Nowadays, microorganisms are considered the best source of transglutaminases, due to the difficulties involved in producing them from mammal tissues an industrial scale. In this chapter is described an analytical method for the transglutaminase (TGase) activity assay. Furthermore, the qualitative and quantitative methods for screening microorganisms that produce this enzyme will also be detailed.

Keratinases are an important group of enzymes with many industrial applications, including the processing of feather residues, feather meal production, detergent, leather and in the pharmaceutical industry. In this chapter the main methods used to detect their activity are described and discussed. Methods to detect and quantify keratinase activity include spectrophotometric analyses with multiple substrates, zymography and SDS-PAGE containing protein substrates. Focus is on the current status of these biochemical analytical methods.

The aim of this chapter is to present the modern analytical methodologies used to study peptidases, such as the colorimetric methods and spectrophotometric analyses. A general method for the detection of peptidases activity includes gelatin and other substrates incorporated into SDS-PAGE using a technique known as zymography. All methodologies presented here are important tools for the discovery and characterization of peptidases.

The enzyme tannase (E.C.3.1.1.20), also known as tannin acyl hydrolase, is a hydrolytic enzyme that acts on tannins. Tannase is an inducible enzyme that catalyses the breakdown of ester linkages in hydrolysable tannins such as tannic acid resulting in the production of gallic acid and glucose.Most of this enzyme has been to produce gallic acid, which is used in the production of trimethoprim and synthesis of esters such as propyl gallate, an antioxidant used in food industry. The enzyme is also applied in the processing of beer and clarification of juices, instantaneous teas processing and treatment of wastewater contaminated with phenolic compounds. The tannase activity determination is extremely important in several areas, mainly in the food industry analysis.

Urease, a nickel-dependent metalloenzyme, is synthesized by a wide variety of organisms, including plants, bacteria and fungi. It catalyzes the hydrolysis of urea into ammonia and carbon dioxide. Although the amino acid sequences of plant and bacterial ureases are closely related, some biological activities differ significantly. To date, the structural information is available only for bacterial ureases although the enzyme extracted from jack bean (Canavalia ensiformis), which is the best-studied plant urease, had been crystallized in 1926. Tests for urease activity determination should be based on direct or indirect methods and most of the methods utilized for urease detection are based on analysis of its activity in urea hydrolysis. The urease activity determination is extremely important in several areas, mainly in agriculture, medicine and clinical analysis.