Supercritical Fluids Technology in Lipase Catalyzed Processes

Sulaiman Al-Zuhair, Hanifa Taher

• Publisher: CRC Press
• Year: 2018
• Print ISBN: 9781498743877
• eBook ISBN: 9781351231428
• Topic: Biological Sciences
• Subtopic: Biotechnology
• Index: Biological Sciences

Enzymes are currently used in various industries, most commonly in food, detergents, and pharmaceuticals production. Lipases are hydrolytic enzymes that demonstrate great potential as an alternative to conventional catalysts in a number of industrial applications. A complete understanding of enzymes, and their proteins structure and environmental behavior, can greatly aid in the further development of industrial applications. Supercritical Fluids Technology in Lipase Catalized Processes provides basic information about enzymes, their sources, reaction kinetics, and main industrial applications. The book focuses in lipases. their main sources, structure, and features, with an emphasis on their specificity and interfacial activity, and presents proven techniques for isolating, extracting, and purifying.

Comprised of six compact chapters, this comprehensive guide introduces:

• Immobilization techniques and immobilized lipases that allow repeated use (which is essential from an economic point of view)
• Different bioreactor configurations using immobilized lipases
• The latest information on the available technologies in lipolytic reactions
• The advantages of nonaqueous media in biochemical synthesis over aqueous and solvent-free systems
• Material on the use of lipases in nonaqueous media to overcome the drawbacks usually encountered with the use of conventional chemical catalysts
• The use of supercritical fluids (SCFs) as a green alternative reaction medium
• Factors affecting the physical properties of lipases in this medium and, hence, their activity and stability
• A case study using supercritical carbon dioxide (SC-CO2) for biodiesel production
• Novel, cutting-edge technology, using immobilized enzymes to reduce the overall production cost

Supercritical Fluids Technology in Lipase Catalized Processes

outlines the main industrial applications of common enzymes and discusses relevant challenges and innovations emerging in the field.

1	Enzymes Fundamentals

Enzymes are catalysts of biological sources. Like other catalysts, they bring reactions to equilibrium more quickly than would occur in their absence. They are proteins and, therefore, understanding enzymes requires an understanding of protein structure and their environmental behavior. This chapter covers basic information about enzymes, and discusses their sources and main features before introducing their classifications based on International Union of Biochemistry and Molecular Biology (IUBMB) standards. Techniques for isolating, extracting, and purifying enzymes are also presented. The chapter concludes with the main industrial applications of common enzymes and genetic protein engineering.

1.1 CATALYSTS AND ENZYMES

Chemical reactions usually require energy, in the form of heat, to make particles move rapidly and increase the collusion frequency between them. The energy required to convert one mole of reactant molecules from their stable state to a transition state is known as the activation energy. As molecules collide with a proper orientation, some bonds are broken and others are formed. The required energy can be lowered by adding catalysts, and, thus, reaction rates are enhanced.

Catalysts are substances that alter the rate of a reaction without being consumed during the reaction. Generally, catalysts increase the rate of reactions by providing alternative and faster paths through which the reaction can proceed. In every chemical reaction, reactants absorb energy and pass through a transitional state between the reactants and the products, thus forming intermediate compounds. These intermediate compounds immediately decompose to give the end product. The catalyst’s role is to reduce the energy required to reach a transitional state by bringing the reactants closer and to allow them to collide more effectively. However, catalysts do not affect the reaction equilibrium. This means that they accelerate both forward and backward reactions by the same factor. Figure 1.1 shows typical reaction progress in the presence and absence of a catalyst. As can be seen, using a catalyst reduces the energy barrier between the reactants and the transition state.

Although chemical catalysts are important and have many industrial applications, their use usually has negative environmental and economic impacts. In addition, they usually result in an increased number of by-products, which must be separated from the desired end product.

Alternatively, enzymes, which are biocatalysts, have many favorable properties compared to conventional chemical catalysts. Enzymes are large proteins that are made up of a sequence of 20 naturally occurring amino acid residues to form a chain connected by peptide bonds, shown in Figure 1.2. As with chemical catalysts, enzymes alter the reaction by providing different pathways and operate effectively in small amounts (Lorenz and Eck, 2005). It has been reported that enzymes are capable of enhancing reaction rates by up to 10⁸- to 10¹⁰-fold (Freifelder and Malacinski, 1993). Table 1.1 shows the enzyme’s effect in reducing the activation energy required for different reactions. The table clearly shows that enzymes have a stronger effect than chemical catalysts.

FIGURE 1.1 Effect of catalysts on activation energy.

FIGURE 1.2 Peptide bond in a protein.

In addition, enzymes are much more specific and can produce only the desired end products without any side effects. They also work well in moderate pH (pH 5 to 8) and temperatures (20°C to 40°C), which makes them less hazardous and less energy intensive.

Typically, enzyme active sites consist of 3 to 12 amino acid residues structured into specific three-dimensional arrangements, as shown in Figure 1.3 for the Candida rugosa enzyme. These active sites have a strong attraction to substrates, where amino acid residues complement certain groups on the substrates. Regiospecificity and stereospecificity are other important characteristics of enzymes, where they are not only specific or selective for certain substrates but also discriminate between similar parts of molecules or optical isomers. For examples, some enzymes, like lipase, can differentiate between stereoisomers that have the same structure but a different atom arrangement. Details of lipase stereo- and regiospecificity are discussed in Chapter 2.

TABLE 1.1
Effect of Enzyme Use on Reaction Activation Energy

Reaction	Catalyst	Activation Energy (cal/mol)	Reference
Hydrogen peroxide decomposition	Without catalyst Platinum surface	18,000 11,700	Campbell and Farrell, 2011; Spencer et al., 2010
	Potassium iodide	13,500
	Catalase	5500
Ethyl butyrate hydrolysis	Hydrochloric acid	16,800	Steward and Bidwell, 1991
	Lipase	4500
Sucrose hydrolysis	Hydrochloric acid	26,000	Goss, 1973
	Invertase	13,000
Casein hydrolysis	Hydrochloric acid	20,600	Braverman and Berk, 1976
	Lipase	12,000

FIGURE 1.3 Three-dimensional structure of Candida rugosa enzyme. (From Cygler, M., and J. D. Schrag, 1999, Biochimica et Biophysica Acta (BBA)—Molecular and Cell Biology of Lipids 1441 (2–3):205–214. With permission.)

Enzymes, on the other hand, have several disadvantages such as high cost of production, isolation, and purification. It has been reported that in enzyme production, 45% of the costs are associated with enzyme recovery, whereas only 14% are attributed to the fermentation process (Fish and Lilly, 1984). Enzymes production and purification are discussed in Section 1.4.

1.2 SOURCES OF ENZYMES

Enzymes can be obtained from different sources such as in the pancreas and liver, plants, and microbial organisms (such as bacteria, fungi, and yeast). Historically, enzymes were initially obtained from animals such as pigs and cows, then from plants, and finally from microorganisms (Panesar et al., 2010; Shanmugam, 2009).

TABLE 1.2
Common Microbial Enzymes and Their Industrial Uses

Microbial enzymes are extracted from fermented bacteria or fungal organisms. Recently, microbial enzymes have been used with increasing frequency. This is because microorganisms can be easily and quickly grown on a large scale. They are relatively inexpensive and provide a continuous and reliable supply as compared to animal and plant sources. Microbial enzymes represent about 90% of all commercially produced enzymes (Godfrey and West, 1996; Margesin et al., 2008). The best known sources of microbial enzymes are Rhizopus nerveus, Bacillus licheniformis, Aspergillus oryzae, and Aspergillus niger. Table 1.2 shows examples of important microbial enzymes, their sources, and industrial uses. Further details on these industrial uses are discussed in Section 1.5.

1.3 CLASSIFICATION OF ENZYMES

An enzyme is often named by adding the suffix -ase to the name of the substrate it works on in nature. For example, the first enzyme discovered was urease, which catalyzes the hydrolysis of urea producing ammonia and carbon dioxide. However, some enzymes have been given uninformative names such as catalase, which catalyzes hydrogen peroxide to water and oxygen. The IUBMB has adopted standards for enzyme nomenclature according to the type of reactions they catalyze and the substrates acted upon.

Six types of reactions catalyzed by enzymes have been identified, and accordingly the enzymes have been classified into six main groups. These are oxidoreductases, transferases, hydrolases, lyases, isomerases, and ligases. These six groups and their respective main functions are summarized in Table 1.3. Of the six groups in the Table 1.3, only the hydrolases have a significant industrial function (Galante and Formantici, 2003), because enzymes of this type can be easily extracted and isolated. They are also capable of working without the need for a coenzyme. Therefore, they can be produced at a relatively low cost and can operate under harsh conditions. Amongst the hydrolysis enzymes that have received a high level of attention are lipases, due to their structural features, favorable properties, and the versatility of the reactions that they catalyze. Details about lipases and respective features are discussed in Chapter 2.

TABLE 1.3
Six Major Classes of Enzymes

Class	Function	Examples
Oxidoreductases	Catalyze oxidoreduction reactions by adding/removing hydrogen bonds	Glucose oxidase, lactate, alcohol dehydrogenase, laccase
Transferases	Transfer of amino, fatty acid, methyl or phosphate functional groups from one molecule to another	Starch phosphorylase, amylosucrase, dextransucrase, levansucrase, aspartate aminotransferase
Hydrolases	Catalyze the hydrolysis of carbohydrates, lipids, proteins, or phosphoric acids esters by breaking single bond and add water across bond	Feruloyl esterases, lipase, chlorophyllase, α-amylases, β-amylases, chymosin
Lyases	Catalyze the breaking/forming of chemical bonds by means other than hydrolysis	Alliinases, cystine lyases, his...

Table of contents

• Cover
• Half Title
• Title Page
• Copyright Page
• Dedication
• Table of Contents
• Preface
• Acknowledgments
• Authors
• Chapter 1 Enzymes Fundamentals
• Chapter 2 Lipases
• Chapter 3 Lipase Immobilization
• Chapter 4 Kinetics of Soluble and Immobilized Enzymes
• Chapter 5 Lipase-Catalyzed Reactions in Nonaqueous Media
• Chapter 6 Lipase-Catalyzed Production of Biodiesel Using Supercritical Technology
• Index