Biocatalysis for Practitioners

Techniques, Reactions and Applications

Gonzalo de Gonzalo, Iván Lavandera, Gonzalo de Gonzalo

• Publisher: Wiley-VCH
• Year: 2021
• Print ISBN: 9783527346837
• eBook ISBN: 9783527824458
• Edition: 1
• Topic: Biological Sciences
• Subtopic: Biochemistry
• Index: Biological Sciences

This reference book originates from the interdisciplinary research cooperation between academia and industry. In three distinct parts, latest results from basic research on stable enzymes are explained and brought into context with possible industrial applications. Downstream processing technology as well as biocatalytic and biotechnological production processes from global players display the enormous potential of biocatalysts. Application of "extreme" reaction conditions (i.e. unconventional, such as high temperature, pressure, and pH value) - biocatalysts are normally used within a well defined process window - leads to novel synthetic effects. Both novel enzyme systems and the synthetic routes in which they can be applied are made accessible to the reader. In addition, the complementary innovative process technology under unconventional conditions is highlighted by latest examples from biotech industry.

Part I
Enzyme Techniques

1
Techniques for Enzyme Purification

Adrie H. Westphal¹ and Willem J. H. van Berkel^1,2

¹ Wageningen University & Research, Laboratory of Biochemistry, Stippeneng 4, 6708WE Wageningen, The Netherlands

² Wageningen University & Research, Laboratory of Food Chemistry, Bornse Weilanden 9, 6708WG, Wageningen, The Netherlands

1.1 Introduction

Biocatalysis is the chemical process through which enzymes or other biological catalysts perform reactions between organic components. Biocatalysis gives an added dimension to synthetic chemistry and offers great opportunities to prepare industrial useful chiral compounds [1, 2]. Depending on the goal of the chemical conversion and the costs involved, biocatalyst‐driven reactions are performed using whole cell systems or isolated enzymes, either in free or immobilized form [3–5].

Initially, industrial applications utilizing isolated enzymes were mainly developed with amylases, lipases, and proteases [6–8]. These hydrolytic enzymes were usually applied in a partially purified form, also because crude enzyme preparations are often more stable than the purified ones. However, for obtaining highly pure products, especially in the pharmaceutical industry, the purity of the enzyme preparation can be a critical factor.

Many enzyme purification methods have been developed over the years. Traditional purification procedures make use of the physicochemical properties of the enzyme of interest. These procedures were developed during the twentieth century for elucidating enzyme mechanisms and solving protein three‐dimensional structures but also appeared to be valuable for the preparation of highly pure biocatalysts. Yet, progress in the preparation of biocatalysts has been given the biggest boost by the amazing developments in recombinant DNA technology and the accompanying revolutionary changes in enzyme production, enzyme purification, and enzyme engineering [9].

Here, we describe our experiences with the contemporary techniques for enzyme purification. For more information about the practical issues of enzyme purification, the reader is referred to the “Guide to Protein Purification” in Methods in Enzymology 463 [10].

1.2 Traditional Enzyme Purification

Before summarizing the traditional enzyme purification methods, it is important to note that the purification of enzymes is made easier by the fact that they are such specific catalysts. This enables the determination of the amount of a given enzyme in units (where 1 unit [U] of enzyme activity is defined as the amount of enzyme that catalyzes the conversion of 1 μmol substrate per minute) and its specific activity (in U mg⁻¹) in crude extracts and after each purification step. The specific activity is a good indication of the purity and quality of the enzyme preparation, especially if the specific activity of the pure enzyme under defined conditions is known. During enzyme purification, the improvement in specific activity and the yield of the enzyme after each purification step can be summarized in a purification scheme. The purification factor (specific activity obtained after a purification step divided by that of the starting material) provides an insight into the “efficiency” of each step. If a pure enzyme is obtained, it also indicates the relative amount of that enzyme present in the starting material. A theoretical example of a purification scheme, comprising three purification steps, is shown in Table 1.1.

Enzymes that are used for biocatalysis are typically purified from microbial cells or from culture media after or during growth of microorganisms (in case of excreted proteins). The enzyme purification generally starts with a cleared cell extract in which the enzyme is present in a soluble form. If the enzyme to be purified is excreted into the culture medium, it is usually sufficient to remove the cells from the medium by centrifugation (for small‐scale purifications) or by filtration (for large‐scale industrial purifications). In the case of an intracellular enzyme, cells should be broken first to release the protein into solution. Depending on the type of cells, different techniques are employed. The microbial cells are first harvested from the culture medium by centrifugation and resuspended in a small amount of buffer. The cells can be broken using a variety of techniques, e.g. by treatment with enzymes that digest cell walls (e.g. lysozyme), followed by osmotic shock, by using lysis buffers containing detergents, by exposure to ultrasound using sonicators, by pushing cells under high pressure through a small orifice using a pressure cell system, or by grinding frozen cells in liquid nitrogen. Extracts thus obtained are cleared from unbroken cells and large, insoluble particles by centrifugation or filtration. To prevent enzyme inactivation during these treatments, and also in the following purification steps, the tem...

Table of contents

• Cover
• Table of Contents
• Title Page
• Copyright
• Foreword
• Part I: Enzyme Techniques
• Part II: Enzymes Handling and Applications
• Part III: Ways to Improve Enzymatic Transformations
• Part IV: Recent Trends in Enzyme-Catalyzed Reactions
• Part V: Industrial Biocatalysis
• Index
• End User License Agreement