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Chapter 30 : Tools for Enzyme Discovery

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Abstract:

This chapter discusses recent remarkable examples in the screening for new enzymes, biocatalysis, and enzyme evolution. A section discusses screening for enzymes by enrichment culture technique from stock cultures, followed by enzyme purification and characterization, coupled with gene cloning. Examples include the discovery of nitrile hydratase and industrial production amides, aldoxime dehydratase and nitrile synthesis, ω-laurolactam hydrolase for enzymatic transcrystallization, and hydroxynitrile lyase from plants. These traditional approaches often yield primary information that can be industrialized successfully. Enrichment culture is a technique to isolate microorganisms having special growth characteristics. Acrylamide synthesis is now the largest enzyme-catalyzed organic synthesis in the world. Metagenomic approaches include sequence-based screening after shotgun large-scale sequencing of environmental DNA, enzyme activity-based screening of expressed libraries of environmental DNA, and a recent methodology called substrate-induced gene expression screening approaches (SIGEX). Reductive amination reaction of α-keto acid to form d-amino acid was made possible with this new enzyme. D-Isoleucine, D-glutamic acid, D-phenylalanine, and D-4-chlorophenylalanine were the substrates on which the wild-type enzyme was completely nonactive. The directed evolution of enzymes has proved to be a very powerful technique, as can be seen in the development of the new enzyme D-amino acid dehydrogenase, a thermostable DCase, and industrial production of nucleic acids with umami flavor by the evolved acid phosphatase.

Citation: Asano Y. 2010. Tools for Enzyme Discovery, p 441-452. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch30

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FIGURE 1

Discovery of industrial enzymes, biocatalysis, and enzyme evolution. These various steps in discovery and development are cited by their respective letters throughout this chapter.

Citation: Asano Y. 2010. Tools for Enzyme Discovery, p 441-452. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch30
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FIGURE 2

Plant and microbial aldoxime-nitrile pathway.

Citation: Asano Y. 2010. Tools for Enzyme Discovery, p 441-452. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch30
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Image of FIGURE 3
FIGURE 3

Synthesis of 12-aminolauric acid from ω-laurolactam.

Citation: Asano Y. 2010. Tools for Enzyme Discovery, p 441-452. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch30
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Image of FIGURE 4
FIGURE 4

Reactions catalyzed by nitrilases.

Citation: Asano Y. 2010. Tools for Enzyme Discovery, p 441-452. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch30
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Image of FIGURE 5
FIGURE 5

Enzyme-catalyzed peptide synthesis. (a) A peptidase from catalyzing ami-nolysis reaction. (b) LAL.

Citation: Asano Y. 2010. Tools for Enzyme Discovery, p 441-452. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch30
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Image of FIGURE 6
FIGURE 6

Biosynthesis of menaquinone in microorganisms.

Citation: Asano Y. 2010. Tools for Enzyme Discovery, p 441-452. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch30
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Image of FIGURE 7
FIGURE 7

Industrial dynamic kinetic resolution of hydantoin by hydantoinase and DCase to produce -hydroxy--phenylglycine.

Citation: Asano Y. 2010. Tools for Enzyme Discovery, p 441-452. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch30
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Image of FIGURE 8
FIGURE 8

Enzymatic synthesis of -amino acids.

Citation: Asano Y. 2010. Tools for Enzyme Discovery, p 441-452. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch30
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Image of FIGURE 9
FIGURE 9

Acid phosphatase-catalyzed 5′-monophosphorylation of nucleoside with PPi as a phosphorylating agent.

Citation: Asano Y. 2010. Tools for Enzyme Discovery, p 441-452. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch30
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Image of FIGURE 10
FIGURE 10

Directed evolution of acid phosphatase as an industrial biocatalyst to produce 5′-IMP and 5′-GMP. ○, wild type; •, mutant.

Citation: Asano Y. 2010. Tools for Enzyme Discovery, p 441-452. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch30
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Image of FIGURE 11
FIGURE 11

Dynamic kinetic resolution of amino acid amide.

Citation: Asano Y. 2010. Tools for Enzyme Discovery, p 441-452. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch30
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Image of FIGURE 12
FIGURE 12

Dynamic kinetic resolution of alanine amide with -aminopeptidase and ACL racemase. •, -alanine; , -alanine amide; , -alanine.

Citation: Asano Y. 2010. Tools for Enzyme Discovery, p 441-452. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch30
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