By Wu-Kuang Yeh, Hsiu-Chiung Yang, James R. McCarthy
An authoritative overview of the newest advancements within the chemical biology of enzymes
within the first decade of the twenty-first century, enzymes and their a number of purposes have performed a serious position within the discovery and improvement of many new healing agents.This publication is a coordinated compilation of analysis services and present opinion uniquely concerned with enzymes and their homes and functions.
Compiled through editors with a mixed pharmaceutical event of over sixty years, the textual content presents in-depth studies of modern advancements in selective subject matters on biosynthesis, biocatalysis, and chemical biology of enzymes because it applies to drug discovery, improvement, and manufacture.
the 1st in a multi-part sequence on enzymes, this quantity beneficial properties 3 sections:
New techniques to discover and regulate Enzymes describes the rising box of metagenomics, offers the sensible purposes of directed evolution to enzymes and pathways, and explores techniques for the invention and layout of biocatalysts
Biocatalytic purposes reports particular functions of other reactions in generating lively pharmaceutical parts and surveys contemporary advancements applying enzymes in natural synthesis
Biosynthetic functions is going over profitable drug discoveries and advancements by means of combinatorial biosynthesis and stories the applying of combinatorial biosynthesis between a number of suitable hosts
those well timed discussions, which conceal every little thing from chemical biology of enzymes, to the redecorate of binding and catalytic specificities of enzymes, make this quantity a invaluable software for maintaining to this point. As such, it really is a big learn for researchers, scholars, and professors within the examine of biotechnology, existence sciences, biochemistry, enzymology, medicinal chemistry, and common products.Content:
Chapter 1 practical Metagenomics as a method for the invention of Novel Enzymes and typical items (pages 1–39): Luke A. Moe, Matthew D. McMahon and Michael G. Thomas
Chapter 2 Directed Enzyme and Pathway Evolution (pages 41–75): Jacob Vick and Claudia Schmidt‐Dannert
Chapter three Combining ordinary Biodiversity and Molecular‐Directed Evolution to increase New commercial Biocatalysts and medicine (pages 77–98): Laurent Fourage, Céline Ayrinhac, Johann Brot, Christophe Ullmann, Denis Wahler and Jean‐Marie Sonet
Chapter four ideas of Enzyme Optimization for the quick production of business Biocatalysts (pages 99–124): Richard J. Fox and Lori Giver
Chapter five Enzyme Catalysis within the Synthesis of lively Pharmaceutical elements (pages 125–184): Animesh Goswami
Chapter 6 Enzymatic techniques for the creation of Pharmaceutical Intermediates (pages 185–198): David Rozzell and Jim Lalonde
Chapter 7 Novel advancements making use of Redox Enzymes: outdated Enzymes in New outfits (pages 199–249): Kurt Faber, Silvia M. Glueck, Birgit Seisser and Wolfgang Kroutil
Chapter eight Drug Discovery and improvement by means of Combinatorial Biosynthesis (pages 251–283): Matthew A. DeSieno, Carl A. Denard and Huimin Zhao
Chapter nine Reprogramming Daptomycin and A54145 Biosynthesis to provide Novel Lipopeptide Antibiotics (pages 285–308): Richard H. Baltz, Kien T. Nguyen and Dylan C. Alexander
Chapter 10 Pathway and Enzyme Engineering and functions for Glycodiversification (pages 309–362): Lishan Zhao and Hung‐Wen Liu
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Extra resources for Enzyme Technologies: Metagenomics, Evolution, Biocatalysis, and Biosynthesis
Such analysis helps to address numerous specific questions about proteases, including: •• Which of the myriad of characterized and postulated proteins exhibit proteolytic activity? •• What type of catalytic function is any given protease likely to support? •• What substrate (or range of substrates) can a given protease process? •• What is the proteolytic mechanism of action? •• What types of chemicals are likely to serve as competitive inhibitors? •• Are there foreseeable mechanisms for noncompetitive inhibition or agonist effects?
The naturally processed substrate site in the gag–pol polyprotein for HIV-1 protease is very well characterized and forms the basis for the design of small peptide substrates for homogeneous FRET assays. This substrate SQNYPIVWL or SQNYPIVQ is labeled at the N-terminus with DABSYL and at the C-terminus with EDANS. The cleavage of the peptide with HIV-1 protease resulted in a 40-fold increase in DABSYL fluorescence , allowing initial reaction velocities to be determined. The same or related peptides have been tagged with chromophores to develop various other assay formats.
However, since high background fluorescence and cell fixation preclude the ability for real-time data imaging, the technical problems were circumvented by using the dye-quenched fluorescent protein substrates. The dye-quenched substrates contain high density of FITC molecules which are quenched due to high molecular proximity . Protease-mediated cleavage of the substrate releases fluorescent degradation products. BioMol has developed cell-based assays which 18 ASSAY TECHNOLOGIES FOR PROTEASES (a) Fluorescent Protein (FP) Wt GFP Green (eGFP) Blue (BFP) Mutations None S65T, F64L Y66H, Y145F, F64L Excitation Max (nm) 395, 495 489 385 Emission Max (nm) 511 509 445 Cyan (CFP) Y66W, S65A, S72A, M153T, V163A 434 477 Yellow (YFP) T203Y, S65G, S72A, 514 V68L 527 T-Sapphire T203I, Q69M, C70P, V163A, S17G 511 399 (b) 527 nm CFP YFP 434 nm Protease 477 nm 434 nm YFP CFP Figure 6 Fluorescent protein-based FRET assays.
Enzyme Technologies: Metagenomics, Evolution, Biocatalysis, and Biosynthesis by Wu-Kuang Yeh, Hsiu-Chiung Yang, James R. McCarthy