Directed Evolution of Lipases and Esterases

Schmidt, Marlen; Baumann, Markus; Henke, Erik; Konarzycka-Bessler, Monika; Bornscheuer, Uwe T.

doi:10.1016/s0076-6879(04)88018-2

Cited by 30 publications

(15 citation statements)

References 21 publications

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“…The value of the Michaelis constant for 1 ( K M = 14 μM) was substantially lower than that for 2 ( K M = 0.54 mM). The active site of the human homologue of PLE is 10–15 Å deep within a hydrophobic pocket, and the increased size and hydrophobicity of 1 could contribute to the decreased K M value [23]. We have noticed a similar inverse relationship between the molecular size and K M value of our fluorogenic esterase substrates [5,6,7].…”

Section: Resultsmentioning

confidence: 61%

“…Adopting this prodrug strategy led to superior chemical stability while maintaining high enzymatic reactivity. These attributes could be useful in numerous contexts, including high-throughput screens and directed evolution of esterases and lipases [23,24], and the de novo discovery of new catalysts for ester-bond hydrolysis [25,26]. The modular design of this substrate enables its tailoring to confer reactivity with other enzymes.…”

Section: Discussionmentioning

confidence: 99%

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Trimethyl Lock: A Stable Chromogenic Substrate for Esterases

Levine

Lavis²,

Raines

2008

Molecules

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p-Nitrophenyl acetate is the most commonly used substrate for detecting the catalytic activity of esterases, including those that activate prodrugs in human cells. This substrate is unstable in aqueous solution, limiting its utility. Here, a stable chromogenic substrate for esterases is produced by the structural isolation of an acetyl ester and p-nitroaniline group using a trimethyl lock moiety. Upon ester hydrolysis, unfavorable steric interactions between the three methyl groups of this o-hydroxycinnamic acid derivative encourage rapid lactonization to form a hydrocoumarin and release p-nitroaniline. This “prochromophore” could find use in a variety of assays.

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Section: Resultsmentioning

confidence: 61%

Section: Discussionmentioning

confidence: 99%

Trimethyl Lock: A Stable Chromogenic Substrate for Esterases

Levine

Lavis²,

Raines

2008

Molecules

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“…Due to their diverse substrate specificities and their stereoselectivity, esterases have been successfully used in the synthesis of optically pure substances. More recently, esterases have also been used in laboratory approaches to obtain tailor-made biocatalysts (94,95,105). It is of striking advantage in the evolutive approach to express the enzyme to be evolved on the surface of a living cell.…”

Section: Biotechnical and Biomedical Applications Of The Autodisplay mentioning

confidence: 99%

The Autodisplay Story, from Discovery to Biotechnical and Biomedical Applications

Jose

Meyer

2007

Microbiol Mol Biol Rev

201

189

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SUMMARY Among the pathways used by gram-negative bacteria for protein secretion, the autotransporter pathway represents a solution of impressive simplicity. Proteins are transported, independent of their nature as recombinant or native passengers, as long as the coding nucleotide sequence is inserted in frame between those of an N-terminal signal peptide and a C-terminal domain, referred to as the β-barrel of the outer membrane translocation unit. The immunoglobulin A1 (IgA1) protease from Neisseria gonorrhoeae was the first identified member of the autotransporter family of secreted proteins. The IgA1 protease was employed in initial experiments investigating autotransporter-mediated surface display of recombinant proteins and to investigate structural and functional requirements. Various other autotransporter proteins have since been described, and the autodisplay system was developed on the basis of the natural Escherichia coli autotransporter protein AIDA-I (adhesin involved in diffuse adherence). Autodisplay has been used for the surface display of random peptide libraries to successfully screen for novel enzyme inhibitors. The autodisplay system was also used for the surface display of functional enzymes, including esterases, oxidoreductases, and electron transfer proteins. Whole E. coli cells displaying enzymes have been utilized to efficiently synthesize industrially important rare organic compounds with specific chirality. Autodisplay of epitopes on the surface of attenuated Salmonella carriers has also provided a novel way to induce immune protection after oral vaccination. This review summarizes the structural and functional features of the autodisplay system, illustrating its discovery and most recent applications. Autodisplay facilitates the export of more than 100,000 recombinant molecules per single cell and permits the oligomerization of subunits on the cell surface as well as the incorporation of inorganic prosthetic groups after transport of apoproteins onto the bacterial surface without disturbing bacterial integrity or viability. We discuss future biotechnical and biomedical applications in the light of these achievements.

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“…In addition, enzymes can be over expressed to make biocatalytic processes economically efficient, and enzymes with modified activity can be tailor-made. Directed evolution of biocatalysts can lead to increased enzyme activity, selectivity and stability [2,3,4,5,6,7,8,9,10,11,12,13,14,15]. A number of review articles [16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31] have been published on the use of enzymes in organic synthesis.…”

Section: Introductionmentioning

confidence: 99%

Biocatalytic Synthesis of Chiral Alcohols and Amino Acids for Development of Pharmaceuticals

Patel

2013

Biomolecules

116

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Chirality is a key factor in the safety and efficacy of many drug products and thus the production of single enantiomers of drug intermediates and drugs has become increasingly important in the pharmaceutical industry. There has been an increasing awareness of the enormous potential of microorganisms and enzymes derived there from for the transformation of synthetic chemicals with high chemo-, regio- and enatioselectivities. In this article, biocatalytic processes are described for the synthesis of chiral alcohols and unntural aminoacids for pharmaceuticals.

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Directed Evolution of Lipases and Esterases

Cited by 30 publications

References 21 publications

Trimethyl Lock: A Stable Chromogenic Substrate for Esterases

Trimethyl Lock: A Stable Chromogenic Substrate for Esterases

The Autodisplay Story, from Discovery to Biotechnical and Biomedical Applications

Biocatalytic Synthesis of Chiral Alcohols and Amino Acids for Development of Pharmaceuticals

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