He evaluates interesting topics in research and development and networks globally with academic and industrial institutions. He did his Ph. His research interests include amino acids, peptides, biological active compounds, asymmetric synthesis, metal and biocatalysis as well as process chemistry. He holds about patents, about scientific publications. Since he is honorary professor for organic chemistry at the University Wurzburg, Germany.
Kunz, after which he completed a postdoctoral appointment with Professor G. He has received numerous awards, e. His research interests lie in bioactive compound development, target identification, and the chemical biology of Ras proteins. Voigt Frances H. Williams Rebecca J. David Rozzell Andreas S.
Using all-atom models and cutting-edge hardware, one can perform nonbiased ligand migration, active site search, and binding studies. In this Letter, we demonstrate and validate by PCR mutagenesis how these techniques, when combined with quantum mechanics, open new possibilities in enzyme engineering. We provide a complete analysis where 1 biophysical simulations produce ligand diffusion and 2 biochemical modeling samples the chemical event.
Using such broad analysis, we engineer a highly stable peroxidase activating the enzyme for new substrate oxidation after rational mutation of two nonconserved surface residues. In particular, we create a new surface-binding site, quantitatively predicting the in vitro change in oxidation rate obtained by mutagenic PCR and achieving a comparable specificity constant to active peroxidases.
Multiple sequence alignment of MnPs from P.
- 1. Introduction;
- Enzyme Engineering.
- Where the wild rose blooms.
- Sigmund Romberg (Yale Broadway Masters Series).
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View Author Information. Cite this: ACS Catal. Article Views Altmetric -. Citations Supporting Information. Cited By. This article is cited by 18 publications. ACS Catalysis , 8 9 , DOI: The Journal of Physical Chemistry B , 25 , Sandra Acebes, Francisco J. The Journal of Physical Chemistry B , 16 , Ernst, Klaus Piontek, Morten J. Journal of Chemical Theory and Computation , 13 3 , Santi, C. The DNA2. The codon-optimized sequence of wild-type Cal-A resulting from assembly of the three parts is reported in the supplemental information S1 Fig.
Transformed E. PCR conditions for the amplification of the parts wild-type and mutated are detailed in the supplementary information S2 Table. All genetic constructs were designed and assembled in silico using the SnapGene software GSL Biotech; available at snapgene. The resulting ligation mixes were transformed into E.
Colonies were picked individually and grown in 96 deep-well plates in LB supplemented with the appropriate antibiotic. The screening of the variants against tributyrin and olive oil was based on previously reported techniques[ 31 , 32 ]. With the help of a Beckman Coulter Biomek NX p robot, we transformed the manual screen into an automated version that allows for higher throughput. We used give the dimensions here rectangular petri dishes to cast the growth media to be inoculated with the library variants. Tributyrin plates were prepared as follows: 1.
Tributyrin oil 2. The media were thoroughly shaken to generate strong emulsions before plating. Each plate was cast with 42 mL of medium and left to set on a smooth and perfectly horizontal surface.
Enzyme Engineering - Volume 3 | E. Pye | Springer
The plates were stacked in the liquid handler, ready to be picked up by the robotic arm. The pipette tips were re-used for the same inoculum. The excess liquid culture was released in an ethanol waste. The incubated plates were visualized by a gel imager and analyzed. As a negative control, a culture of E. This kit allows for the elimination of any bias during mutation.
This is achieved by using very little template DNA 0. Once part2 was randomly mutated, we assembled it with the wild-type part1 and part3 and screened for positive clones. DNA sequencing of a number of those clones served to assess the library quality refer to text in main paper and Table 2. By sequencing approximately 50 clones from each library, we identified 11 of the 12 codons expected for the Tyr93 libraries, 10 of 12 for the Tyr libraries and 9 of 12 for the Phe libraries. The left-hand map shows the entire gene, the right-hand map shows the gene divided into the three parts.
The expected band is consistent with the size of the complete Cal-A gene, which is about bp. In this simplified one-pot assembly procedure the three mother vectors carrying the parts are restricted and ligated together with the daughter vector in a single reaction 1 and the ligated product is then directly transformed 2. It is worth noting that the use of type IIS restriction enzymes should, in principle, allow to directly cut and ligate the parts and the vector together without the need of PCR steps for pre-amplification.
Furthermore, the use of different selective antibiotic markers on the mother and daughter vectors eliminates the worry of carrying forward the mother constructs. In this scenario, the three parts carried in the circular mother vectors are directly pooled with the daughter vector, cut at the respective restriction sites at the same time and assembled by adding the ligase directly to the mixture. We used this simplified assembly strategy during our experiments to generate the wild-type Cal-A construct. DNA sequencing of three of the clones confirmed that no undesired events had occurred.
However, we also noticed that when we applied this simplified strategy to achieve the recombination of the mutant parts, a reduced success rate of ligation was achieved. Even though we cannot at present explain this phenomenon, the use of our standard method Fig 2 in main article solved the problem.
Methods and Protocols
We thank Prof. We would also like to thank Mr. Harold P. Le Bris for his help with the design of the figures reported in this paper, and Dr. David M. Charbonneau for sharing his knowledge on lipase enzymes. Formal analysis: DQ JP. Funding acquisition: JP PM.
Investigation: DQ ME. Project administration: DQ JP. Resources: JP.
International Journal of Food Engineering
Software: DQ ME. Supervision: JP. Validation: DQ JP. Writing — original draft: DQ JP.
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