Screening biocatalysts

Rhone-Poulenc Industrialization has developped new automated techniques for screening biocatalysts. A special laboratory was devoted to the screening of microorganisms. It contains two robots which are able to carry out automaticaly all the basic operations involved in screening procedures. 

When starting our first experiments with available ionic liquids, in screening programs to identify suitable systems, we encountered several difficulties such as pH shifts or precipitation. More generally, the following aspects should be taken into account when ionic liquids are used with biocatalysts ... 

Other biocatalysts were also used to perform the dynamic kinetic resolution through reduction. For example, Thermoanaerobium brockii reduced the aldehyde with a moderate enantioselectivity [30b,c], and Candida humicola was found, as a result of screening from 107 microorganisms, to give the (Jl)-alcohol with 98.2% ee when ester group was methyl [30dj. 

Biocatalysts these are essential for life and play a vital role in most processes occurring within the body as well as in plants. In the laboratory biocatalysts are usually natural enzymes or enzymes produced in situ from whole cells. They offer the possibility of carrying out many difficult transformations under mild conditions and are especially valuable for producing enantiomerically pure materials. Their huge potential is currently largely untapped, partially due to the time and expense of isolating and screening enzymes. 

A combinatorial approach for biocatalytic production of polyesters was demonstrated. A library of polyesters were synthesized in 96 deep-well plates from a combination of divinyl esters and glycols with lipases of different origin. In this screening, lipase CA was confirmed to be the most active biocatalyst for the polyester production. As acyl acceptor, 2,2,2-trifluoroethyl esters and vinyl esters were examined and the former produced the polymer of higher molecular weight. Various monomers such as carbohydrates, nucleic acids, and a natural steroid diol were used as acyl acceptor. 

Chapters 1-4 serve as an introduction to emerging biocatalysts, modern expression hosts, state of the art of directed evolution, high-throughput screening, and bioprocess engineering for industrial applications. 

Becker, S., Schmoldt, H.U., Adams, T.M. et al. (2004) Ultra-high-throughput screening based on cell-surface display and fluorescence-activated cell sorting for the identification of novel biocatalysts. Current Opinion in Biotechnology, 15, 323-329. 

Woodyer, R., van der Donk, W.A. and Zhao, H.M. (2006) Optimizing a biocatalyst for improved NAD(P)H regeneration directed evolution of phosphite dehydrogenase. Combinatorial Chemistry High Throughput Screening, 9, 237-245. 

By screening 53 Rhodococcus and Pseudomonas strains, an NHase-amidase biocatalyst system was identified for the production of the 2,2-dimethylcyclopropane carboxylic acid precursor of the dehydropeptidase inhibitor Cilastatin, which is used to prolong the antibacterial effect of Imipenem. A systematic study of the most selective of these strains, Rhodococcus erythropolis ATCC25 544, revealed that maximal product formation occurs at pH 8.0 but that ee decreased above pH 7.0. In addition, significant enantioselectivity decreases were observed above 20 °C. A survey of organic solvent effects identified methanol (10% v/v) as the... 

A BDS patent [106] was awarded for the use of biocatalysts belonging to the group of Pseudomonas, Flavobacterium, Enterobacter, Aeromonas, Bacillus, or Corynebac-terium. One of the strains P. putida was further developed by mutation of the parent strain to obtain organic solvent-resistant mutants [107], The mutated strains were screened by selective cultivation in the presence of 0.1% to 10% by volume (v/v) of concentrations of a toxic organic solvent. The specific mutated strains obtained were P. putida No. 69-1 (PERM BP-4519), P. putida No. 69-2 (PERM BP-4520), and P. putida No. 69-3 (PERM BP-4521). 

In addition to desulfurization activity, several other parameters are important in selecting the right biocatalyst for a commercial BDS application. These include solvent tolerance, substrate specificity, complete conversion to a desulfurized product (as opposed to initial consumption/removal of a sulfur substrate), catalyst stability, biosurfactant production, cell growth rate (for biocatalyst production), impact of final desulfurized oil product on separation, biocatalyst separation from oil phase (for recycle), and finally, ability to regenerate the biocatalyst. Very few studies have addressed these issues and their impact on a process in detail [155,160], even though these seem to be very important from a commercialization point of view. While parameters such as activity in solvent or oil phase and substrate specificity have been studied for biocatalysts, these have not been used as screening criteria for identifying better biocatalysts. 

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