Tolerance, protein engineering

In addition to protein engineering, the substrate mimicking approach was also appHed for RhuA catalyst It was uncovered that RhuA can perform the aldol addition of DHA to aldehyde at remarkably high rates when the reactions were carried out in the presence of borate [29]. Indeed, when sodium borate was added, the rates of aldol formation improved between 35- and 100-fold [25]. Besides the intrinsic tolerance of RhuA for DHA, the measured retroaldol rates for some aldol adducts in the presence of borate were low or negligible as compared with the synthetic ones, making the process virtually irreversible [29, 30]. Therefore, it was further suggested that the aldol adduct may be trapped by the formation of borate complexes which would be less active substrates for the aldolase [29, 30]. 

Aldolase redesign by protein engineering is mainly focused on enhancing the acceptor and donor tolerance toward a wide stractural spectrum of nucleophiles and electrophiles. In this connection, FSA-Hke enzymes constitute an attractive platform for modification because of their intrinsic tolerance toward structurally diverse donors. 

The desymmetrization of l-alkylbicyclo[3.3.0]octane-2,8-diones can be achieved in a facile coenzyme-independent enzymatic reaction in buffer. Alkyl chains in the 1 -position of up to at least five carbon atoms are tolerated. The yields of the crude keto-acids are essentially quantitative, and the enantiotopic discrimination by the enzyme is usually excellent." Work remains to be done on the optimization of this biocatalyst with respect to protein stability and reaction engineering, but it remains a unique and intriguing possibility for the generation of interesting intermediates bearing multiple chiral centres. 

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