H2O2, is an attractive oxidant for synthetic chemistry, especially if activated as percarboxylic acid. H2O2, however, is also a potent inactivator of enzymes. Protein engineering efforts to improve enzyme resistance against H2O2 in the past have mostly focused on tedious probabilistic directed evolution approaches. Here we demonstrate that a rational approach combining multiscale MD simulations and Born-Oppenheimer ab initio QM/MM MD simulations is an efficient approach to rapidly identify improved enzyme variants. Thus, the lipase from Penicillium camembertii was redesigned with a single mutation (I260R), leading to drastic improvements in H2O2 resistance while maintaining the catalytic activity. Also the extension of this methodology to other enzymes is demonstrated.
Title: Engineering a lipase B from Candida antactica with efficient perhydrolysis performance by eliminating its hydrolase activity Wang XP, Zhou PF, Li ZG, Yang B, Hollmann F, Wang YH Ref: Sci Rep, 7:44599, 2017 : PubMed
A Ser105Ala mutant of the lipase B from Candida antarctica enables 'perhydrolase-only' reactions. At the example of the chemoenzymatic Baeyer-Villiger oxidation of cyclohexanone, we demonstrate that with this mutant selective oxidation can be achieved in deep eutectic solvent while essentially eliminating the undesired hydrolysis reaction of the product.
Title: Engineering of Candida antarctica lipase B for hydrolysis of bulky carboxylic acid esters Juhl PB, Doderer K, Hollmann F, Thum O, Pleiss J Ref: J Biotechnol, 150:474, 2010 : PubMed
Candida antarctica lipase B (CALB) is a widely used biocatalyst with high activity and specificity for a wide range of primary and secondary alcohols. However, the range of converted carboxylic acids is more narrow and mainly limited to unbranched fatty acids. To further broaden the biotechnological applications of CALB it is of interest to expand the range of converted carboxylic acid and extend it to carboxylic acids that are branched or substituted in close proximity of the carboxyl group. An in silico library of 2400 CALB variants was built and screened in silico by substrate-imprinted docking, a four step docking procedure. First, reaction intermediates of putative substrates are covalently docked into enzyme active sites. Second, the geometry of the resulting enzyme-substrate complex is optimized. Third, the substrate is removed from the complex and then docked again into the optimized structure. Fourth, the resulting substrate poses are rated by geometric filter criteria as productive or non-productive poses. Eleven enzyme variants resulting from the in silico screening were expressed in Escherichia coli BL21 and measured in the hydrolysis of two branched fatty acid esters, isononanoic acid ethyl ester and 2-ethyl hexanoic acid ethyl esters. Five variants showed an initial increase in activity. The variant with the highest wet mass activity (T138S) was purified and further characterized. It showed a 5-fold increase in hydrolysis of isononanoic acid ethyl ester, but not toward sterically more demanding 2-ethyl hexanoic acid ethyl ester.
Title: Enzyme engineering for enantioselectivity: from trial-and-error to rational design? Otten LG, Hollmann F, Arends IW Ref: Trends Biotechnol, 28:46, 2010 : PubMed
The availability of tailored enzymes is crucial for the implementation of biocatalysis in organic chemistry. Enantioselectivity is one key parameter defining the usefulness of an enzyme and, therefore, the competitiveness of the corresponding industrial process. Hence, identification of enzymes with high enantioselectivity in the desired transformation is important. Currently, this is achieved by screening collections and libraries comprising natural or man-made diversity for the desired trait. Recently, a variety of improved methods have been developed to generate and screen this diversity more efficiently. Here, we present and discuss the most important advances in both library generation and screening. We also evaluate future trends, such as moving from random evolution to more rational.
