Carbon−Carbon Bonds by Hydrolytic Enzymes

Abstract

Enzymes are efficient catalysts in synthetic chemistry, and their catalytic activity with unnatural substrates in organic reaction media is an area attracting much attention. Protein engineering has opened the possibility to change the reaction specificity of enzymes and allow for new reactions to take place in their active sites. We have used this strategy on the well-studied active-site scaffold offered by the serine hydrolase Candida antarctica lipase B (CALB, EC 3.1.1.3) to achieve catalytic activity for aldol reactions. The catalytic reaction was studied in detail by means of quantum chemical calculations in model systems. The predictions from the quantum chemical calculations were then challenged by experiments. Consequently, Ser105 in CALB was targeted by site-directed mutagenesis to create enzyme variants lacking the nucleophilic feature of the active site. The experiments clearly showed an increased reaction rate when the aldol reaction was catalyzed by the mutant enzymes as compared to the wild-type lipase. We expect that the new catalytic activity, harbored in the stable protein scaffold of the lipase, will allow aldol additions of substrates, which cannot be reached by traditional aldolases.

Biocatalytic preparation of enantioenriched 3,4-dihydroxypiperidines and theoretical study of Candida antarctica lipase B enantioselectivity

“…These reports have been very useful to make rational design of this biocatalyst 18 with improved selectivities, 17b,19 or catalyzing through novel reaction pathways. 20 Thus, engineered CAL-B has been utilized to perform aldol, 21 Michael-type additions, 22 or Baeyer-Villiger reactions. 23 The restricted volume of the active site of this lipase has been used to rationalize its special properties.…”

Computational design of a lipase for catalysis of the Diels-Alder reaction

“…Building on previous studies of Candida Antarctica Lipase B (CALB), performed by us and collaborators, [19,20,22,21] we set out to test this versatile enzyme for Diels-Alder activity. CALB was chosen because of its high documented catalytic promiscuity and tolerance for a broad range of solvents and reaction conditions.…”

“…the ability of an enzyme to exhibit catalysis of other than its native reaction) towards e.g. aldol condensations, [19,20] epoxidations [21] and Michael additions, [22,23,24,25,26] not seldom enhanced by point mutations. [27] An often-seen feature in promiscuous catalysis is that only parts of the catalytic machinery are used, so that removal of one or more of For Production's Reference Only Click here to download Manuscript: Brinck_2010-DielsAlder_revised.pdf 2 …”

Application of Deep Eutectic Solvents in Promiscuous Lipase‐Catalysed Aldol Reactions

“…The mixture composed by choline chloride and glycerol (ChCl:Gly, 1:1.5 mol/mol) was selected as initial starting point based on the suitability of this hydrogen bond donor and acceptor in previous studied biotransformations. Three enzymes were initially selected based on their already demonstrated activity as efficient catalyst in the formation of aldol products: Candida antarctica lipase type B (CAL-B), 19 protease from Bacillus lincheniformis (Alcalase-CLEA ® ) 20 and porcine pancreas lipase (PPL). 21 Attempts to follow the biotransformations by GC analysis were unsuccessful since the retro-aldol reaction occurred at the high temperatures used in the analysis, so we decided to fix a 1 day reaction time and measure the conversions using 1 H-NMR analysis.…”