Highly Focused Library‐Based Engineering of Candida antarctica Lipase B with (S)‐Selectivity Towards sec‐Alcohols

Abstract

Candida antarctica lipase B (CALB) is one of the most extensively used biocatalysts in both academia and industry and exhibits remarkable (R)-enantioselectivity for various chiral sec-alcohols. Considering the significance of tailor-made stereoselectivity in organic synthesis, a discovery of enantiocomplementary lipase mutants with high (R)-and (S)-selectivity is valuable and highly desired. Herein, we report a highly efficient directed evolution strategy, using only 4 representative amino acids, namely, alanine (A), leucine (L), lysine (K), tryptophan (W) at each mutated site to create an extremely small library of CALB variants requiring notably less screening. The obtained best mutant with three mutations W104V/A281L/ A282K displayed highly reversed (S)-selectivity towards a series of sec-alcohol with E values up to 115 (conv. 50%, ee 94%). Compared with the previously reported (S)-selective CALB variant, W104A, a single mutation provided less selectivity, while the synergistic effects of three mutations in the best variant endow better (S)-selectivity and a broader substrate scope than the W104A variant. Structural analysis and molecular dynamics simulation unveiled the source of reversed enantioselectivity.

Electronic Effect‐Guided Rational Design of Candida antarctica Lipase B for Kinetic Resolution Towards Diarylmethanols

“…In the KR of sec ‐alcohols containing phenyl and alkyl groups, CALB mutant W104V/A281L/A282K gave the best reversed selectivity compared to WT, according to our previous study, [11c] showing the high affinity between phenyl group and the reshaped stereospecificity pocket. We then further tested this mutant for the KR of substrate rac ‐ 1 b , and obtained a reversed enantioselectivity (Figure 5, entries 17–19 in Table S4), expectedly.…”

<p>Immobilization studies of Candida Antarctica lipase B on gallic acid resin-grafted magnetic iron oxide nanoparticles</p>

“…Candida Antarctica lipase B (Cal-B) is an enzyme with numerous applications in a broad range of catalytic reactions and is often used in industrial chemical processes1–4 such as kinetic resolutions, aminolysis, esterification, transesterification, hydrolysis, and stereoisomeric transformations3,5–8 and even the synthesis of glucolipids 9,10. Cal-B is highly selective, offering stability in an acidic pH environment, as high-quality end-product, and with fewer side-products from chemical reactions.…”

Chemoenzymatic Sequential Catalysis with Carbonic Anhydrase for the Synthesis of Chiral Alcohols from Alkanes, Alkenes, and Alkynes

“…Chiral alcohols are valuable building blocks used in the synthesis of, particularly, pharmaceuticals, agrochemicals, and fine chemicals and have drawn extensive attention in the field of catalysis. − A considerable number of significant active intermediates and top-selling drugs contain chiral alcohol moieties. − In the context, ( S )-1-phenylethanol can be employed as a synthetic precursor to prepare sertraline for treating depression, asmalar for treating asthma, and levamisole for enhancing the immune system. − ( R )-2-chloro-1-phenylethanol and ( S )-1-(2,6-dichloro-3-fluorophenyl)-ethanol are the intermediates for synthesizing fluoxetine and crizotinib, respectively. , (+)-Bacillamide exhibits anti-HIV activities and lovastatin displays potential for the prevention and treatment of various types of cancers and cholesterolemic and neurological disorders (Figure ). , The classical synthetic procedures of chiral alcohol compounds usually rely on harsh reaction conditions such as high pressure and temperature, expensive chiral ligands, and environmentally unfriendly organic reagents. − Therefore, developing efficient, economical, and sustainable routes for chiral alcohol synthesis is urgently needed owing to the rising shortage of resources and environmental concerns. , Various oxidoreductases such as ketoreductases (KREDs), also known as alcohol dehydrogenases (ADHs), have been employed for the asymmetric reduction of ketones to produce chiral alcohols for their merits of excellent enantioselectivity, specificity, and benign conditions. ,− However, more than 80% of enzymatic reductions from ketones catalyzed by oxidoreductases require a cofactor NAD­(P)H as the hydrogen source to be consumed for accomplishing catalytic functions. , In the last decades, extensive efforts have been made toward exploring simple and suitable methods for cofactor regeneration. ,, Despite the significant progress that has been made in the regeneration of NAD­(P)H via biocatalytic, electrochemical, and photochemical methods, biocatalysts that do not rely on cofactors for the fabrication of chiral alcohols still remain to be further explored. , Recently, a human carbonic anhydrase II (hCAII) was reported that exhibited excellent catalytic activity and enantioselectivity in the asymmetric reduction of ketones with silanes as the reductant .…”