Stereocomplementary Bioreduction of β‑Ketonitrile without Ethylated Byproduct

α-Ethylation is competing with the biocatalytic reduction of aromatic β-ketonitriles in a whole-cell system. Use of two newly mined robust and stereocomplementary carbonyl reductases in a biphasic system has completely eliminated the competing byproduct. For the first time, both enantiomers of fluoroxetine precursors were obtained at 0.5 M with >99% <i>ee</i> and excellent chemoselectivity, without addition of any external cofactors

Molecular Dynamics Investigation of the Substrate Binding Mechanism in Carboxylesterase

A recombinant carboxylesterase, cloned from <i>Pseudomonas putida</i> and designated as rPPE, is capable of catalyzing the bioresolution of racemic 2-acetoxy-2-(2′-chlorophenyl)­acetate (<i>rac</i>-AcO-CPA) with excellent (<i>S</i>)-enantioselectivity. Semirational design of the enzyme showed that the W187H variant could increase the activity by ∼100-fold compared to the wild type (WT) enzyme. In this study, we performed all-atom molecular dynamics (MD) simulations of both apo-rPPE and rPPE in complex with (<i>S</i>)-AcO-CPA to gain insights into the origin of the increased catalysis in the W187H mutant. Our results show differential binding of (<i>S</i>)-AcO-CPA in the WT and W187H enzymes, especially the interactions of the substrate with the two active site residues Ser159 and His286. The replacement of Trp187 by His leads to considerable structural rearrangement in the active site of W187H. Unlike in the WT rPPE, the cap domain in the W187 mutant shows an open conformation in the simulations of both apo and substrate-bound enzymes. This open conformation exposes the catalytic triad to the solvent through a water accessible channel, which may facilitate the entry of the substrate and/or the exit of the product. Binding free energy calculations confirmed that the substrate binds more strongly in W187H than in WT. On the basis of these computational results, we further predicted that the mutations W187Y and D287G might also be able to increase the substrate binding and thus improve the enzyme’s catalytic efficiency. Experimental binding and kinetic assays on W187Y and D287G show improved catalytic efficiency over WT, but not W187H. Contrary to our prediction, W187Y shows slightly decreased substrate binding coupled with a 100-fold increase in turnover rate, while in D287G the substrate binding is 8 times stronger but with a slightly reduced turnover rate. Our work provides important molecular-level insights into the binding of the (<i>S</i>)-AcO-CPA substrate to carboxylesterase rPPEs, which will help guide future development of more efficient rPPE variants

Access to Optically Active Aryl Halohydrins Using a Substrate-Tolerant Carbonyl Reductase Discovered from Kluyveromyces thermotolerans

By genome data mining, a carbonyl reductase tool box was designed and developed for chiral alcohol synthesis. On the basis of systematic comparison of the specific activity and substrate tolerance toward α-chloroacetophenone among reductases in this tool box, KtCR, a highly substrate-/product-tolerant carbonyl reductase from Kluyveromyces thermotolerans, was identified. The reduction of a series of substituted aryl ketones was investigated using this newly mined biocatalyst. Almost all of the ketones tested were asymmetrically reduced into corresponding chiral alcohols in 99% ee. Substrates with substituents adjacent to the carbonyl group or those with substituents on the para position of the phenyl ring were easier to reduce. For α-choloacetophenone as a representative substrate, as much as 154 g/L (1.0 M) of the substrate was asymmetrically reduced within merely 12 h by lyophilized cells of Escherichia coli/pET28-KtCR, resulting in an isolated yield of 92%, an enantiopurity of >99% ee, and a total turnover number of 5000, which was five times higher than the highest record reported so far. These results indicate the great potential of KtCR in practical synthesis of valuable aryl halohydrins as versatile chiral synthons

Identification of an Imine Reductase for Asymmetric Reduction of Bulky Dihydroisoquinolines

A new imine reductase from <i>Stackebrandtia nassauensis</i> (<i>Sn</i>IR) was identified, which displayed over 25- to 1400-fold greater catalytic efficiency for 1-methyl-3,4-dihydro­isoquinoline (1-Me DHIQ) compared to other imine reductases reported. Subsequently, an efficient <i>Sn</i>IR-catalyzed process was developed by simply optimizing the amount of cosolvent, and up to 15 g L^–1 1-Me DHIQ was converted completely without a feeding strategy. Furthermore, the reaction proceeded well for a panel of dihydroisoquinolines, affording the corresponding tetrahydroisoquinolines (mostly in <i>S</i>-configuration) in good yields (up to 81%) and with moderate to excellent enantioselectivities (up to 99% ee)

Boletín de Segovia: Número 103 - 1893 agosto 28

Copia digital. Madrid : Ministerio de Cultura. Subdirección General de Coordinación Bibliotecaria, 200

Highly Efficient Synthesis of (<i>R</i>)‑3-Quinuclidinol in a Space–Time Yield of 916 g L^–1 d^–1 Using a New Bacterial Reductase <i>Ar</i>QR

A new keto reductase (<i>Ar</i>QR), identified from <i>Agrobacterium radiobacter</i> ECU2556, can efficiently reduce 3-quinuclidinone in excellent enantioselectivity and high space–time yield for the synthesis of (<i>R</i>)-3-quinuclidinol, a chiral building block of many antimuscarinic agents. This is the first time that a high yield of (<i>R</i>)-3-quinuclidinol up to 916 g L^–1 d^–1 using a bioreduction approach is reported

MOESM1 of Marked enhancement of Acinetobacter sp. organophosphorus hydrolase activity by a single residue substitution Ile211Ala

Additional file 1. In the Supplemental Material Section results from the crystallization of the purified AbOPH protein and selection of the mutational amino acids as well as the activities comparison of AbOPH with its variants are presented

Efficient Degradation of Malathion in the Presence of Detergents Using an Engineered Organophosphorus Hydrolase Highly Expressed by <i>Pichia pastoris</i> without Methanol Induction

The biodegradation of pesticides by organophosphorus hydrolases (OPHs) requires an efficient enzyme production technology in industry. Herein, a <i>Pichia pastoris</i> strain was constructed for the extracellular expression of <i>Po</i>OPH_M9, an engineered malathion-degrading enzyme. After optimization, the maximum titer and yield of fermentation reached 50.8 kU/L and 4.1 g_protein/L after 3 days, with the highest space-time yield (STY) reported so far, 640 U L^–1 h^–1. <i>Po</i>OPH_M9 displayed its high activity and stability in the presence of 0.1% (w/w) plant-derived detergent. Only 0.04 mg/mL enzyme could completely remove 0.15 mM malathion in aqueous solution within 20 min. Furthermore, 12 μmol malathion on apples and cucumbers surfaces was completely removed by 0.05 mg/mL <i>Po</i>OPH_M9 in tap water after 35 min washing. The efficient production of the highly active <i>Po</i>OPH_M9 has cleared a major barrier to biodegradation of pesticide residues in food industry

Dramatically Improved Performance of an Esterase for Cilastatin Synthesis by Cap Domain Engineering

Whole-protein random mutation and substrate tunnel evolution have recently been applied to the pharmaceutically relevant esterase <i>Rh</i>Est1 for the synthesis of a cilastatin precursor. The mutant <i>Rh</i>Est1_M1 (=<i>Rh</i>Est1_{A147I/V148F/G254A}) was identified from a large library consisting of 1.5 × 10⁴ variants. Though the activity of this mutant was improved 5-fold, the enantioselectivity for biohydrolysis decreased at the same time. Herein a smart library (3.0 × 10³) focused on the cap domain of <i>Rh</i>Est1 was constructed to improve its catalytic performance comprehensively. As a result, a variant designated as <i>Rh</i>Est1_M2 (=<i>Rh</i>Est1_M1‑A143T), showed a 6-fold increase in specific activity compared with the wild type. Meanwhile, the decreased enantioselectivity for enzymatic resolution was recovered to the native enzyme level. The melting temperature of <i>Rh</i>Est1_M2 was nearly 11 °C higher than that of the wild type. This work provides detailed insight into the vital role of α/β hydrolase cap domains in influencing all aspects of enzyme characteristics. Furthermore, the commercial resin ESR-1 with free amino groups was used for enzyme immobilization to enhance the operational performance of <i>Rh</i>Est1_M2. No obvious activity loss was observed when the immobilized enzyme was incubated at 30 °C for 200 h. The immobilized enzyme could be repeatedly used for up to 20 batches, and the total turnover number (TTN) reached up to 8.0 × 10⁵

Increased Catalyst Productivity in α‑Hydroxy Acids Resolution by Esterase Mutation and Substrate Modification

Optically pure α-hydroxy acids and their derivatives are versatile chiral building blocks in the pharmaceutical industry. In this study, the potential of a recombinant Pseudomonas putida esterase (rPPE01) for the enzymatic resolution of α-acetoxy acids was significantly improved by combinatorial engineering of both the biocatalyst and substrate. Semirational design based on homologous modeling and molecular docking provided a single-point variant, W187H, whose <i>k</i>_cat/<i>K</i>_M for sodium 2-acetoxy-2-(2′-chlorophenyl)­acetate (Ac-CPA-Na) was increased 100-fold, from 0.0611 to 6.20 mM^–1 s^–1, while retaining its excellent enantioselectivity and broad substrate spectrum. Biocatalyst deactivation under the operating conditions was decreased by using the potassium salt of Ac-CPA instead of Ac-CPA-Na. With 0.5 g L^–1 of lyophilized cells containing rPPE01-W187H, 500 mM (<i>R</i>,<i>S</i>)-Ac-CPA-K was selectively deacylated with 49.9% conversion within 15 h, giving satisfactory enantiomeric excesses (ee) for both the <i>S</i> product (>99% ee) and the remaining <i>R</i> substrate (98.7% ee). Consequently, the amount of (<i>S</i>)-2-hydroxy-2-(2′-chlorophenyl)­acetate prepared per unit weight of lyophilized cells was improved by a factor of 18.9 compared with the original productivity of the wild-type esterase. Further enzymatic resolution of other important hydroxy acids at the 100 mL scale demonstrated that the rPPE01-W187H-based bioprocess is versatile and practical for the large-scale preparation of chiral α-hydroxy acids