Carboxylesterases (CarEs) is an important detoxification enzyme system in phase Ⅰ participating in insecticides resistance. In our previous study, SlCarE054, a CarEs gene from lepidoptera class, was screened out to be upregulated in a pyrethroids and organophosphates resistant population. Its overexpression was verified in two field-collected populations of Spodoptera litura (Lepidoptera: Noctuidae) resistant to pyrethroids and organophosphates by qRT-PCR. Spatiotemporal expression results showed that SlCarE054 was highly expressed in the pupae stage and the digestive tissue midgut. To further explore its role in pyrethroids and organophosphates resistance, its metabolism activity to insecticides was determined by UPLC. Its recombinant protein showed significant metabolism activity to cyhalothrin and fenvalerate, but not to phoxim or chlorpyrifos. The metabolic activity of SlCarE054 to β-cypermethrin showed stereoselectivity, with higher metabolic activity to θ-cypermethrin than the enantiomer α-cypermethrin. The metabolite of β-cypermethrin was identified as 3-phenoxybenzaldehyde. Further modelling and docking analysis indicated that β-cypermethrin, cyhalothrin and fenvalerate could bind with the catalytic triad of the 3D structure of SlCarE054. The interaction of β-cypermethrin with SlCarE054 also showed the lowest binding energy. Our work provides evidence that SlCarE054 play roles in β-cypermethrin resistance in S. litura.

Attempts at optical resolution and asymmetric syntheses using smectite clay minerals are described. Use of the method was prompted by the discovery that the saturated adsorption of a tris(chelated) metal complex, [Ru(1,10-phenanthroline)3]2+, by Na-montmorillonite depended heavily on the stereochemical properties. The pure enantiomer was adsorbed by cation exchange at negative surface sites of the clay mineral, while the racemic mixture was adsorbed to two times excess of the cation exchange capacity. The chelate takes a uniform orientation on a clay mineral surface due to the matching between the molecular symmetry and the two-dimensional network of a phyllosilicate layer. On a clay mineral surface covered with the enantiomeric chelates, a vacant space capable of chiral discrimination was generated. Based on this, an ion-exchange adduct of smectite and the chiral chelate was used as an adsorbent for separating racemic mixtures or selectively producing either one of the optical isomers.

The lipases from Rhizopus and Rhizomucor are members of the family of Mucorales lipases. Although they display high sequence homology, their stereoselectivity toward triradylglycerols (sn-2 substituted triacylglycerols) varies. Four different triradylglycerols were investigated, which were classified into two groups: flexible substrates with rotatable O′-C1′ ether or ester bonds adjacent to C2 of glycerol and rigid substrates with a rigid N′-C1′ amide bond or a phenyl ring in sn-2. Although Rhizopus lipase shows opposite stereopreference for flexible and rigid substrates (hydrolysis in sn-1 and sn-3, respectively), Rhizomucor lipase hydrolyzes both groups of triradylglycerols preferably in sn-1. To explain these experimental observations, computer-aided molecular modeling was applied to study the molecular basis of stereoselectivity. A generalized model for both lipases of the Mucorales family highlights the residues mediating stereoselectivity: (1) L258, the C-terminal neighbor of the catalytic histidine, and (2) G266, which is located in a loop contacting the glycerol backbone of a bound substrate. Interactions with triradylglycerol substrates are dominated by van der Waals contacts. Stereoselectivity can be predicted by analyzing the value of a single substrate torsion angle that discriminates between sn-1 and sn-3 stereopreference for all substrates and lipases investigated here. This simple model can be easily applied in enzyme and substrate engineering to predict Mucorales lipase variants and synthetic substrates with desired stereoselectivity.