11 potential α-glucosidase inhibitors in leaves of Cyclocarya paliurus were quickly identified by UF-UPLC-Q/TOF-MS/MS, and their inhibitory activities were verified in vitro and in vivo, as well as docked with homology model.

Feruloyl or ferulic acid esterase (Fae, EC 3.1.1.73) catalyzes the hydrolysis of ester bonds between polysaccharides and phenolic acid compounds in xylan side chain. In this study, the thermostability of a type A feruloyl esterase (AuFaeA) from Aspergillus usamii was increased by iterative saturation mutagenesis (ISM). Two amino acids, Ser33 and Asn92, were selected for saturation mutagenesis according to the B-factors analyzed by B-FITTER software and ΔΔG values predicted by PoPMuSiC algorithm. After screening the saturation mutagenesis libraries constructed in Pichia pastoris, 15 promising variants were obtained. The best variant S33E/N92-4 (S33E/N92R) produced a T m value of 44.5 °C, the half-lives (t1/2) of 35 and 198 min at 55 and 50 °C, respectively, corresponding to a 4.7 °C, 2.33- and 3.96-fold improvement compared to the wild type. Additionally, the best S33 variant S33-6 (S33E) was thermostable at 50 °C with a t1/2 of 82 min, which was 32 min longer than that of the wild type. All the screened S33E/N92 variants were more thermostable than the best S33 variant S33-6 (S33E). This work would contribute to the further studies on higher thermostability modification of type A feruloyl esterases, especially those from fungi. The thermostable feruloyl esterase variants were expected to be potential candidates for industrial application in prompting the enzymic degradation of plant biomass materials at elevated temperatures.

The contribution of disulfide bridges to the thermostability of a type A feruloyl esterase (AuFaeA) from Aspergillus usamii E001 was studied by introducing an extra disulfide bridge or eliminating a native one from the enzyme. MODIP and DbD, two computational tools that can predict the possible disulfide bridges in proteins for thermostability improvement, and molecular dynamics (MD) simulations were used to design the extra disulfide bridge. One residue pair A126-N152 was chosen, and the respective amino acid residues were mutated to cysteine. The wild-type AuFaeA and its variants were expressed in Pichia pastoris GS115. The temperature optimum of the recombinant (re-) AuFaeAA126C-N152C was increased by 6°C compared to that of re-AuFaeA. The thermal inactivation half-lives of re-AuFaeAA126C-N152C at 55 and 60°C were 188 and 40 min, which were 12.5- and 10-folds longer than those of re-AuFaeA. The catalytic efficiency (k cat/K m) of re-AuFaeAA126C-N152C was similar to that of re-AuFaeA. Additionally, after elimination of each native disulfide bridge in AuFaeA, a great decrease in expression level and at least 10°C decrease in thermal stability of recombinant AuEaeA variants were also observed.

To perfect the enzymatic properties of AuMan5A, a mesophilic glycoside hydrolase (GH) family 5 β-mannanase from Aspergillus usamii, its loop-structure substitution was carried out by rational design and followed by megaprimer PCR. Based on the structural analysis and enzymatic property comparison of various β-mannanases, a piece of loop-structure with seven amino acids between two β-strands (βD and βE) in the substrate-binding groove, named "Loop DE," was speculated to be correlative to the thermostability and catalytic efficiency of GH family 5 β-mannanases. Therefore, three AuMan5A's mutants, AuMan5A-Af, AuMan5A-An, and AuMan5A-Th, were designed by substituting a Loop DE sequence ((316)KSPDGGN(322)) of AuMan5A with the corresponding sequences of other three family 5 β-mannanases, respectively. Then, the mutant-encoding genes, Auman5A-Af, Auman5A-An, and Auman5A-Th, were constructed as designed theoretically and then expressed in Pichia pastoris GS115. The expressed recombinant AuMan5A-Af (re-AuMan5A-Af) displayed the temperature optimum (T opt) of 75 °C, T m value of 76.6 °C and half-life (t 1/2) of 480 min at 70 °C, which were 10 and 12.1 °C higher and 48-fold longer than those of re-AuMan5A, respectively. Its catalytic efficiency (k cat/K m) was 12.7-fold that of re-AuMan5A. What is more, the site-directed mutagenesis of D320G in AuMan5A-Af was performed. The T opt and t 1/2 of expressed re-AuMan5A-Af(D320G) decreased to 70 °C and 40 min, respectively, while its k cat/K m was only 35 % of that of re-AuMan5A-Af. These results demonstrated that the mutation of G320 (in AuMan5A) into D320 (in AuMan5A-Af) through Loop DE substitution was mainly responsible for the thermostability and catalytic efficiency improvement of AuMan5A-Af.