Die Hu

Contribution of Disulfide Bridges to the Thermostability of a Type A Feruloyl Esterase from Aspergillus usamii

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.

Kinetic resolution of racemic styrene oxide at a high concentration by recombinant Aspergillus usamii epoxide hydrolase in an n-hexanol/buffer biphasic system.

Using the cell-free extract of engineered E. coli/Aueh2, expressing the recombinant Aspergillus usamii epoxide hydrolase (reAuEH2), as a biocatalyst, the kinetic resolution technique of racemic styrene oxide (rac-SO) was examined. In a phosphate buffer system (50mM, pH 7.0), 200mM rac-SO was efficiently resolved, obtaining (S)-SO with 98.1% enantiomeric excess (e.e.), whereas (S)-SO only with 45.2% e.e. was obtained from 750mM rac-SO. The analytical results verified that reAuEH2 shows tolerance towards high substrate concentration but is inactivated at a product concentration of 300mM. To produce (S)-SO with the high concentration, e.e. and volumetric productivity, n-hexanol was selected from a variety of water-miscible and water-immiscible organic solvents to construct an n-hexanol/buffer biphasic system. The optimal phase volume ratio, substrate over enzyme ratio and temperature were 1:1 (v/v), 6:1 (w/w) and 25°C, respectively. In an optimized biocatalytic system, a gram-scale resolution of rac-SO at a high concentration of 1M (120g/L) was performed at 25°C for 2h, obtaining (S)-SO with 98.2% e.e., 34.3% yield (maximum yield of 50%). The substrate concentration and volumetric productivity (1M, 20.6g/L/h) in a biphasic system significantly increased compared with those (0.2M, 3.1g/L/h) in a phosphate buffer system. The efficient resolution of rac-SO at a high concentration in a biphasic system makes it a promising technique for preparing a highly value-added enantiopure (S)-SO with high volumetric productivity.

Exploration of Disulfide Bridge and N-Glycosylation Contributing to High Thermostability of a Hybrid Xylanase

A comparison between three-dimensional structures of a wild-type xylanase AoXyn11A and a hybrid xylanase AEx11A revealed that a disulfide bridge (Cys(5)-Cys(32)) and an N-glycosylation site (Asn(42)) were imported into AEx11A by N-terminal substitution of AoXyn11A with EvXyn11(TS). Two mutant genes AEx11A(C5T) and AEx11A(N42Q) were constructed by mutating Cys(5)- and Asn(42)-encoding codons of AEx11A into Thr(5)- and Gln(42)-encoding ones, and heterologously expressed in Pichia pastoris GS115, respectively. The temperature optimum of the recombinant AEx11A(C5T) (reAEx11A(C5T)) was decreased to 60°C from 80°C of reAEx11A, while its thermal inactivation half-lives at 70 and 80°C shortened to 3 and 1 min from 197 and 25 min of reAEx11A, respectively. However, there was no obvious alteration between reAEx11A and reAEx11A(C5T) in pH characteristics and kinetic parameters. Furthermore, both reAEx11A(N42Q) and reAEx11A displayed no significant difference in all enzymatic properties tested, except for the apparent molecular weight. We concluded based on this study that the disulfide bridge of AEx11A was vital to its high thermostability, but the N-glycosylation had no effect on.

Multiple site-directed mutagenesis of a Phaseolus vulgaris epoxide hydrolase to improve its catalytic performance towards p-chlorostyrene oxide based on the computer-aided re-design

To improve the activity and regioselectivity of a Phaseolus vulgaris epoxide hydrolase (PvEH3) towards p-chlorostyrene oxide (pCSO), the site-directed mutagenesis was conducted based on the computer-aided re-design. Firstly, seven single-site variants of a PvEH3-encoding gene (pveh3) were constructed as designed theoretically and expressed in E. coli BL21(DE3), respectively. One transformant, E. coli/pveh3G170E, had the higher EH activity towards racemic pCSO, while both E. coli/pveh3F187L and /pveh3P237L with enhanced regioselectivity coefficient αS values. Secondly, to combine their respective merits, the double- and triple-site variants, pveh3G170E/F187L, pveh3G170E/P237L and pveh3G170E/F187L/P237L, were also constructed. Among all E. coli transformants, E. coli/pveh3G170E/F187L/P237L simultaneously had the highest EH activity of 20.3 U/g wet cell and αS value of 95.2%, by which the hydrolysis of rac-pCSO enantioconvergently produced (R)-p-chlorophenylethane-1,2-diol with an enantiomeric excess of 93.2%. Furthermore, PvEH3G170E/F187L/P237L expressed in E. coli/pveh3G170E/F187L/P237L was purified. Its specific activity and catalytic efficiency towards rac-pCSO were 4.1 U/mg protein and 1.81 mM-1 s-1, which were 3.0- and 3.1-fold those of PvEH3. Finally, the molecular docking simulation analysis indicated that PvEH3G170E/F187L/P237L preferentially attacks the more hindered benzylic carbon of (S)-pCSO over PvEH3, which was consistent with their αS values measured experimentally.

Stereoselective Hydrolysis of Epoxides by reVrEH3, a Novel Vigna radiata Epoxide Hydrolase with High Enantioselectivity or High and Complementary Regioselectivity

To provide more options for the stereoselective hydrolysis of epoxides, an epoxide hydrolase (VrEH3) gene from Vigna radiata was cloned and expressed in Escherichia coli. Recombinant VrEH3 displayed the maximum activity at pH 7.0 and 45 °C and high stability at pH 4.5-7.5 and 55 °C. Notably, reVrEH3 exhibited high and complementary regioselectivity toward styrene oxides 1a-3a and high enantioselectivity (E = 48.7) toward o-cresyl glycidyl ether 9a. To elucidate these interesting phenomena, the interactions of the three-dimensional structure between VrEH3 and enantiomers of 1a and 9a were analyzed by molecular docking simulation. Using E. coli/vreh3 whole cells, gram-scale preparations of (R)-1b and (R)-9a were performed by enantioconvergent hydrolysis of 100 mM rac-1a and kinetic resolution of 200 mM rac-9a in the buffer-free water system at 25 °C. These afforded (R)-1b with >99% eep and 78.7% overall yield after recrystallization and (R)-9a with >99% ees, 38.7% overall yield, and 12.7 g/L/h space-time yield.

Improved temperature characteristics of an Aspergillus oryzae GHF11 xylanase, by in silico design and site-directed mutagenesis

To improve the temperature characteristics of a mesophilic glycoside hydrolase family (GHF) 11 xylanase AoXyn11A from Aspergillus oryzae, both introduction of a disulfide bridge and the substitution of a specific amino acid were carried out by in silico design and site-directed mutagenesis. Based on the analysis of a known crystal structure of thermophilic xylanase TlXynA from Thermomyces lanuginosus, and the alignment of primary structures between AoXyn11A and TlXynA, one mutant AoXyn11AM with a disulfide bridge (Cys108–Cys152) was designed by replacing the Ser108 and Asn152 of AoXyn11A with Cys residues, respectively. Additionally, based on the analysis of amino acid B-factor values, another mutant AoXyn11AM-G22A was predicted by substituting Gly22 of AoXyn11AM (having the maximum B-factor value of 69.25 Å, with the corresponding Ala23 of TlXynA. Thereafter, two mutant xylanase-encoding genes, Aoxyn11AM and Aoxyn11AM-G22A, were constructed by site-directed mutagenesis. Aoxyn11A and two mutant genes were expressed in E. coli BL21(DE3) respectively, and three expressed recombinant xylanases, reAoXyn11A, reAoXyn11AM and reAoXyn11AM-G22A, were purified to homogeneity. The temperature optima of reAoXyn11AM and reAoXyn11AM-G22A were 60 and 65°C, respectively, being 5 and 10°C higher than that of reAoXyn11A. Their thermal inactivation half-lives at 50°C were 1.8- and 8.4-folds longer than that of reAoXyn11A. There were no obvious alterations after mutations in specific activity and enzymatic properties, except for the temperature characteristics.