The β-glycoside hydrolases (LXYL-P1−1 and LXYL-P1−2) from Lentinula edodes (strain M95.33) can specifically hydrolyze 7-β-xylosyl-10-deacetyltaxol (XDT) to form 10-deacetyltaxol for the semi-synthesis of Taxol. Our previous study showed that both the I368T mutation in LXYL-P1−1 and the T368E mutation in LXYL-P1−2 could increase the enzyme activity, which prompted us to investigate the effect of the I368E mutation on LXYL-P1−1 activity. In this study, the β-xylosidase and β-glucosidase activities of LXYL-P1−1^I368E were 1.5 and 2.2 times higher than those of LXYL-P1−1. Most importantly, combination of I368E and V91S exerted the cumulative effects on the improvement of the enzyme activities and catalytic efficiency. The β-xylosidase and β-glucosidase activities of the double mutant LXYL-P1−1^V91S/I368E were 3.2 and 1.7-fold higher than those of LXYL-P1−1^I368E. Similarly, the catalytic efficiency of LXYL-P1−1^V91S/I368E on 7-β-xylosyl-10-deacetyltaxol was 1.8-fold higher than that of LXYL-P1−1^I368E due to the dramatic increase in the substrate affinity. Molecular docking results suggest that the V91S and I368E mutation might positively promote the interaction between enzyme and substrate through altering the loop conformation near XDT and increasing the hydrogen bonds among Ser⁹¹, Trp³⁰¹, and XDT. This study lays the foundation for exploring the relationship between the structure and function of the β-glycoside hydrolases.

The glycoside hydrolase LXYL-P1–2 from Lentinula edodes can specifically hydrolyze 7-β-xylosyltaxanes to form 7-β-hydroxyltaxanes for the semi-synthesis of paclitaxel. In order to improve the catalytic properties of the enzyme, we performed error-prone PCR to construct the random mutant library of LXYL-P1–2 and used the methanol-induced plate method to screen the mutants with improved catalytic properties. Two variants, LXYL-P1–2-EP1 (EP1, S91D mutation) and LXYL-P1–2-EP2 (EP2, T368E mutation), were obtained from the library and exhibited 17% and 47% increases in their catalytic efficiencies on 7-β-xylosyl-10-deacetyltaxol. Meanwhile, compared with LXYL-P1–2, EP1 and EP2 showed elevated stabilities in the range of pH ≥ 6 conditions. After treatment at pH 12 for 48 h, EP1 and EP2 retained 77% and 63% activities, respectively, while the wild-type only retained 33% activity under the same condition. Molecular docking results revealed that the S91D mutation led to a shorter distance between the R-chain and the substrate, while the T368E mutation increased negative charge at the surface of the enzyme, and may introduce alterations of the loop near the active pocket, both of which may result in improved stabilities and catalytic activities of enzymes. This study provides a practical directed evolution method for exploring catalytically improved glycoside hydrolase.

Eight cadinane-type sesquiterpenes, including a new cadinane-type sesquiterpene, named as mappianiodene (1), and seven known analogues (2–8), were isolated and identified from the stems and leaves of Mappianthus iodoides. The chemical structure and absolute configurations of 1 was elucidated by extensive spectral methods and the known compounds were identified by comparing their experimental spectral data with the reported spectral data in the literature. The anti-inflammatory and anti-HIV activities of those isolated cadinane-type sesquiterpenes were tested. As a result, cadinane-type sesquiterpenes 1–8 displayed notable inhibitory effects on NO (nitric oxide) production with IC₅₀ values equivalent to that of the hydrocortisone. Moreover, compounds 1–8 exhibited pronounced anti-HIV-1 reverse transcriptase (RT) activities with the EC₅₀ values in range of 0.17 to 9.28 µM. Graphical abstract

Strain preservation to maintain stable vitality and the recombinant enzyme activity plays a crucial role in industrial fermentation. A Pichia pastoris strain is routinely stored at −80 °C in a glycerol vial and activated on an antibiotic-containing YPD agar plate before being used for fermentation. Alternatively, the activated strain should be preserved in the agar slant at 2~4 °C (low-temperature storage) for a short period before use. To maximize the utilization of the low-temperature storage for fermentation, we evaluated this method by observing the capacity of both the vitality and the recombinant enzyme activity of the strain at different preservation durations. We found that engineered yeast could be preserved by low-temperature storage for at least 30 months without losing its vitality and biomass enzyme activity by the end of fermentation and could be directly used for the seed cultivation of fermentation, which is more time-saving than strain recovery from −80 °C in a glycerol vial. Moreover, the antibiotic added to the agar slant could be omitted if the heterologous gene was integrated into the host chromosome. Our approach may greatly elevate the production efficiency of the strain.

The β-glycosidase LXYL-P1-2 identified from Lentinula edodes can be used to hydrolyze 7-β-xylosyl-10-deacetyltaxol (XDT) into 10-deacetyltaxol (DT) for the semi-synthesis of Taxol. Recent success in obtaining the high-resolution X-ray crystal of LXYL-P1-2 and resolving its three-dimensional structure has enabled us to perform molecular docking of LXYL-P1-2 with substrate XDT and investigate the roles of the three noncatalytic amino acid residues located around the active cavity in LXYL-P1-2. Site-directed mutagenesis results demonstrated that Tyr²⁶⁸ and Ser⁴⁶⁶ were essential for maintaining the β-glycosidase activity, and the L220G mutation exhibited a positive effect on increasing activity by enlarging the channel that facilitates the entrance of the substrate XDT into the active cavity. Moreover, introducing L220G mutation into the other LXYL-P1-2 mutant further increased the enzyme activity, and the β-d-xylosidase activity of the mutant EP2-L220G was nearly two times higher than that of LXYL-P1-2. Thus, the recombinant yeast GS115-EP2-L220G can be used for efficiently biocatalyzing XDT to DT for the semi-synthesis of Taxol. Our study provides not only the prospective candidate strain for industrial production, but also a theoretical basis for exploring the key amino acid residues in LXYL-P1-2.