%0 Journal Article
%T Combinatorial mutation on the ¦Â-glycosidase specific to 7-¦Â-xylosyltaxanes and increasing the mutated enzyme production by engineering the recombinant yeast
%A Fen Wang
%A Jin-Ling Yang
%A Jing-Jing Chen
%A Tian-Jiao Chen
%A Ting Gong
%A Wan-Cang Liu
%A Xiao Liang
%A Yan-Hua Wen
%J Archive of "Acta Pharmaceutica Sinica. B".
%D 2019
%R 10.1016/j.apsb.2018.11.003
%X Taxol is a ¡°blockbuster¡± antitumor drug produced by Taxus species with extremely low amount, while its analogue 7-¦Â-xylosyl-10-deacetyltaxol is generally much higher in the plants. Both the fungal enzymes LXYL-P1£¿1 and LXYL-P1£¿2 can convert 7-¦Â-xylosyl-10-deacetyltaxol into 10-deacetyltaxol for Taxol semi-synthesis. Of them, LXYL-P1£¿2 is twice more active than LXYL-P1£¿1, but there are only 11 significantly different amino acids in terms of the polarity and acidic-basic properties between them. In this study, single and multiple site-directed mutations at the 11 sites from LXYL-P1£¿1 to LXYL-P1£¿2 were performed to define the amino acids with upward bias in activities and to acquire variants with improved catalytic properties. Among all the 17 mutants, E12 (A72T/V91S) was the most active and even displayed 2.8- and 3-fold higher than LXYL-P1£¿2 on ¦Â-xylosidase and ¦Â-glucosidase activities. The possible mechanism for such improvement was proposed by homology modeling and molecular docking between E12 and 7-¦Â-xylosyl-10-deacetyltaxol. The recombinant yeast GS115-P1E12-7 was constructed by introducing variant E12, the molecular chaperone gene pdi and the bacterial hemoglobin gene vhb. This engineered yeast rendered 4 times higher biomass enzyme activity than GS115-3.5K-P1£¿2 that had been used for demo-scale fermentation. Thus, GS115-P1E12-7 becomes a promising candidate to replace GS115-3.5K-P1£¿2 for industrial purpose
%K ¦Â-Glycosidases
%K Combinatorial mutation
%K Improved catalytic property
%K Molecular docking
%K Engineered yeast
%K Taxol
%U https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6542770/