Studies on the enzymatic synthesis of a new PPT-type ginsenoside via UDP-glycosyltransferase PgUGT74AE2 from Panax ginseng / 药学学报

UDP-glycosyltransferase PgUGT74AE2 from Panax ginseng can transfer a glucose moiety to the free C-3 hydroxyl of protopanaxadiol (PPD) to produce ginsenoside Rh2. However, no report demonstrates that PgUGT74AE2 can transfer a glucose moiety to the free C-3 hydroxyl of protopanaxatriol (PPT) to produce a PPT-type ginsenoside. In this study, the expression plasmid pET-32a-PgUGT74AE2 was constructed for expression of the recombinant protein and transferred into Escherichia coli Transetta (DE3) to generate the recombinant strain Transetta-PgUGT74AE2. The recombinant enzyme PgUGT74AE2 was expressed by induction of isopropyl-β-D-thiogalactoside (IPTG). An in vitro enzymatic reaction system was established with the recombinant enzyme PgUGT74AE2 and the substrate PPT. PgUGT74AE2 catalyzed the glycosylation of the free C-3 hydroxyl of PPT to produce 3-O-β-D-glucopyranosyl-dammar-24-ene-3β,6α,12β,20S-tetraol, a new PPT-type ginsenoside. This study provides an efficient approach for the biosynthesis of a new PPT-type ginsenoside through in vitro enzymatic reaction, which may pave a way to produce promising lead in drug discovery.

Studies on site-directed mutagenesis of BmK AngM1 from scorpion venom and its anti-inflammatory activity / 药学学报

Scorpion toxin BmK AngM1 has been reported to have a strong analgesic effect. However, its anti-inflammatory activity was unknown. In this study, the recombinant BmK AngM1 (rBmK AngM1) was expressed in Escherichia coli BL21 trxB (DE3). The purified rBmK AngM1 was obtained efficiently through the IMPACTTM-TWIN system. The anti-inflammatory activity of the recombinant protein was investigated. In order to improve the anti-inflammatory activity of rBmK AngM1, the potential active sites (Y5, Y42, R58) were substituted with different amino acids. The results showed that rBmK AngM1 and its mutants all have significant anti-inflammatory activity. The activities were significantly increased in the single mutant R58N and mutants Y5F/R58N, Y42F/R58N over the wild type protein. The data suggest that position 58 in BmK AngM1 plays a functional role in the anti-inflammatory activity. This study lays a foundation for the protein engineering design of BmK AngM1 to improve its pharmacological activity.

Expression, subcellular localization and characterization of dammarenediol-Ⅱ synthase of Panax ginseng in Saccharomyces cerevisiae / 药学学报

To study the expression and subcellular localization of recombinant dammarenediol-Ⅱ synthase (DS) in Saccharomyces cerevisiae, the dammarenediol-Ⅱ synthase gene ds was cloned from Panax ginseng, and the gene ds was fused with the gene of green fluorescent protein to obtain the fusion gene ds-gfp. The recombinant expression plasmids pESC-HIS-DS and pESC-HIS-DS-GFP were constructed and transformed into S. cerevisiae INVSc1 to obtain recombinant strains INVSc1-DS and INVSc1-DS-GFP. Microsomes of recombinant strains were prepared by differential centrifugation and observed by fluorescence microscope. The green fluorescence was only detected in INVSc1-DS-GFP microsomes, which indicated that DS was a membrane protein. It was also proved that dammarenediol-Ⅱ was produced from the cyclization of 2, 3-oxidosqualene catalyzed by DS through in vitro enzymatic reaction. In addition, our results revealed that the fusion expression of ds with gfp significantly improved the production of dammarenediol-Ⅱ from 7.53 mg·g-1 to 12.24 mg·g-1. This study provides a new strategy in the optimization of the pathway of ginsenosides biosynthesis in S. cerevisiae.