Abstract
Rare ginsenoside Rh2 exhibits diverse pharmacological effects. UDP-glycosyltransferase (UGT) catalyzed glycosylation of protopanaxadiol (PPD) has been of growing interest in recent years. UDP-glycosyltransferase Bs-YjiC coupling sucrose synthase in one-pot reaction was successfully applied to ginsenoside biosynthesis with UDP-glucose regeneration from sucrose and UDP, which formed a green and sustainable approach. In this study, the his-tagged UDP-glycosyltransferase Bs-YjiC mutant M315F and sucrose synthase AtSuSy were co-immobilized on heterofunctional supports. The affinity adsorption significantly improved the capacity of specific binding of the two recombinant enzymes, and the dual enzyme covalently cross-linked by the acetaldehyde groups significantly promoted the binding stability of the immobilized bienzyme, allowing higher substrate concentration by easing substrate inhibition for the coupled reaction. The dual enzyme amount used for ginsenoside Rh2 biosynthesis is Bs-YjiC-M315F: AtSuSy = 18 mU/mL: 25.2 mU/mL, a yield of 79.2% was achieved. The coimmobilized M315F/AtSuSy had good operational stability of repetitive usage for 10 cycles, and the yield of ginsenoside Rh2 was kept between 77.6% and 81.3%. The high titer of the ginsenoside Rh2 cumulatively reached up to 16.6 mM (10.3 g/L) using fed-batch technology, and the final yield was 83.2%. This study has established a green and sustainable approach for the production of ginsenoside Rh2 in a high level of titer, which provides promising candidates for natural drug research and development.
Highlights
Ginsenosides are the major bioactive compounds from Chinese medicine Panax ginseng
UGT73C5 from A. thaliana was able to selectively glycosylate the C3-OH of PPD and ginsenoside CK to form ginsenosides Rh2 and F12, and 3.2 g/L ginsenoside Rh2 with a yield of 87% was attained by UGT73C5-AtSuSy coupled reactions [14]
The M315F and AtSuSy directly adsorbed by specifical binding with Ni2+-chelate groups were easy to lose the total enzyme activity using buffer wash over twice, which both M315F and AtSuSy retained less than 60% of activities (Figure 1B,C) by desorption test, suggesting the enzymes through direct affinity adsorption were easy to leach from the resin and
Summary
Ginsenosides are the major bioactive compounds from Chinese medicine Panax ginseng. PPD-type ginsenoside Rh2, an important saponin, showed diverse pharmacological activities, such as anti-oxidation, hepatoprotection, anti-diabetes [1,2,3], and it is a promising candidate for cancer prevention and therapy [4,5,6,7]. The co-localized multiple enzymes in close proximity of the active sites to one another with a suitable orientation by first step specifical adsorption may be beneficial to control the Catalysts 2021, 11, x FOR PEER REVcIEoWvalent cross-linking in the step, which leads to high activity expression, facilita5tionfg the access of substrates between one enzyme and another in an adequate concentration to perform improved reaction [27]. The M315F and AtSuSy directly adsorbed by specifical binding with Ni2+-chelate groups were easy to lose the total enzyme activity using buffer wash over twice, which both M315F and AtSuSy retained less than 60% of activities (Figure 1B,C) by desorption test, suggesting the enzymes through direct affinity adsorption were easy to leach from the resin and showed the low ability to bear mild conditions. The co-immobilization of M315F and AtSuSy with the two-step ways significantly improved the binding stability of the co-immobilized bi-enzyme, though histagged enzymes specifical binding with Ni2+-chelate groups and covalently cross-linking between the glyoxyl groups and the adsorbed proteins [29]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
