Biocatalysis for Rare Ginsenoside Rh2 Production in High Level with Co-Immobilized UDP-Glycosyltransferase Bs-YjiC Mutant and Sucrose Synthase AtSuSy

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.

Evaluation of double expression system for co-expression and co-immobilization of flavonoid glucosylation cascade

“…Low pH values significantly disfavor the transfer of the glucose moiety to the acceptor molecule. The optimum operating temperature and thermostability attained for both Gm SuSy and YjiC correspond with other research investigations, as well as the influence of the co-immobilization on the characteristics and stability of the enzymes (Zhang et al 2019 ; Chu et al 2021 ; Liu et al 2021 ). Temperature above 40 °C displayed denaturing impact on the enzymes in both free and immobilized form (a increased turbidity of the reaction mixture with soluble enzymes was observed after the catalysis (data not shown)).…”

“…Current studies are focused mainly on the enhancement of robustness, increasing reaction yields, and application of a biocatalyst in an industrial environment. The co-immobilization of the enzymes is one of the employed strategies (Chu et al 2021 ; Liu et al 2021 ). Apart from the assets of single immobilization, such as stabilization, reusability, and facilitated downstream processing (Sheldon 2007 ), co-immobilization offers additional benefits.…”

“…Among available pathways for the formation of NDP-sugars, sucrose synthase (SuSy) enables the reversible transfer of glucose from cheap sucrose to nucleotide (NDP), delivering the simplest and tailored solution for a continuous supply of required activated glucoside (Schmölzer et al 2016 ). The SuSy-GT two-step glucosylation cascade has been already successively employed for the glucosylation of macrolides (Rupprath et al 2007 ), diterpenes (Zhang et al 2020 ), saponins (Ali et al 2021 ; Chu et al 2021 ), and flavonoids (Gutmann and Nidetzky 2016 ; Pei et al 2020 ; Liu et al 2021 ), both in soluble and heterogeneous (immobilized) form.…”

Characterization of Carboxylic Acid Reductase from Mycobacterium phlei Immobilized onto Seplite LX120

“…As seen in Figure 6 G,H, the support with adsorbed enzyme shows a compact and continuous structure after the enzyme immobilization, as compared with the structure of an empty support at 5000× and 10,000× magnifications. Similar SEM images were observed when UDP-glucosyltransferase and sucrose synthase were co-immobilized onto a heterofunctional resin [ 49 ]. The decrease in surface roughness and visibility of the spatial position of the support, as shown by SEM images, verified the successful enzyme immobilization [ 50 ].…”

Enzyme Immobilization and Biocatalysis

“…A field of application of choice for enzymatic catalysis, thanks to its high racemic resolution power, the purity of the products, and the use of substances that are not toxic to human health, is undoubtedly the pharmaceutical industry. For example, UDPglycosyltransferase Bs-YjiC mutant M315F and sucrose synthase AtSuSy were co-immobilized on heterofunctional supports (resin LX1000HG modified with glyoxyl-metal-chelate bifunctional groups) to promote the one-pot reaction of ginsenoside biosynthesis from fructose and protopanaxadiol (PPD) [6]. Rare ginsenoside Rh2 exhibits diverse pharmacological effects, such as anti-oxidation, hepatoprotection, and anti-diabetes and it is a promising candidate for cancer prevention and therapy.…”