Characterization of a ginsenoside-transforming β-glucosidase from Paenibacillus mucilaginosus and its application for enhanced production of minor ginsenoside F₂

Abstract

A novel β-glucosidase (BglPm) was identified from Paenibacillus mucilaginosus KCTC 3870(T) which has ginsenoside converting activity. The gene, termed bglPm, consists of 1,260 bp and belongs to glycoside hydrolase family 1 (GH1). After being overexpressed and purified from Escherichia coli, the enzymatic properties of BglPm were investigated. The enzyme exhibited an optimal activity at 45°C and pH 7.5 and showed high bioconversion ability for major ginsenoside Rb1 and Rd into ginsenoside F2. Thus, it was used for mass production of relatively high pure F2 from relatively abundant protopanaxadiol type ginsenosides mixture (PPDGM) with combined usage of ginsenoside Rc-hydrolyzing enzyme. Scale-up of production using 250 g of the PPDGM resulted in 152 g of F2 with 80.1% chromatography purity and 95.7% recovery. These results suggest that this enzyme would be useful in the preparation of pharmacologically active ginsenoside F2 in the functional food and pharmaceutical industries.

Figure 1. Sequence alignment of BglPm and related ginsenoside-transforming or characterized glycoside hydrolase family 1 enzymes.

The conserved regions for the catalytic central in glycoside hydrolase family 1 are boxed, and the conserved catalytic amino acids are marked with an asterisk. The two putative conserved motifs were shown in box, and the predicted GH1 active site residues (general acid/base and nucleophile residue) were marked by an arrowhead. Full species names and Genbank IDs of the glycoside hydrolases family 1 are as follows, Paenibacillus mucilaginosus KCTC 3870^T β-glucosidase [P. muc (This study)], AEI42200; Uncultured bacteria β-glucosidase (U. bac), ABI18350; Thermus caldophilus β-glycosidase (T. cal), AAO15361; Sulfolobus solfataricus P2 β-glycosidase (S. sol), AAK43121; Sulfolobus acidocaldarius β-glycosidase (S. aci), WP_011278657; Sphingomonas sp. 2F2 β-glucosidase (S. 2F2), ADY18331; Pyrococcus furiosus β-glucosidase (P. fur), AAC25555; Micrococcus antarcticus β-glucosidase (M. ant), ACM66669; Uncultured bacterium β-glucosidase (U. bact), AFN70956; Cellulomonas biazotea cellobiase (C. bia), AEM45802.

Figure 2. SDS-PAGE analysis of the recombinant BglPm after purification using the GST-bind agarose resin.

Lanes: M, molecular weight standard; 1, insoluble fraction of the crude extract of the induced recombinant BL 21 (DE3) cells; 2, soluble fraction of the crude extract of the induced recombinant BL 21 (DE3); 3, GST-BglPm after purification with the GST-bind agarose resin.

Figure 3. Effects of pH (A) and temperature (B) on the activity and stability of recombinant BglPm.

Figure 4. Thin layer chromatography (TLC) analyses of biotransformation of Rb₁, Rd, Gyp XVII, Rg₃(S) in (A) and Rb₂, Rb₃, Rc in (B) by recombinant BglPm.

The reaction time was 30: CHCl₃-CH₃OH-H₂O (65∶35∶10, lower phase). (S, S1, S2, S3), ginsenoside standards (PPD type ginsenoside mixtures); Standards: 1, 3, 5, 7, 9, 11, 13; Reaction mixture: 2, 4, 6, 8, 10, 12, 14; 1, Rb₁; 2, reaction mixture of Rb₁; 3, Rd; 4, reaction mixture of Rd; 5, Gyp XVII; 6, reaction mixture of Gyp XVII; 7, Rg₃(S); 8, reaction mixture of Rg₃(S); 9, Rb₂; 10, reaction mixture of Rb₂; 11, Rb₃; 12, reaction mixture of Rb₃; 13, Rc; 14, reaction mixture of Rc. Abbreviations: C-K, compound K.

Figure 5. Biotransformation pathways of ginsenosides Rb₁, Rd, Rb₂, Rb₃, and Rc by recombinant BglPm and incorporated pathways for F₂ production by two glycoside hydrolases.

Figure 6. Effect of the concentration of PPDGM and crude BglPm on the production of ginsenoside F₂.

Figure 7. Relative abundance of ginsenosides Rb₁, Rc, Rd, Gyp XVII and F₂ in scaled-up production reactor in time course.

Figure 8. HPLC results of the transformation of PPDGM via Abf22-3 and followed by GST-BglPm; (a) ginsenoside peaks of PPDGM, (b) reaction mixture after 6 hour treatment with Abf-22-3, (c) reaction mixture after 1 hour treatment with GST-BglPm, (d) reaction mixture after 7 hour treatment with GST-BglPm.

Abbreviations: PPDGM, protopanaxadiol type ginsenoside mixture.