Antimicrobial activity and enzymatic analysis of endophytes isolated from Codonopsis pilosula

Abstract

The roots of the medicinal plant Codonopsis pilosula (Franch.) Nannf (C. pilosula) possess most medicinal supplements. In current research on C. pilosula root endophytes were isolated, identified, and evaluated for their antimicrobial activity against human pathogens such as Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Salmonella typhi, and Pseudomonas aeruginosa and the fungi Candida albicans and Aspergillus niger. Endophytes C.P-8 and C.P-20 exhibited very significant antimicrobial activity, the secondary metabolite of C.P-8 registered at retention time 24.075 by HPLC analysis. Significant minimum inhibitory concentration (MIC) of C.P-8 was exhibited at 250 µg/ml against S. aureus and 500 µg/ml against B. subtilis. Qualitative, quantitative analyses, and partial purification of enzymes and purity was analysed by molecular weight determined by SDS‒PAGE of enzymes produced by C.P-20, amylase-64 kDa, protease-64 kDa, chitinase-30 kDa, and cellulase-54 kDa. Optimum pH and temperature of the partially purified enzymes, was carried out. The partially purified enzymes from C.P-20 displayed maximum activity at pH 6-7 and temperatures of 40°C-45°C. Moreover, the above endophytes will be useful tools for producing active enzymes and active bioantimicrobial agents against human pathogens.

Keywords: Codonopsis pilosula; antimicrobial activity; endophytes; enzymatic activity; secondary metabolites.

Figure 1.

Bacterial isolates of C. pilosula. The endophytic bacteria isolate from the root of C. pilosula on trypticase soy agar (TSA). (A) The root of C. pilosula was washed several times and the last wash distilled sterile water 100 µl was inoculated on to the TSA plate, there is no growth of bacteria. (B) Endophytic bacteria CP-8 grown on the TSA. (C) Endophytic bacteria CP-20 grown on the TSA.

Figure 2.

Morphological identification of active endophytes of C. pilosula. The Gram’s staining and microscopic analysis of C.P-8 (A) and C.P-20 (B) under 100x lenses represents that C.P-8 was the Gram-negative, rod-shaped bacteria and C.P-20 was the Gram-positive, rod-shaped bacteria.

Figure 3.

Preliminary analyses of the antibacterial activities of C. pilosula endophytic bacteria. Codonopsis pilosula endophytic bacteria C.P-8 agar plug evinced antibacterial activity against B. subtilis, S. aureus, and E. coli. Negative control (C-ve)—LB broth, positive control (C +ve)—40 µg/ml ampicillin.

Figure 4.

Antibacterial activity by fungal endophyte. Antibacterial activity evinced significant by C.P-F-11 (P. commune) against S. aureus and B. subtilis.

Figure 5.

Antibacterial activity of endophytic bacteria. Antibacterial activity of the bacterial endophytes of C. pilosula. Negative control (C-ve)—LB broth, positive control (C +ve)—40 µg/ml ampicillin. C.P-8 cell free supernatant exhibited significant antibacterial activity against B. subtilis (A) and Streptococcus aureus (B). C.P-F-11 cell-free supernatant exhibited antibacterial activity against S. aureus. C.P-14 and C.P-24 cell-free supernatant has no antibacterial activity.

Figure 6.

Antifungal activity of the bacterial endophytes. C.P-20 (B. velezensis) displayed the most significant antifungal activity, and C.P-14 (P. terrae strain IHBB 9910) exhibited mild antifungal activity against A. niger.

Figure 7.

Antibacterial analysis of C.P-8 crude secondary metabolite extract. Antimicrobial activity of endophytes of C.P-8 was analysed against human pathogens, here, blank—Muller–Hinton broth, control negative (C –ve)—Muller–Hinton broth, and DMSO at 1:1 ratio inoculated with human pathogen with 1 × 10⁴ CFU/ml), control positive (C +ve)—Muller–Hinton broth, and DMSO at 1:1 ratio inoculated with human pathogen with 1 × 10⁴ CFU/ml) and antibiotic ampicillin—0.01, 0.05, 0.5, 1, and 5 µg/ml. The significant MIC of positive control evinced at 0.5 µg/ml against B. subtilis and 0.05 µg/ml against S. aureus. 100–5000 µg/ml concentration of C.P-8 endophytic bacteria crude extracts of secondary metabolites against B. subtilis and S. aureus. Significant MIC of the crude extract was exhibited at 250 µg/ml against S. aureus and 500 µg/ml against B. subtilis.

Figure 8.

Comparison of antibacterial activity of C.P-8 with other endophytes: the antibacterial activity of C.P-8 was significant compared to other endophytes. (A) Antibacterial activity against B. subtilis was analysed, positive control—antibiotic ampicillin with 0.5 µg/ml concentration evinced antibacterial activity. C.P-8 represented its activity at 500 µg/ml concentration, whereas, C.P-F-11 exhibited activity at 750 µg/ml concentration. (B) Antibacterial activity against S. aureus was analysed, the positive control evinced activity at 0.05 µg/ml concentration. C.P-8 represented its MIC at 250 µg/ml concentration. Whereas, C.P-F-11 represented significant activity at 750 µg/ml concentration compared to the other endophytes.

Figure 9.

HPLC analysis of secondary metabolites produced by C.P-8: The major secondary metabolite produced by C.P-8 was registered at retention time 24.075.

Figure 10.

Activities of the C. pilosula endophyte exoenzymes: The exoenzymes activity of the endophytes is presented. Blank—broth medium, control—substrate reagent without enzyme or cell-free supernatant. (A) C.P-4 (B. subtilis) and C.P-20 (B. velezensis) showed significant amylase activity (6 U/ml and 5.6 U/ml, respectively). (B) C.P-20 (B. velezensis) exhibited significant protease activity (0.3 U/ml). (C) C.P-20 (B. velezensis) exhibited significant chitinase activity (3583.67 U/ml). (D) C.P-20 (B. velezensis) and C.P-4 (B. subtilis) evinced significant cellulase activity (8.9 U/ml and 7.3 U/ml, respectively).

Figure 11.

SDS- PAGE analysis of partially purified enzymes from C.P-20 and C.P-8: Lane-1: protein marker, Lanes-2–5: molecular weights of partially purified enzymes from C.P-20 (Lane-2: amylase, Lane-3: protease, Lane-4: cellulase, and Lane-5: chitinase). Lanes-6–9: molecular weights of partially purified enzymes from C.P-8 (Lane-6: amylase, Lane-7: protease, Lane-8: cellulase, and Lane-9: chitinase.

Figure 12.

Biochemical characterization of partially purified enzymes: (A) and (B): represents optimum pH and temperature for relative amylase activity. (C) and (D): represents optimum pH and temperature for relative protease activity. (E) and (F): represents optimum pH and temperature for relative cellulase activity. (G) and (H): represents optimum pH and temperature for relative chitinase activity.