Potential anti-fungal enzymes from drumstick tree callus (moringa oleifera Lam.)

Abstract

The root of Moringa oleifera (drumstick tree) was selected for the investigation of anti-fungal enzymatic activity of chitinase and glucanase. Natural root and root-derived calli were extracted with 0.02 M sodium acetate buffer, pH 5.6. Results showed that activity of chitinase was not detectable in native root of drumstick tree, but was detected in root-derived calli in 0-80%sat. protein precipitate at 0.026 ± 0.008 µu/mg. Glucanase activity in native root was also undetectable, but observed in crude extract of root-derived calli, 0-80%sat. protein precipitate and supernatant at 0.043 ± 0.009, 0.008 ± 0.000 and 0.002 ± 0.002 mu/mg, respectively. The specific activity of the enzymes from root-derived calli were found higher than that from native root of drumstick tree. The calli were cultured for future screening of anti-fungal enzymes. The seeds of moringa were used to produce seedlings for the moringa callus culture. Seedling of drumstick tree was produced by culturing on MS (Murashige & Skoog) agar medium at pH 5.8 for 6 weeks. The leaf, shoot and root segments of the seedling were cultured separately on MS agar medium containing 0.5, 1.0, 3.0 and 7.0 mg/L of 2,4-D (2,4-dichlorophenoxy acetic acid). The callus culture was havested at 10-13 weeks after subculturing in MS medium containing 1.0 mg/mL of 2,4-D. The enzymatic production in calli was studied in the elicited callus culture with elicitors including chitosan, methyl jasmonate and salicylic acid at various concentrations (10.0, 15.0, 30.0 and 45.0 mg/mL) in MS suspension with constant shaking at 90 rpm for 48 hr. The activity screening was performed using 0.02 M sodium acetate buffer, pH 5.6 at 25 °C. Root derived calli showed the specific activity without elicitation at 1.217 ± 0.216 pu/µg. Leaf-derived calli showed the highest chitinase specific activity of 0.721 ± 0.055, 1.586 ± 0.706 and 0.606 ± 0.143 pu/µg when elicitated with salicylic acid, methyl jasmonate and chitosan at 45.0, 30.0 and 15.0 mg/mL, respectively. Root-derived calli exhibited glucanase specific activity at 0.074 ± 0.012 and 0.448 ± 0.039 µu/µg for 10.0 and 15.0 mg/mL of methyl jasmonate and chitosan, respectively, while leaf-derived calli showed 0.010 ± 0.004 µu/µg at 10.0 mg/mL of methyl jasmonate and 0.013 ± 0.004 µu/µg at 15.0 mg/mL for chitosan elicitation. Production of all studied enzymes in shoot-derived calli was not affected by elicitors. Protease activity was not detectable in all callus culture at these conditions. This investigation revealed that methyl jasmonate and chitosan significantly enhanced the production of anti-fungal enzymes in callus culture of drumstick tree, while salicylic acid only slightly affected such production. All callus culture derived from shoot, root and leaf showed high activity of chitinase and glucanase in root and leaf-derived calli. The leaf-derived calli with higher activity were elicited bymethyl jasmonate (10.0 and 30.0 mg/mL) and chitosan (10.0 and 15.0 mg/mL) for 6 days. The calli were collected and extracted at different times of elicitation. The specific activity of chitinase and glucanase were the highest at 10.0 and 30.0 mg/mL of methyl jasmonate, respectively, on the third day. The methyl jasmonate was able to enhance the specific activity better than chitosan. The enhancement of chitinase and glucanase production by elicitors is an alternative mean to obtain the enzymes for future applications.