Biological Characteristics of Recombinant Arthrobotrys oligospora Chitinase AO-801

Abstract

Chitinase AO-801 is a hydrolase secreted by Arthrobotrys oligospora during nematode feeding, while its role remained elusive. This study analyzed the molecular characteristics of recombinant chitinase of Arthrobotrys oligospora (reAO-801). AO-801 belongs to the typical glycoside hydrolase 18 family with conserved chitinase sequence and tertiary structure of (α/β)8 triose-phosphate isomerase (TIM) barrel. The molecular weight of reAO-801 was 42 kDa. reAO-801 effectively degraded colloidal and powdered chitin, egg lysate, and stage I larval lysate of Caenorhabditis elegans. The activity of reAO-801 reached its peak at 40˚C and pH values between 4-7. Enzyme activity was inhibited by Zn2+, Ca2+, and Fe3+, whereas Mg2+ and K+ potentiated its activity. In addition, urea, sodium dodecyl sulfate, and 2-mercaptoethanol significantly inhibited enzyme activity. reAO-801 showed complete nematicidal activity against C. elegans stage I larvae. reAO-801 broke down the C. elegans egg shells, causing them to die or die prematurely by hatching the eggs. It also invoked degradation of Haemonchus contortus eggs, resulting in apparent changes in the morphological structure. This study demonstrated the cytotoxic effect of reAO-801, which laid the foundation for further dissecting the mechanism of nematode infestation by A. oligospora.

Keywords: Arthrobotrys oligospora; biological activity; chitinase; enzyme characteristic.

Fig. 1

Substrate determination of recombinant chitinase AO-801. (A) Standard curve line of NAG. (B) Effects of different substrate on reAO-801. Error bars represents the standard deviation (n=3).

Fig. 2

Effects of temperature, pH, metal ions and inhibitors on reAO-801. (A) Effects of different temperature on reAO-801. (B) Effects of different pH on reAO-801. (C) Effects of different metal ions on reAO-801. (D) Effects of different inhibitors on reAO-801.Relative activity was expressed as a percentage of the standard activity. All values were the average of 3 replications. Error bars represents the standard deviations. **P< 0.001.

Fig. 3

The C. elegans larva of stage I treated with reAO-801. Control: (A–C) C. elegans larva of stage I treated with the inactivated reAO-801 after 0, 6, and 12 h, respectively. Experiment: (D–F) C. elegans larva of stage I treated with the reAO-801 after 0, 6, and 12 h, respectively.

Fig. 4

The degradation role of reAO-801 against C. elegans eggs. Control: (A–C) C. elegans eggs treated with the inactivated reAO-801 after 0, 6, and 12 h, respectively. Experiment: (D–F, G–I) C. elegans eggs treated with the reAO-801 after 0, 6, and 12 h, respectively.

Fig. 5

The degradation role of reAO-801 against H. contortus eggs. Control: (A–C) H. contortus eggs treated with the inactivated reAO-801 after 0, 6, and 12 h, respectively. Experiment: (D–F) H. contortus eggs treated with the reAO-801 after 0, 6, and 12 h, respectively.