Purification and characterization of phenoloxidase from the hemolymph of healthy and diseased Antheraea assamensis Helfer (Lepidoptera: Saturniidae): Effects of certain biological components and chemical agents on enzyme activity

In the current study, a dimeric phenoloxidase (PO) from the hemolymph of healthy and diseased (pebrine infected) larvae of Antheraea assamensis Helfer was extracted and purified. The protein was subjected to purification using Sephacryl S‐100 and CM Sepharose chromatography. The enzyme comprised of two subunits of ~76.8 and 76 kDa that showed PO activity in 6 mM l ‐3,4‐dihydroxyphenylalanine ( L ‐DOPA) and 8 mM catechol but not in hydroquinone. Optimum temperature for PO activity was 30°C in l ‐DOPA and 37°C in catechol. Optimum pH ranged from 6.8 to 7.0 in L ‐DOPA and 7.0–7.2 in catechol. Specific activity of the purified PO from healthy larvae was 53.9 µM/min per mg of protein per ml in L ‐DOPA and 50.77 µM/min per mg of protein per ml in catechol. Specific activity of PO from diseased larvae was 30.0 µM/min per mg of protein per ml in L ‐DOPA and 28.55 µM/min per mg of protein per ml in catechol. Purification fold was 3.27–4.21 for healthy and 2.38–2.56 for diseased fractions. The enzyme showed the Michaelis constant ( K m ) of 2.46–2.85 mM for healthy and diseased fractions in L ‐DOPA. In catechol K m of 9.23–17.71 mM was observed. Peptidoglycan was the best activator of purified PO from both healthy and diseased fractions. Interactions between controls and activators appeared statistically significant ( F  = 767.5; df  = 3; P  < 0.0001). Na + , K + , and Cu 2+ increased, whereas Ca 2+ , Zn 2+ , Mg 2+ , and Co 2+ decreased PO activity. The overall interactions appeared highly significant ( F  = 217.0; df  = 27; P  < 0.0001). Kojic acid, dithiothreitol, thiourea, phenylthiourea, carbendazim, N ‐bromosuccinimide, N , N , N ′, N ′‐tetraacetic acid, and diethyldithiocarbamate inhibited PO activity.