Purification, characterisation, and thermal denaturation of polyphenoloxidase from prawns (Penaeus vannamei)

Figures & data

Figure 1. Purification and profiles of PPO from Penaeus vannamei. (A) Ammonium sulphate precipitation. (B) DEAE-Sepharose fast flow anion exchange chromatography. (C) Gel filtration chromatography on Sephadex G-100. (D) SDS–PAGE, M: Standard protein markers; lane 1: Purified PPO. (E) Native-PAGE, M: Native-markers; lane 1: Purified PPO stained with Coomassie brilliant R-250; lane 2: Purified PPO actively stained with L-DOPA.

Table 1. Purification of polyphenoloxidase from Penaeus vannamei using L-DOPA as the substrate

	Total activity	Volume		Total protein		Yield	Fold
Purification step	(Units)	(mL)	Protein concentration (mg /mL)	(mg)	Specific activity(U /mg protein)	(%)	purification
Crude extract	283288.32	400	11.71	4684	60.48	100	1
50% ammonium sulphate precipitation	114893.75	66	10.16	670.56	171.34	40.56	2.83
DEAE Sepharose Fast Flow Concentrated	39755.07	42	1.83	76.86	517.24	14.03	8.55
Sephadex G100, Concentrated	25318.57	37	1.19	44.03	575.03	8.94	9.51

Figure 2. Effects of pH (A) and temperature (B) on the PPO activity of Penaeus vannamei.

Figure 3. The K_m value was determined by using L-DOPA as substrate. According to the Lineweaver–Burk model, the K_m was calculated to be 2.5 mM.

Figure 4. Effects of heat treatments on residual activity (A) and DSC curve (B) of purified PPO from Penaeus vannamei.

Table 2. Inactivation parameters of Penaeus vannamei PPO

Temperature(°C)	k(min^−1)	r^2	t1/2 (min)	D(min)
40	0.0066 ± 0.0013	0.9832	105	348.9
50	0.0175 ± 0.0031	0.9984	39.6	131.6
60	0.0516 ± 0.0037	0.9972	13.4	44.6
70	0.0782 ± 0.0081	0.9842	8.9	29.4
80	0.2257 ± 0.0132	0.9995	3.1	10.2

Figure 5. Effects of thermal treatments on the tertiary structure of purified PPO. (A) Changes in the intrinsic fluorescence spectra of PPO by thermal treatments. Labels 1–6 indicate native, 40, 50, 60, 70, and 80°C, respectively. (B) The maximum wavelength of PPO intrinsic fluorescence at different temperatures.

Figure 6. FTIR spectra of purified PPO during the thermal treatments from 40 to 80°C for 10 min.

Table 3. Secondary structure change (%) of PPO obtained by curve-fitting during thermal treatment

Temperature (°C)	α-Helix (%)	β-Turn (%)	Random coil (%)	β-Sheet (%)
native	18.18 ± 1.20^a	21.15 ± 0.34^d	30.79 ± 0.25^d	29.88 ± 1.40^a
40	16.22 ± 0.90^a	20.81 ± 0.33^d	37.20 ± 0.60^c	25.77 ± 1.80^b
50	12.66 ± 0.31^bc	33.49 ± 0.89^bc	43.04 ± 0.23^a	10.81 ± 0.60^e
60	14.05 ± 0.10^b	33.06 ± 0.21^c	40.71 ± 0.16^b	12.18 ± 0.11^de
70	12.59 ± 1.89^bc	34.21 ± 0.55^b	38.45 ± 1.67^c	14.75 ± 0.47^c
80	11.82 ± 1.22^c	35.83 ± 0.41^a	38.81 ± 1.93^bc	13.54 ± 0.60^cd

Values are means ± standard deviation, and the small letters in the same column with the same letter are not significantly (p < 0.05).

Figure 7. AFM images of PPO during the thermal treatments from 40 to 80°C for 10 min.