Isolation and in vitro identification of proteinase inhibitors from soybean seeds inhibiting helicoverpa gut proteases

Figures & data

Figure 1.  Graph showing IC50 value Soybean varieties for HGP.

Table 1. Percent inhibition of trypsin and chymotrypsin activity.

		Inhibition (%)
Proteinase class	Fraction	MAUS-158	MAUS-61
Trypsin	Crude	61.68±0.61	63.22±1.16
	F0–60%	85.61±0.57	82.22±1.50
	F60–90%	33.75±1.02	32.47±1.07
Chymotrypsin	Crude	33.88±0.79	32.62±0.87
	F0–60%	42.74±1.66	40.20±1.12
	F60–90%	16.46±0.60	13.21±0.58

Figure 2.  Isoforms of proteases from crude HGP.

Table 2. Percent inhibition of HGP by crude and F1 fraction.

Proteinase class from HGP	Soybean PI fraction	Inhibition of HGP action (%)
		MAUS-158	MAUS-61
Trypsin like (BApNA assay)	Crude	48.46±0.53	52.07±0.78
	F0–60%	70.64±0.84	68.15±0.59
Chymotrypsin like (SAALPpNA assay)	Crude	31.58±0.71	32.45±0.58
	F0–60%	42.19±1.55	39.36±0.67
Total Protease inhibition (Azocasein assay)	Crude	56.57±0.72	52.10±0.64
	F0–60%	91.34±1.49	89.95±0.96

Table 3. Specific activity and yield of proteinase inhibitors after each step of partial purification.

Varieties	Steps	Total protein (mg)	PI units per mg	Specific activity per mg	Yield
MAUS-158	Crude	60	34.1	0.56	100
	0–60%	27	20.32	0.75	59.59
	Heat (60%)	22.5	18.54	0.82	54.36
MAUS-61	Crude	75	29.24	0.38	100
	0–60%	32	17.72	0.55	60.60
	Heat (60%)	25.5	15.1	0.59	51.24

Figure 3.  Isoforms of specific trypsin and chymotrypsin proteases using overlay technique. (a) Trypsin like proteases (using BApNA substrate) and (b) chymotrypsin like proteases (using SAALpNA substrate).

Figure 4.  (a) Gel–X-ray film contact technique and (b) zymography. Lanes 1 and 3 corresponds to the F1 and F2 saturated fraction of soybean cultivar MAUS-158. Lanes 2 and 4 corresponds to the F1 and F2 saturated fraction of soybean cultivar MAUS-61.

Figure 5.  Visualization of chymotrypsin-inhibiting isoforms from soybean using zymography. Lane 1, crude; Lane 2, F1 fraction; Lane 3, F2 fraction soybean cultivar MAUS-158; Lane 4, crude; Lane 5, F1 fraction; and Lane 6, F2 fraction from MAUS-61 cultivar.

Figure 6.  Visualization HGPI isoforms of soybean using Gel–X-ray film contact technique. (1) F1 fraction of MAUS 158 and (2) F1 fraction of MAUS 61.