Aqueous two-phase partitioning of liver proteinase from albacore tuna (Thunnus alalunga): Application to starry triggerfish (Abalistes stellaris) muscle hydrolysis

Figures & data

Table 1. Effect of phase composition in PEG1000-salt ATPS on partitioning of liver proteinase from albacore tuna.

Phase composition (%, w/w)	VR	KP	KE	SA	PF	Yield
20% PEG1000-15% NaH2PO4	–	–	–	–	–	–
20% PEG1000-20% NaH2PO4	1.83	0.53	7.01	8.56	2.31	68.75
20% PEG1000-25% NaH2PO4	0.83	1.14	7.56	3.00	0.81	41.84
20% PEG1000-15% (NH4)2SO4	1.07	1.02	5.70	3.72	1.00	46.49
20% PEG1000-20% (NH4)2SO4	0.90	1.00	3.47	3.03	0.82	37.74
20% PEG1000-25% (NH4)2SO4	0.64	1.08	4.97	3.12	0.84	39.83
20% PEG1000-15% MgSO4	2.45	0.65	0.37	3.61	0.98	41.32
20% PEG1000-20% MgSO4	1.36	0.79	0.44	2.67	0.72	31.65
20% PEG1000-25% MgSO4	0.92	0.90	0.53	1.39	0.37	16.50
20% PEG1000-15% K2HPO4	0.97	1.09	1.98	4.10	1.11	64.12
20% PEG1000-20% K2HPO4	0.57	1.04	2.10	3.23	0.87	52.30
20% PEG1000-25% K2HPO4	0.67	1.08	4.25	3.09	0.83	50.97
20% PEG1000-15% Na3C6H5O7	1.09	0.75	3.27	3.68	0.99	48.21
20% PEG1000-20% Na3C6H5O7	0.86	0.96	3.34	2.49	0.67	38.52
20% PEG1000-25% Na3C6H5O7	0.72	0.86	1.13	1.62	0.44	22.53
20% PEG1000-15% Na2SO4	1.08	1.47	1.08	3.74	1.01	47.85
20% PEG1000-20% Na2SO4	0.85	1.31	0.81	2.41	0.65	35.87
20% PEG1000-25% Na2SO4	0.67	1.42	0.87	1.74	0.47	30.10

(-) No phase separation.

V_R: volume ratio (upper/lower); K_P: partition coefficient of protein; K_E: partition coefficient of proteinases; SA: specific activity (U/(g protein) in the upper phase; PF: purification factor in the upper phase; Yield: recovery yield in the upper phase.

Table 2. Effect of PEG molecular mass and concentration in a PEG-NaH₂PO₄ ATPS on partitioning of liver proteinase from albacore tuna.

Phase composition (%, w/w)	VR	KP	KE	SA	PF	Yield
10% PEG1000-20% NaH2PO4	–	–	–	–	–	–
15% PEG1000-20% NaH2PO4	1.04	0.87	1.97	7.45	2.01	70.88
20% PEG1000-20% NaH2PO4	1.05	0.77	3.93	9.34	2.52	66.62
25% PEG1000-20% NaH2PO4	1.18	0.44	8.88	17.03	4.60	73.70
30% PEG1000-20% NaH2PO4	1.51	0.45	2.27	12.21	3.30	53.28
10% PEG4000-20% NaH2PO4	0.53	0.73	1.05	4.32	1.17	16.52
15% PEG4000-20% NaH2PO4	0.77	0.89	1.14	6.75	1.82	28.30
20% PEG4000-20% NaH2PO4	0.87	0.98	1.64	4.91	1.33	21.15
25% PEG4000-20% NaH2PO4	1.25	1.34	1.66	5.07	1.37	25.95
30% PEG4000-20% NaH2PO4	1.40	1.67	1.01	3.30	0.46	20.13

(-) No phase separation.

V_R: volume ratio (upper/lower); K_P: partition coefficient of protein; K_E: partition coefficient of proteinases; SA: specific activity (U/(g protein) in the upper phase; PF: purification factor in the upper phase; Yield: recovery yield in the upper phase.

Figure 1. Effect of system pH on the purification factor (a) and proteinase recovery (b) of liver proteinase partitioning in 25% PEG1000-20% NaH₂PO₄ ATPS. Bars represented the standard deviation from triplicate determinations. Different letters within the same parameter indicate the significant differences (P<0.05).

Figure 2. Effect of NaCl concentration on the purification factor (a) and proteinase recovery (b) of liver proteinase partitioning in 25% PEG1000-20% NaH₂PO₄ ATPS at pH 7.0. Bars represented the standard deviation from triplicate determinations. Different letters within the same parameter indicate the significant differences (P<0.05).

Figure 3. SDS-PAGE of liver extract and ATPS fraction from albacore tuna. M, molecular weight standard; lane 1, liver extract; lane 2, 20% PEG1000-20% NaH₂PO₄ ATPS fraction; lane 3, 25% PEG1000-20% NaH₂PO₄ ATPS fraction; lane 4, 25% PEG1000-20% NaH₂PO₄, pH 7.0 ATPS fraction.

Figure 4. Activity staining of liver extract and ATPS fraction from albacore tuna. M, molecular weight standard; lane 1, liver extract; lane 2, 20% PEG1000-20% NaH₂PO₄ ATPS fraction; lane 3, 25% PEG1000-20% NaH₂PO₄ ATPS fraction; lane 4, 25% PEG1000-20% NaH₂PO₄, pH 7.0 ATPS fraction.

Figure 5. Degree of hydrolysis (DH) of starry triggerfish muscle during hydrolysis with liver enzyme from ATPS fraction (top phase of system 25% PEG1000-20% NaH₂PO₄, pH 7.0). The hydrolytic reaction was performed at pH 8.5, 55°C. Bars represented the standard deviation from triplicate determinations.

Figure 6. SDS-PAGE patterns of starry triggerfish muscle with liver enzyme from ATPS fraction (top phase of system 25% PEG1000-20% NaH₂PO₄, pH 7.0). The hydrolytic reaction was performed at pH 8.5, 55°C. Numbers designate the enzyme level (unit). M: molecular weight standard; C: control MHC: myosin heavy chain; AC: actin.