TABLE 4.

Purification of recombinant EntL50A and EntL50B produced by P. pastoris X-33A and P. pastoris X-33B, respectively^a

Strain and purification stage	Volume (ml)	Total A254b	Total activity (103 BU)c	Sp act (BU/A254)d	Increase in sp act (fold)e	Total activity (%)	Enterocin yield (ng)f	Enterocin yield (%)	Enterocin sp act (BU/ng)g
P. pastoris X-33A
Culture supernatant	400	320.8	670	2,100	1	100	114,500	100	5.9
Ammonium sulfate precipitation	40	29.2	865	29,600	14	129	74,100	65	11.7
Gel filtration chromatography	80	10.9	550	50,500	24	82	68,000	60	8.1
Cation-exchange chromatography	50	2.3	490	213,000	102	73	57,300	50	8.6
Hydrophobic-interaction chromatography	10	0.9	450	500,000	239	67	46,200	41	9.7
Reversed-phase chromatography	3.5	0.9	290	322,200	154	43	24,000	21	12.1
P. pastoris X-33B
Culture supernatant	400	321.2	40	124	1	100	89,600	100	0.4
Ammonium sulfate precipitation	40	29.3	100	3,400	27	250	60,900	68	1.6
Gel filtration chromatography	80	15.2	24	1,600	12	60	57,300	64	0.4
Cation-exchange chromatography	50	2.1	3	1,400	12	7.5	49,300	55	0.06
Hydrophobic-interaction chromatography	10	1.0	6	6,000	48	15	29,600	33	0.2
Reversed-phase chromatography	1.6	0.1	2	20,000	161	5	6,500	7.2	0.3

^aCultures were grown in BMMY broth at 30°C.

^bAbsorbance at 254 nm multiplied by the volume in milliliters.

^cAntimicrobial activity against Pc. damnosus CECT4797 in bacteriocin units per milliliter (BU/ml), as determined by an MPA, multiplied by the total volume.

^dSpecific activity expressed as the total activity (BU) divided by the total A₂₅₄.

^eThe specific activity of a fraction (BU/A₂₅₄) divided by the specific activity of the culture supernatant (BU/A₂₅₄).

^fEntL50A and EntL50B concentration as determined by an NCI-ELISA using specific polyclonal antibodies for EntL50A or EntL50B.

^gSpecific activity expressed as the total activity (BU) divided by the enterocin yield (ng).