TABLE 5.

Purification of recombinant EntL50A and EntL50B produced by S. cerevisiae L50A-20(pYABD02) and S. cerevisiae L50B-4(pYABD03), respectively^a

Purification stage	Vol (ml)	Total A254b	Total activity (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
S. cerevisiae L50A-20(pYABD02)
Culture supernatant	1000	24,580	142,100	6	1	100	18,500	100	7.7
Ammonium sulfate precipitation	100	221	15,700	71	12	11	ND	ND	ND
Gel filtration chromatography	200	37.4	12,800	342	57	9	ND	ND	ND
Cation exchange chromatography	50	3.9	1,100	282	47	0.8	ND	ND	ND
Hydrophobic-interaction chromatography	10	0.8	14,200	17,750	2,958	10	ND	ND	ND
Reversed-phase chromatography	2.6	0.7	1,900	2,714	452	1.3	8,600	46.5	0.2
S. cerevisiae L50B-4(pYBAD03)
Culture supernatant	1000	14,400	46,000	3	1	100	51,300	100	0.9
Ammonium sulfate precipitation	100	157	10,000	64	21	22	ND	ND	ND
Gel filtration chromatography	200	29.6	6,400	216	72	14	ND	ND	ND
Cation exchange chromatography	50	0.5	0	0	0	0	ND	ND	ND
Hydrophobic-interaction chromatography	10	3.0	2,700	900	300	5.9	ND	ND	ND
Reversed-phase chromatography	1.9	0.2	1,200	6,000	2,000	2.6	23,500	45.8	0.05

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

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

^cAntimicrobial activity 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. ND, not determined.

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