Overexpression and characterization of medium-chain-length polyhydroxyalkanoate granule bound polymerases from Pseudomonas putida GPo1

Table 2 Purification of PhaC1 and PhaC2 from P. putida GPp104 [pGEc405] and GPp104 [pGEc404].

Fraction		Total protein (mg)	PhaC (mg)	Recovery of PhaC (%)	Total activity (U)	Specific activity (U/mg protein)	Specific activity (U/mg PhaC)	Recovery of total activity (%)
Crude cell extract	PhaC1	750	1.8^a	-^c	67.5^b	0.09^b	37.5	100
	PhaC2	850	-^c	-^c	3.2^b	0.0375^b	-^c	100
Isolated PHA granules	PhaC1	2.4	1.3	100	52.8	22	40.6	78
	PhaC2	2.6	1.4	100	2.9	1.1	2.1	91
Released proteins from PHA granules	PhaC1	1.5	1.1	85	<0.01	<0.01	<0.01	<0.01
	PhaC2	1.2	0.9	64	<0.01	<0.01	<0.01	<0.01
Source 15Q and ammonia precipitation	PhaC1	0.9	0.8	61	< 0.01	< 0.01	<0.01	<0.01
	PhaC2	0.7	0.7	50	<0.01	<0.01	<0.01	<0.01

a) The amount of PhaC1 was estimated by Western blotting using anti-PhaC1 antibodies. In all other fractions, PhaC1 was estimated by densitometric scanning.
b) Activity was assayed by measuring depletion of 3-hydroxyoctanoyl-CoA (HPLC) in the presence of 1 mM CoA (to reduce background activity). In all other fractions PhaC1 activity was measured spectrophotometrically (A412) by following the release of CoA using DTNB.
c) Not determined.

ISSN: 1475-2859