Molecular and biochemical characterization of recombinant cel12B, cel8C, and peh28 overexpressed in Escherichia coli and their potential in biofuel production

Table 2 Purification steps of cel12B, cel8C, and peh28 overexpressed in E. coli ^a

Enzyme	Purification method	Fractions	Total enzyme activity (units)^c	Total protein (mg)^d	Specific activity (U/mg protein)	Purification fold	Yield (%)
cel12B	Crude extract^b		4.49	73.2	0.061	0	100
	UF^e-PES^f—MWC^g (100 kDa)	Permeate	3.73	38.0	0.098	1.6	83.1
	UF-PES—MWC (50 kDa)	Permeate	2.76	11.0	0.251	4.1	61.4
	UF-PES—MWC (30 kDa)	Permeate	1.91	2.32	0.823	13.4	42.6
	Gel filtration^h		1.32	1.24	1.06	17.4	29.3
cel8C	Crude extract		28.7	75.3	0.381	0	100
	UF-PES—MWC (100 kDa)	Permeate	25.8	44.5	0.579	1.5	89.7
	UF-PES—MWC (50 kDa)	Retentate	23.1	27.4	0.844	2.2	80.5
	UF-PES—MWC (30 kDa)	Permeate	18.2	7.39	2.47	6.5	63.4
	Gel filtration		14.4	6.08	2.36	6.2	50.0
peh28	Crude extract		1.58	88.8	0.0178	0	100
	UF-PES—MWC (100 kDa)	Permeate	1.37	52.2	0.0262	1.5	86.5
	UF-PES—MWC (50 kDa)	Retentate	1.31	31.2	0.0419	2.4	82.6
	UF-PES—MWC (30 kDa)	Permeate	1.00	11.3	0.0888	5.0	63.4
	Gel filtration		0.708	6.56	0.108	6.0	44.7

All values are given as a mean of triplicates ± SE
^aCel12B, cel8C, and peh28 are clones of Pcc for genes encoding cellulase B, cellulase C, and polygalacturonase, respectively, that were transformed into E. coli using pTAC-MAT expression vector
^bCrude extracts are cell-free extracts of E. coli cell-free lysates. The cultures were stimulated for enzyme induction for 5 h, for cel12B and cel8C, and for 7 h, for peh28, at 37 °C using 10 mM IPTG
^cOne Unit of enzymatic activity is defined as the amount of enzyme releasing 1 µmol of reducing sugars per minute from the substrate under the assay conditions (pH 5.0 at 40 °C, for cel12B and cel8C, and pH 5.0 at 40 °C, for peh28)
^dAll protein concentrations are in mg per ml of enzyme solution at each fractionation stage
^eUltrafiltration
^fPolyethersulfone
^gMolecular weight cut-off
^hGel filtration was carried out using Sephadex G-100

ISSN: 2731-3654