Fig 1.
Vector map of CPXpETDuet-1 (Plotted using SimVector4.0 software).
Table 1.
Primer sequences used for amplification of CotA, Pel and Xyl genes.
Fig 2.
Predicted restriction digestion pattern of CPXpETDuet-1 plasmid construct when treated with different restriction enzyme combinations.
Fig 3.
Western blotting analysis of the expression of recombinant cocktail in E. coli BL21(DE3).
Lane 1: Protein MW marker; Lane 2: Supernatant of induced culture; Lane 3: Cell biomass of induced culture; Lane 4–5: Western blot of CS and cell biomass showing no signal in the supernatant which might be due to removal of N-terminal signal sequence along with His tag during secretion.
Fig 4.
Comparison of CPX expression when cultivated in LB, wheat bran and terrific broth.
Significant amount of pectate lyase and endoxylanase was observed in CS. Lane 1: Protein MW marker; Lane 2: LB uninduced control culture; Lane 3–4: LB induced culture cell biomass and CS; Lane 5–7: LB+wheat bran induced culture CF pellet, CF sup and CS; Lane 8–10: terrific broth induced culture CF pellet, CF sup and CS.
Table 2.
Activity of CotA (a), Xyl (b) and Pel (c) in CS, PF and CF when expressed in E. coli BL21(DE3), E. coli BL21(DE3) pTUM4 and E. coli BL21(DE3) Arctic.
Table 3.
Comparison of the Activities of Pel, CotA and Xyl between cocktail and individual gene expression.
Table 4.
Activity of recombinant enzymes from various sub-cellular fractions obtained during scale-up.
Fig 5.
Bioreactor level optimization of the recombinant enzyme cocktail production.
(a) Change in DO concentration with respect to change in RPM; (b) Increase in dry cell weight (DCW) with respect to change in OD600nm, and DO concentration; (c) Increase in the activities of CotA, Pel and Xyl with respect to DCW; and (d) Change in concentration of glucose and glycerol with respect to DCW and OD600nm.
Fig 6.
Comparison of the hydrolyzing efficiency of CPX enzyme cocktail and Xyl alone by sugar release estimation at uniform time intervals.