Extractive cultivation of recombinant Escherichia coli using aqueous two phase systems for production and separation of extracellular xylanase.
Recombinant Escherichia coli (pATBX 1.8) secreting extracellular xylanase was used as a model system to study the application of an aqueous two phase system for extractive cultivation. An increase in the polymer concentrations from 6 to 20% in the polyethylene glycol phosphate aqueous two phase system resulted in an increase in the phase volume ratio with a concomitant decrease in the partition coefficient (K) and recovery of xylanase in the top phase. However, varying phosphate concentrations from 8 to 16% decreased both the phase volume ratio and the partition coefficient of xylanase. The polyethylene glycol (6%) and phosphate (12%) system was found to be optimum for extracellular cultivation of E. coli, where extracellular xylanase was selectively partitioned to the top phase giving a purification ratio of above 1.0. The process was extended to a semicontinuous operating mode at the optimal condition, wherein the top phase containing xylanase was recovered and the surviving cells were recycled together with the new top phase. The maximum recovery of xylanase was obtained after 12 h in the top phase with a twofold increase in the specific activity as compared to the one obtained in the reference fermentation. In the present work, we report for the first time the use of the two phase system for the extractive cultivation of recombinant E. coli (pATBX 1.8) with the purpose of obtaining a simple and inexpensive separation procedure and achieving the maximal extraction of xylanase to one phase.
Biochemical Sciences Division, National Chemical Laboratory, Pune, India.
N
N
Kulkarni
A
A
Vaidya
M
M
Rao
D001431:Bacteriological Techniques; D003470:Culture Media; D043364:Endo-1,4-beta Xylanases; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development*; D005285:Fermentation; D010710:Phosphates; D010940:Plant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D011092:Polyethylene Glycols; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D011995:Recombination, Genetic; D013997:Time Factors; D014867:Water; D014995:Xylosidases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
D003470:Culture Media; D010710:Phosphates; D010940:Plant Proteins; D011994:Recombinant Proteins; D014867:Water; D011092:Polyethylene Glycols; D014995:Xylosidases; D043364:Endo-1,4-beta Xylanases