Inexpensive, rapid procedure for bulk purification of cellulase-free beta-1,4-D-xylanase of high specific activity.
A process has been developed for the bulk purification of cellulase-free beta-1,4-D-xylanase from the fungus Trichoderma harzianum E58. The process involved the primary step of ultrafiltering the culture filtrate via a 10,000-molecular-weight cut-off membrane to separate the cellulase (retentate) and xylanase (permeate) fractions. The cellulase component was concentrated by 40- to 60-fold, resulting in an enzyme complex that could effectively hydrolyze high concentrations of cellulose and xylan to glucose and xylose. The xylanase was concentrated and solvent exchanged by adsorption to a cationic exchanger, SP-ZetaPrep 250, followed by elution with a pH change in the buffer to give a purified and concentrated xylanase complex dissolved in a low-salt buffer. The resultant xylanase system was pure by the criteria of sodium dodecyl sulfate polyacrylamide electrophoresis, had a very high specific activity of 2400 IU/mg protein, was virtually free of filter paper activity, and had a ratio of contaminating filter paper activity of 2 x 10(-6) (0.009% endoglucanase activity). Approximately 3.3 g protein, which contained in excess of 7 x 10(6) IU xylanase activity, was obtained from 17 L original culture filtrate. The process scheme was designed to facilitate scale-up to an industrial level of production.
Tan
L U
LU
Biotechnology and Chemistry Department, Forintek Canada Corp., 800 Montreal Road, Ottawa, Canada.
Yu
E K
EK
Louis-Seize
G W
GW
Saddler
J N
JN