Antibody preparation, cellulase

NG14. This mutant is capable of elaborating 15 FP units/mL with a productivity of 45 units/L/hr. The final concentration of soluble extracellular protein is 21.2 mg/mL. Samples of this enzyme preparation were sent to G. Pettersson at the University of Uppsala, Sweden, for quantification of each of the enzymes in the cellulase complex using purified antibodies to the individual enzymes. The quantitative antigen-antibody reaction showed that 600 mg/g of this enzyme preparation was one enzyme, cellobiohydrolase. This represents a yield of 13 g/L of cellobio-hydrolase, which is a 100-fold increase over the amount of cellobiohydrolase obtained with strain QM 9414 (130 mg/L) the best previously existing cellulase mutant (G. Pettersson, personal communication). 

A number of selective-screening methodologies have been devised that have allowed isolation of a series of hyperproducing and catabolite repression-resistant mutants of T. reesei. Yields of cellulase of 15 units/ mL under controlled fermentor conditions have been achieved with both Rut-NG14 and Rut-C30. Quantitative reaction of Rut-NG14 enzyme preparation with purified antibodies to cellobiohydrolase shows that in this mutant, the cellobiohydrolase is specifically hyperproduced relative to the rest of the enzymes in the cellulase complex. Rut-C30, which was derived from Rut-NG14, shows resistance to catabolite repression for... 

Preparation of antibody. The purified cellulase CIV (1 mg) was dissolved in 1 ml of 0.2% sodium dodecyl sulfate (SDS), boiled for 10 min, and emulsified with 1 ml of Freund complete adjuvant (Difco Co.) by sonlcatlon. It was subcutaneously injected on the back of two New Zealand white rabbits. After 2 weeks, 2 subcutaneous injections of the enzyme emulsion prepared with Freund incomplete adjuvant (Difco Co.) were carried out at an 8-day interval. One week after the last injection, the blood was withdrawn and the antiserum was obtained. 

We thank Mr Miwa of the Ajinomoto Co. for preparing the crude cellulase of S. cellulophilum and cellulase CIV antibody. 

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