Production of chitinolytic enzymes with Trichoderma longibrachiatum IMI 92027 in solid substrate fermentation.

Abstract

Thirty Trichoderma strains representing 15 species within the genus were screened for extracellular production of chitinolytic enzymes in solid substrate fermentation. Trichoderma longibrachiatum IMI 92027 (ATCC 36838) gave the highest yield (5.0 IU/g of dry matter of substrate) after 3 d of fermentation on wheat bran-crude chitin (9:1 mixture) medium. The optimal moisture content (66.7%), chitin content (20%), initial pH of the medium (2.0-5.0), and time course (5 d) of solid substrate fermentation were determined for strain IMI 92027. Cellulase, xylanase, alpha-amylase, and beta-xylosidase activities were also detected. The pH and temperature optima of the chitinase complex of T. longibrachiatum IMI 92027 were 4.5 and 55 degrees C, respectively. The enzyme totally lost its activity at 70 degrees C in 5 min in the absence of the substrate but retained about 15% of its initial activity even at 70 degrees C after a 60-min incubation in the presence of solid substrate fermentation solids. Purification of protein extract from the solid substrate fermentation material revealed high chitinolytic activities between pI 5.9 and 4.8, where N-acetyl-beta-D-hexosaminidase and chitinase peaks have been found in the same pI range. Two chitinases of 43.5 and 30 kDa were purified at acidic pI.