Thirty Trichoderma strains representing 15 species within the genus have been screened for extracellular production of chitinolytic enzymes in solid substrate fermentation (SSF). T.longibrachiatum IMI 92027 (=ATCC 368...Thirty Trichoderma strains representing 15 species within the genus have been screened for extracellular production of chitinolytic enzymes in solid substrate fermentation (SSF). T.longibrachiatum IMI 92027 (=ATCC 36838) gave the highest yield (5.0 IU/g dry matter of substrate) after 3 days of fermentation on wheat bran-crude chitin (9:1 mixture) medium. The optimum moisture content (66.7%), chitin content (20%), initial pH of the medium (2-5) and time course (5 d) of SSF were determined for strain IMI 92027. No significant effect of different N and P additives was found on the chitinase yield in wheat bran-chitin mixture medium. Cellulase, xylanase, alpha-amylase and beta-xylosidase activities were also detected. The pH and temperature optima of chitinase complex of T.longibrachiatum IMI 92027 was found to be at 4.5 and 55 ℃, respectively. The enzyme totally lost its activity at 70 ℃ in 5 min in the absence of the substrate but retained about 15% of its initial activity even at 70 ℃ after incubation of 60 min in the presence of SSF solids (residual substrate and fungal mycelium). Purification of protein extract from the SSF material revealed high chitinolytic activities between pI 5.9.-4.8 where N-acetyl-β-D-hexosaminidase and chitinase peaks have been found in the same pI range. Chitinase peaks could be described at least at four pI values: pI 5.9; 5.6; 5.3 and 4.8 while at least four main N-acetyl-β-D-hexosaminidase peaks could be separated at pI 6.0 and 5.1 and at more basic isoelectric points of 7.2 and 8.0. Two chitinases with 43.5 kDa and 30 kDa were purified at acidic isoelectric point.展开更多
Over forty Trichoderma harzianum isolates have been screened in solid substrate fermentation (SSF) for chitinase production. Strains were isolated from Asian soil and tree bark samples. Identification was performed in...Over forty Trichoderma harzianum isolates have been screened in solid substrate fermentation (SSF) for chitinase production. Strains were isolated from Asian soil and tree bark samples. Identification was performed in Canada and Austria by classical and molecular taxonomical methods. Four SSF media were used for the screening. They contained wheat bran, crude chitin from crab shells (SIGMA) and different salt solutions for wetting of the substrate. In a five day fermentation at 30°C the best chitinase producers were T. harzianum TUB F-691, TUB F-693, TUB F-699, TUB F-700, TUB F-927, TUB F-947 and TUB F-972 isolates (TUB = Technical University of Budapest culture collection) . The best producers yielded between 3.5-5.5 International Unit/g dry matter (DM) chitinase. T. harzianum TUB F-947 was selected for further optimization. This strain was isolated from a soil of Thailand. The optimum moisture content (67%), optimal alternative substrate-carrier for SSF (wheat straw) and chitin content of the substrate (30%) were determined. Temperature and pH optima of chitinase produced by T. harzianum TUB F-947 were found to be at 50°C and 5.0, respectively. The enzyme complex was thermally not stable at 50 ℃. Medium optimization experiments to enhance the chitinase production using statistical methods also have been performed.展开更多
文摘Thirty Trichoderma strains representing 15 species within the genus have been screened for extracellular production of chitinolytic enzymes in solid substrate fermentation (SSF). T.longibrachiatum IMI 92027 (=ATCC 36838) gave the highest yield (5.0 IU/g dry matter of substrate) after 3 days of fermentation on wheat bran-crude chitin (9:1 mixture) medium. The optimum moisture content (66.7%), chitin content (20%), initial pH of the medium (2-5) and time course (5 d) of SSF were determined for strain IMI 92027. No significant effect of different N and P additives was found on the chitinase yield in wheat bran-chitin mixture medium. Cellulase, xylanase, alpha-amylase and beta-xylosidase activities were also detected. The pH and temperature optima of chitinase complex of T.longibrachiatum IMI 92027 was found to be at 4.5 and 55 ℃, respectively. The enzyme totally lost its activity at 70 ℃ in 5 min in the absence of the substrate but retained about 15% of its initial activity even at 70 ℃ after incubation of 60 min in the presence of SSF solids (residual substrate and fungal mycelium). Purification of protein extract from the SSF material revealed high chitinolytic activities between pI 5.9.-4.8 where N-acetyl-β-D-hexosaminidase and chitinase peaks have been found in the same pI range. Chitinase peaks could be described at least at four pI values: pI 5.9; 5.6; 5.3 and 4.8 while at least four main N-acetyl-β-D-hexosaminidase peaks could be separated at pI 6.0 and 5.1 and at more basic isoelectric points of 7.2 and 8.0. Two chitinases with 43.5 kDa and 30 kDa were purified at acidic isoelectric point.
文摘Over forty Trichoderma harzianum isolates have been screened in solid substrate fermentation (SSF) for chitinase production. Strains were isolated from Asian soil and tree bark samples. Identification was performed in Canada and Austria by classical and molecular taxonomical methods. Four SSF media were used for the screening. They contained wheat bran, crude chitin from crab shells (SIGMA) and different salt solutions for wetting of the substrate. In a five day fermentation at 30°C the best chitinase producers were T. harzianum TUB F-691, TUB F-693, TUB F-699, TUB F-700, TUB F-927, TUB F-947 and TUB F-972 isolates (TUB = Technical University of Budapest culture collection) . The best producers yielded between 3.5-5.5 International Unit/g dry matter (DM) chitinase. T. harzianum TUB F-947 was selected for further optimization. This strain was isolated from a soil of Thailand. The optimum moisture content (67%), optimal alternative substrate-carrier for SSF (wheat straw) and chitin content of the substrate (30%) were determined. Temperature and pH optima of chitinase produced by T. harzianum TUB F-947 were found to be at 50°C and 5.0, respectively. The enzyme complex was thermally not stable at 50 ℃. Medium optimization experiments to enhance the chitinase production using statistical methods also have been performed.
