Meng-Chun Lin1,2, Hsion-Wen Kuo3, Mu-Rong Kao1,2, Wen-Dar Lin1, Chen-Wei Li1, Kuo-Sheng Hung1, Sheng-Chih Yang4, Su-May Yu2,5, Tuan-Hua David Ho6,7. 1. Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan, ROC. 2. Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan, ROC. 3. Department of Environmental Science and Engineering, Tunghai University, Taichung, Taiwan, ROC. 4. Institute of Tropical Plant Science, National Cheng Kung University, Tainan, Taiwan, ROC. 5. Biotechnology Research Center, National Chung Hsing University, Taichung, Taiwan, ROC. 6. Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan, ROC. tho@gate.sinica.edu.tw. 7. Biotechnology Research Center, National Chung Hsing University, Taichung, Taiwan, ROC. tho@gate.sinica.edu.tw.
Abstract
BACKGROUND: Lignocellulolytic enzymes are essential for agricultural waste disposal and production of renewable bioenergy. Many commercialized cellulase mixtures have been developed, mostly from saprophytic or endophytic fungal species. The cost of complete cellulose digestion is considerable because a wide range of cellulolytic enzymes is needed. However, most fungi can only produce limited range of highly bioactive cellulolytic enzymes. We aimed to investigate a simple yet specific method for discovering unique enzymes so that fungal species producing a diverse group of cellulolytic enzymes can be identified. RESULTS: The culture medium of an endophytic fungus, Daldinia caldariorum D263, contained a complete set of cellulolytic enzymes capable of effectively digesting cellulose residues into glucose. By taking advantage of the unique product inhibition property of β-glucosidases, we have established an improved zymography method that can easily distinguish β-glucosidase and exoglucanase activity. Our zymography method revealed that D263 can secrete a wide range of highly bioactive cellulases. Analyzing the assembled genome of D263, we found over 100 potential genes for cellulolytic enzymes that are distinct from those of the commercially used fungal species Trichoderma reesei and Aspergillus niger. We further identified several of these cellulolytic enzymes by mass spectrometry. CONCLUSIONS: The genome of Daldinia caldariorum D263 has been sequenced and annotated taking advantage of a simple yet specific zymography method followed by mass spectrometry analysis, and it appears to encode and secrete a wide range of bioactive cellulolytic enzymes. The genome and cellulolytic enzyme secretion of this unique endophytic fungus should be of value for identifying active cellulolytic enzymes that can facilitate conversion of agricultural wastes to fermentable sugars for the industrial production of biofuels.
BACKGROUND: Lignocellulolytic enzymes are essential for agricultural waste disposal and production of renewable bioenergy. Many commercialized cellulase mixtures have been developed, mostly from saprophytic or endophytic fungal species. The cost of complete cellulose digestion is considerable because a wide range of cellulolytic enzymes is needed. However, most fungi can only produce limited range of highly bioactive cellulolytic enzymes. We aimed to investigate a simple yet specific method for discovering unique enzymes so that fungal species producing a diverse group of cellulolytic enzymes can be identified. RESULTS: The culture medium of an endophytic fungus, Daldinia caldariorumD263, contained a complete set of cellulolytic enzymes capable of effectively digesting cellulose residues into glucose. By taking advantage of the unique product inhibition property of β-glucosidases, we have established an improved zymography method that can easily distinguish β-glucosidase and exoglucanase activity. Our zymography method revealed that D263 can secrete a wide range of highly bioactive cellulases. Analyzing the assembled genome of D263, we found over 100 potential genes for cellulolytic enzymes that are distinct from those of the commercially used fungal species Trichoderma reesei and Aspergillus niger. We further identified several of these cellulolytic enzymes by mass spectrometry. CONCLUSIONS: The genome of Daldinia caldariorumD263 has been sequenced and annotated taking advantage of a simple yet specific zymography method followed by mass spectrometry analysis, and it appears to encode and secrete a wide range of bioactive cellulolytic enzymes. The genome and cellulolytic enzyme secretion of this unique endophytic fungus should be of value for identifying active cellulolytic enzymes that can facilitate conversion of agricultural wastes to fermentable sugars for the industrial production of biofuels.
Authors: Daniel Klein-Marcuschamer; Piotr Oleskowicz-Popiel; Blake A Simmons; Harvey W Blanch Journal: Biotechnol Bioeng Date: 2011-11-21 Impact factor: 4.530
Authors: Christian P Kubicek; Marianna Mikus; André Schuster; Monika Schmoll; Bernhard Seiboth Journal: Biotechnol Biofuels Date: 2009-09-01 Impact factor: 6.040