Abstract
Cellulose is the most abundant biomass on earth. The major players in cellulose degradation in nature are cellulases produced by microorganisms. Aerobic filamentous fungi are the main sources of commercial cellulase. Trichoderma reesei has been explored extensively for cellulase production; however, its major limitations are its low β-glucosidase activity and inefficiency in biomass degradation. The aim of this work was to isolate new fungal strains from subtropical and tropical forests in China, which produce high levels of cellulase in order to facilitate development of improved commercial cellulases. We isolated 305 fungal strains from 330 samples collected from subtropical and tropical virgin forests in China. Of these, 31 strains were found to have Avicelase activity of more than 0.2 U/ml in liquid batch cultivation. Molecular analyses of the 31 strains based on internal transcribed spacer sequences revealed that 18 were Trichoderma and 13 were Penicillium species. The best-performing isolate was Trichoderma koningiopsis FCD3-1, which had similar Avicelase activity to T. reesei Rut-C30. Most interestingly, strain FCD3-1 exhibited extracellular β-glucosidase activity of 1.18 U/ml, which was approximately 17 times higher than that of Rut-C30. One β-glucosidase secreted by FCD3-1 was purified, and its gene was cloned and identified. The β-glucosidase belonged to glycosyl hydrolase (GH) family 3, sharing the highest identity of 94% with a GH family 3 protein from Trichoderma atroviride IMI 206040, and was designated TkBgl3A. The optimal pH and temperature of TkBgl3A were 4.5 and 65°C, respectively. The enzyme retained over 90% activity for 360 hours at pH 4.0 and 30°C, which are the usual conditions used for simultaneous saccharification and fermentation (SSF) of cellulose to ethanol. The enzyme showed significantly higher specific activity toward natural substrate cellobiose (141.4 U/mg) than toward artificial substrate p-nitrophenyl-beta-D-glucopyranoside (108.0 U/mg). Strains of Trichoderma and Penicillium were the predominant cellulolytic fungi in subtropical and tropical forests in China. T. koningiopsis FCD3-1 was the most efficient producer of cellulase, and also produced a high level of β-glucosidase. The high specific activity toward cellobiose and stability under SSF conditions of the purified β-glucosidase from FCD3-1 indicates its potential application in SSF of cellulose to bioethanol.
Highlights
Cellulose is the most abundant biomass on earth
Isolation, screening, and identification of fungal strains with higher cellulase activity from forests in subtropical and tropical regions of China In total, 330 soil and rotten wood samples were collected from sampling sites in virgin forests in 8 natural reserves in Guangxi and Yunnan Provinces, China (Table 1)
The optimum pH and temperature (Table 3) of the cellulases produced by the 51 fungal strains were measured against Avicel
Summary
The major players in cellulose degradation in nature are cellulases produced by microorganisms. Cellulose can be hydrolyzed by cellulase into D-glucose, which can be further transformed into renewable fuel and various valuable chemicals [2]. These characteristics have stimulated studies on the degradation and utilization of cellulose. Cellulose degradation and utilization are part of the global carbon cycle, with the major degradation power of cellulose in nature derived from cellulases produced by microorganisms [4,5,6,7]. Endo-1,4-β-glucanase randomly cuts cellulose chains, yielding cellulose ends, while cellobiohydrolase attacks the cellulose chains from the reducing or non-reducing ends, yielding cellobiose, and β-glucosidase hydrolyzes cellobiose and cello-oligosaccharides to form glucose [8]
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