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Secretome analysis of Trichoderma reesei and Aspergillus niger cultivated by submerged and sequential fermentation processes: Enzyme production for sugarcane bagasse hydrolysis
Institution:1. Laboratory of Renewable Resources Engineering, Department of Agricultural and Biological Engineering, Purdue University, IN 47907, West Lafayette, IN, USA;2. Graduate Program of Biotechnology, Federal University of Sao Carlos, 13565-905, Sao Carlos, SP, Brazil;3. Embrapa Instrumentation, 1452 XV de Novembro Street, 13560-970, Sao Carlos, SP, Brazil;4. Graduate Program of Chemical Engineering, Federal University of Sao Carlos, 13565-905, Sao Carlos, SP, Brazil;1. Department of Microbiology, Guru Nanak Dev University, Amritsar 143005, Punjab, India;2. Division of Microbiology, ICAR-Indian Agricultural Research Institute, New Delhi, India;3. Centre for Structural and Functional Genomics, Concordia University, 7141 Sherbrooke Street West, Montreal, Quebec H4B 1R6, Canada;3. Department of Science and Environment, Roskilde University, Universitetsvej 1, Building 28, DK-4000 Roskilde, Denmark;4. Novozymes A/S, Krogshøjvej 36, DK-2880 Bagsværd, Denmark;5. Department of Biotechnology and Biomedicine, Technical University of Denmark, Building 224, DK-2800 Kgs. Lyngby, Denmark;1. Brazilian Bioethanol Science and Technology Laboratory (CTBE), Brazilian Center for Research in Energy and Materials, Rua Giuseppe Máximo Scolfaro, 10000, Post Code: 6192, Zip Code: 13083-970, Campinas, São Paulo, Brazil;2. School of Chemical Engineering, University of Campinas (Unicamp), Av. Albert Einstein, 500, Post Code: 6066, Zip Code: 13083-852, Campinas, São Paulo, Brazil;1. Section for Sustainable Biotechnology, Aalborg University Copenhagen, A. C. Meyers Vænge 15, DK-2450 Copenhagen SV, Denmark;2. Bioproducts, Sciences and Engineering Laboratory (BSEL), Washington State University Tri-Cities, 2710 Crimson Way, Richland 99354, WA, USA
Abstract:Cellulases and hemicellulases from Trichoderma reesei and Aspergillus niger have been shown to be powerful enzymes for biomass conversion to sugars, but the production costs are still relatively high for commercial application. The choice of an effective microbial cultivation process employed for enzyme production is important, since it may affect titers and the profile of protein secretion. We used proteomic analysis to characterize the secretome of T. reesei and A. niger cultivated in submerged and sequential fermentation processes. The information gained was key to understand differences in hydrolysis of steam exploded sugarcane bagasse for enzyme cocktails obtained from two different cultivation processes. The sequential process for cultivating A. niger gave xylanase and β-glucosidase activities 3- and 8-fold higher, respectively, than corresponding activities from the submerged process. A greater protein diversity of critical cellulolytic and hemicellulolytic enzymes were also observed through secretome analyses. These results helped to explain the 3-fold higher yield for hydrolysis of non-washed pretreated bagasse when combined T. reesei and A. niger enzyme extracts from sequential fermentation were used in place of enzymes obtained from submerged fermentation. An enzyme loading of 0.7 FPU cellulase activity/g glucan was surprisingly effective when compared to the 5–15 times more enzyme loadings commonly reported for other cellulose hydrolysis studies. Analyses showed that more than 80% consisted of proteins other than cellulases whose role is important to the hydrolysis of a lignocellulose substrate. Our work combined proteomic analyses and enzymology studies to show that sequential and submerged cultivation methods differently influence both titers and secretion profile of key enzymes required for the hydrolysis of sugarcane bagasse. The higher diversity of feruloyl esterases, xylanases and other auxiliary hemicellulolytic enzymes observed in the enzyme mixtures from the sequential fermentation could be one major reason for the more efficient enzyme hydrolysis that results when using the combined secretomes from A. niger and T. reesei.
Keywords:Secretome  Fermentation  Cellulases  Sugarcane bagasse
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