High cell density co-culture for production of recombinant hydrolases |
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Affiliation: | 1. Laboratório Nacional de Ciência e Tecnologia do Bioetanol – CTBE/CNPEM, Caixa Postal 6170, 13083-970 Campinas, São Paulo, Brazil;2. Biotechnology Center, Butantan Institute, 05503-000 São Paulo, São Paulo, Brazil;1. School of Applied Biosciences, Kyungpook National University, Daegu 702-701, South Korea;2. Department of Cellular and Molecular Medicine, Chosun University School of Medicine, Gwangju 501-759, South Korea;3. School of Food Sciences & Biotechnology, Kyungpook National University, Daegu 702-701, South Korea;4. Major in Food Biotechnology, Silla University, Busan 617-736, South Korea;5. Infection & Immunity Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-806, South Korea;1. School of Biotechnology and Key laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, 1800 Lihu Road, Wuxi 214122, China;2. State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Wuxi 214122, China;1. College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, China;2. Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, NY, USA;1. Industrial Enzymes National Engineering Laboratory, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China;2. Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan;3. Biofuels Institute, School of Environment, Jiangsu University, Zhenjiang 212013, China;1. Walther-Straub-Institute of Pharmacology and Toxicology, Nussbaumstr. 26, D-80336 Munich, Germany;2. Department of Operative/Restorative Dentistry, Periodontology and Pedodontics, Ludwig-Maximilians-University of Munich, Goethestr. 70, D-80336 Munich, Germany;3. KU Leuven Biomat, Department of Oral Health Sciences, KU Leuven, Kapucijnenvoer 7, B-3000 Leuven, Belgium |
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Abstract: | Sugarcane bagasse is a residue with great potential as a feedstock for second-generation ethanol production. One of the approaches studied for making use of this material is the utilization of enzymes to hydrolyze the cell wall carbohydrates and generate fermentable sugars. These enzymes can be produced by cultivation of filamentous fungi or bacteria; however, the high production cost still represents a bottleneck to second-generation ethanol production. Expression of recombinant hydrolases through a co-culture strategy could be an interesting alternative for producing a recombinant cocktail at high levels of productivity that is tailor-made for each material to be hydrolyzed. In this study we evaluate the production of hydrolases by co-culturing two recombinant Escherichia coli, each expressing a specific hydrolase, β-1,3-1,4-glucanase or β-1,4-xylanase, both isolated from Bacillus subtilis. The cultures were conducted in bioreactors in batch and fed-batch mode in order to reach high cell densities. Co-culture in batch cultivation reached a dry cell weight of 10.4 g/L and volumetric activities of 31.96 U/mL and 11.89 U/mL for xylanase and endoglucanase, respectively. Fed-batch cultivation reached a dry cell weight of 60 g/L and the volumetric activities of xylanase and endoglucanase were respectively up to 5 and 1.3 times higher than those in batch mode. A competition assay indicates that no clone predominates over the other during cultivation. These results suggest that co-culture is a potential technique for producing recombinant hydrolase cocktails at lower cost than those associated with the production of a single culture. |
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