2016 Fiscal Year Research-status Report
Ecological function of wood insect associated microbes（国際共同研究強化）
| Project/Area Number |
15KK0269
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| Research Institution | Hokkaido University |
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Principal Investigator |
高須賀 太一 北海道大学, 農学研究院, 助教 (70748409)
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| Project Period (FY) |
2016 – 2018
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| Keywords | cellulose deconstruction |
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| Outline of Annual Research Achievements |
The purpose for this research is to obtain best possible combination of enzymes to deconstruct cellulose by using three different enzyme families, endocellulase, reducing and non-reducing cellobiohydrolases.
During my visit at Prof. Fox laboratory, Department of Biochemistry, University of Wisconsin - Madison, USA, I expressed over 100 endocellulases, 25 each reducing and non-reducing cellobiohydrolases using cell-free protein synthesis. The activity of each enzyme was tested by using cellulose substrate. The optimum reaction for each enzyme and substrate specificities will be tested at my laboratory at Hokkaido University.
Most recently, in agreement with Prof. Fox, we started a combinatorial assays with two enzyme families in order to achieve efficient decomposition of kelp. I expressed 38 laminarinases and 7 polysaccharide lyases from different organisms. The best laminarinase was chosen based on laminarin-degrading assay, and I performed a combination between the laminarinase and 7 different polysaccharide lyases for kelp degradation. Currently, end product formation has been analyzed by the NMR.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
50 different genes that encode either reducing end or non-reducing end cellobiohydrolases were chosen for the gene synthesis. In the first year, we designed the genes, synthesized them, and cloned into the protein expression plasmid vectors. The non-reducing enzymes were synthesized as soluble form, and initial enzymatic screens were performed for various polysaccharide substrates including cellulose, xylan, modified xylan, mannan, modified mannan and others. Out of 30 non-reducing end cellobiohydrolases, all were successfully expressed and 20 were determined to react at least one substrate shown above. 20 reducing end cellobiohydrolases were expressed successfully, and they were all soluble.
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| Strategy for Future Research Activity |
20 reducing end cellobiohydrolases will be subjected for enzyme assays. In collaboration with the United States, Department of Energy - Joint Genome Institute, I will test them in the nano-initiator mass spectrometry experimental context to determine the activity for synthesized soluble polysaccharide substrates. Once activities are measured, then results from 30 non-reducing end cellobiohydrolases and 20 reducing end cellobiohydrolases will be compared to find the best enzymes that might give the highest specific activity at similar reaction conditions, temperature and pH.
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