成果報告書詳細
管理番号20160000000239
タイトル*平成27年度中間年報 バイオマスエネルギー技術研究開発/バイオ燃料製造の有用要素技術開発事業/可溶性糖質源培養による木質系バイオマス由来パルプ分解用酵素生産の研究開発
公開日2016/6/1
報告書年度2015 - 2015
委託先名株式会社Biomaterial in Tokyo 国立大学法人信州大学 国立研究開発法人森林総合研究所
プロジェクト番号P13011
部署名新エネルギー部
和文要約
英文要約Title: Bio-Energy Technology Development, Project on the Development of Useful Elemental Technology for Biofuel Production, Research and Development on Producing Woody-Biomass Pulp Degrading Enzymes by Microbial Cultivation on Soluble Sugars (FY2013-FY2016) FY2015 Annual Report
    Enzymes obtained by cultivating Trichoderma reesei M2-1 with soluble sugars were examined using gel electrophoresis, and the amount of protein and efficiency in pulp saccharification were evaluated. Changing the cellobiose:glucose ratio from 1:4 to 1:8 did not affect the enzyme’s ability to saccharify Kraft pulp nor the composition of the enzymes produced, but when saccharified eucalyptus pulp solution was used as the carbon source, the expression of xylan-degrading enzymes (ex: β-xylosidase, exo-/endo-xylanase, acetyl xylan esterase) were 5 times higher, with glucose and xylose release from pulp being 10% and 20% higher respectively. Further tests showed that optimal enzymes for eucalyptus pulp saccharification could be obtained by using saccharified eucalyptus pulp as the carbon source in the culture medium. In preparation for scaling up the production, repeatability is currently being tested with small-scale replicates, and the effect of other cultivation factors on enzyme composition are also being examined.
    Transcriptome analysis from the Irpex lacteus NK-1 research that was carried out last year showed genetic information for several enzymes (ex: GH115, GH131, CE1) that are not found in T. reesei. Transformation work is being done with these genes and the effect of adding these enzymes to the T. reesei M2-1 enzyme solution is being analyzed. Furthermore, the overexpression of xylanase (Xyn1) was successfully done using a cellulose medium, allowing for simultaneous production of cellulase and xylanase. It was also found that the use of Cel3B promoter significantly increased Xyn1 and cellulase production. The exact reason is still under investigation but a patent is expected to be filed for this finding.
    Since CBH2 is a factor in limiting saccharification efficiency, a modified version was created to investigate its stability. Modifications with either proline insertion or replacement showed higher temperature stability. Particularly with proline replacement, the modified enzymes maintained 60% of its enzyme activity at 55°C, compared to 10% with the wild type. The glucose-resistant β-glucosidase (Cel1A) produced by T. reesei was also closely examined, and the glucose resisting mechanism was identified. An increase in temperature and pH stabilities were also observed.
    Improvements to the cultivation methods were also attempted. The current system with continuous glucose-cellobiose feed that produces 30 FPU/mL enzymes in 10 days in a small scale was modified with cheaper and more stable methods to reduce cultivation time to 6 days. However, this method only produced 25 FPU/mL and 19 FPU/mL enzymes when replicated at 200 L and 3,000 L scales. In both cases, the consumption of the initial glucose content was slowed, and adjustments to certain factors (ex: agitation) were needed. Therefore, the oxygen transfer coefficients will be examined closely to optimize agitation settings at different scales of production.
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