成果報告書詳細
管理番号20150000000313
タイトル*平成26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2015/6/23
報告書年度2014 - 2014
委託先名株式会社Biomaterial in Tokyo 国立大学法人信州大学 独立行政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) FY2014 Annual Report

    The PC-3-7 strain, the parent strain of Trichoderma reesei M2-1, was cultivated using various cellulase-inducing substrates, including Kraft pulp made from broad-leaf trees (LUKP), to identify the important enzymes and cofactors needed to effectively saccharify LUKP. The proteins expressed in the enzyme solution were compared, and distinct proteins that were only found in the enzyme solution cultured with LUKP were identified. The enzyme solution produced with LUKP was also tested for its effectiveness in saccharifying LUKP.
Xylan-degrading enzymes (β-xylosidase, exo-xylanase, endo-xylanase, and co-factor acetyl xylan esterase) were well-expressed when using LUKP compared to cellobiose (G2) or glucose. Improvement in saccharifying LUKP with xylanase addition shows that xylan-degrading enzymes are vital in effectively saccharifying LUKP. To make the ideal enzyme cocktail, CBH1, CBH2, EG1, EG2, EG5, swollenin and polysaccharide mono-oxygenase (PMO) enzymes from T. reesei were expressed in koji molds and isolated, as were Ex-1 (CBH1), Ex-3 (CBH2), En-1 (EG) and cellobiose dehydrogenase from Irpex lacteus. Cel5A, Xyn10A and CE15 from Pestalotopsis sp. were expressed in Pichia pastoris. Adding these isolates to the T. reesei M2-1 enzyme solution showed that adding swollenin improved saccharification, as did endo-/exo-type cellulases and BGL. CBH1 and CBH2 showed the most improvement. PMO did not improve saccharification efficiency but its rate increased in the presence of BGL. Improvement in hemicellulose breakdown was also seen with the addition of CE15.
Enzyme kinetics analysis showed that the synthesis of sophorose, an inducing substrate, required β-glucosidase (BGL, Cel1A), as a variant of T. reesei without the gene for Cel1A was very slow in reaching the enzyme production stage and in transcript regulator expression. To prevent the non-specific adsorption of CBH2 from T. reesei, structural modification and improvements to its temperature resilience and productivity were carried out. Adding CBH2 from the wild type to the T. reesei M2-1 enzyme solution improved its efficiency, and the effect of CBH2 from the modified strains are currently being analyzed. Cel1A was also altered by inserting a modification in two areas of the amino acid near the active site, increasing its glucose tolerance by 200% relative to the wild type in conditions with 50% activity. High activity for breaking down G2 and induction of sophorose synthesis were also seen.
After cultivating T. reesei M2-1 on a 30L scale for 10 days, enzyme activity of 27.4 FPU/mL was obtained, more than 80% of the small scale cultivation. On a small scale, using saccharified biomass solution could cut production cost to JPY 460/kg of enzyme proteins, or, by using corn steep liquor (CSL) as the nitrogen source, down to JPY 700/kg. Trials are currently being done on the 30L scale to replicate the small scale results.
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