成果報告書詳細
管理番号20140000000255
タイトル*平成25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2014/7/5
報告書年度2013 - 2013
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
和文要約木質系バイオマス(広葉樹)の蒸解パルプの酵素糖化に特化した酵素開発を行うため、木質系バイオマス由来パルプの糖化に最適なセルラーゼの成分酵素の組成を明らかにし、可溶性糖質源培養による大規模生産技術を確立することを目標とする。具体的には、以下の3項目を実施する。
1. 木質系バイオマス由来パルプに最適なセルラーゼ成分酵素の探索・評価
1) T. reesei ATCC66589株(入手可能な高セルラーゼ生産変異株)、Aspergillus aculeatus F50(株式会社Biomaterial in Tokyo保有の高活性BGL生産株)、Irpex lacteus、Pestalotiopsis sp. AN-7などチーム内で入手可能なセルラーゼ生産菌、2) 炭素源として使用する糖の組成を変えることで、高いセルラーゼ活性を維持したまま成分酵素組成が変わることが報告されているT. reesei M2-1を用いて、種々の広葉樹クラフトパルプ基質について、糖化に重要な酵素及び糖化補助因子の成分組成を検討する。
2. 酵素生産力及び酵素性能の改良
バイオマス(広葉樹クラフトパルプ)によってセルラーゼ生産誘導を実施するセルラーゼ酵素を分裂酵母S. pombeに異種発現させる。各酵素遺伝子をS. pombe用に最適化し、酵素タンパク質生産性を最適化し、研究項目1で明らかになった酵素組成を基に酵素カクテルを作成し、前処理バイオマスに対する酵素糖化試験により最終評価する。
また、S. pombeに導入する酵素遺伝子に、酵素の耐熱性、グルコース耐性、エタノール耐性を付加すべく変異を導入する事により、酵素性能の改良検討を行う。
3. 可溶性糖質源培養による大規模酵素生産の技術開発
ラボスケール(5L及び30L容ジャー)によるS. pombe及びT. reesei M2-1の流加培養法・繰り返し発酵生産において、酵素活性・生産量を一定水準に安定させた状態で安価な培地成分への代替を検討する。
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) FY2013 Annual Report

Several key enzymes that are necessary for effective cellulose digestion have been identified, and the associated genes from Trichoderma reesei, Irpex lacteus and Pestalotiopsis sp. were decoded. These enzymes are important for pulp degradation, but the necessity and ideal ratio of each component have not yet been determined. Genes for CBH1, CBH2, EG1, EG2, EG4, EG7, and swollenin enzymes from T. reesei have been successfully integrated into Aspergillus oryzae for expression, but as enzyme productivity is very poor, the S. pombe expression system will be tested next to improve productivity. Other genes from Pestalotiopsis sp. AN-7 that code for enzymes like glucoside hydrolases and carbohydrate esterases have been identified and expressed in E. coli and A. oryzae. A mutant strain that over-expresses the Cel1A enzyme will also be developed, as Cel1A converts 10% of cellobiose into sophorose, and was found to be a key enzyme in inducing cellulase production. If successful, the enzyme productivity of this mutant strain will also be improved.
It is known that catalytic efficiency decreases in the early stages of biomass digestion, although the reason is not clear. It was found that CBH2, an important component of cellulase, was easily deactivated due to non-specific adsorption; 37% of the enzymes bound to the substrate, but the majority bound to the container, particularly to glass. This is considered to be the primary cause of the inefficiency, so the hydrophobicity of CBH2’s molecular surface should be improved to prevent such adsorption.
Maxiumum filter paper unit activity (FPU) of commercially available enzymes was improved with the addition of 20-40 pNP-Glucose units of AaBGL1. Saccharification tests with continual pulp addition resulted in about 2 mg/g-sugars produced, so follow-up tests will be done using cellulases produced by woody biomass induction for comparison.
In order to produce enzymes that hydrolyze woody biomass efficiently, carbon sources used in the cultivation medium for T. reesei were examined. The culture supernatant with Japanese cedar pulp hydrolyzed pulp more efficiently compared to cellulose powder. Protein expression was compared by two-dimensional polyacrylamide gel electrophoresis, and many protein spots were isolated. Spots specific to cedar pulp are being identified by protein sequence, and the data will be used to determine the ideal enzyme combination. Culture trials of T. reesei M2-1 using 5L jar-fermenters also suggested the conditions for the seed culture, particularly glucose consumption, were a key factor. A system for consistent enzyme production in 5L jar-fermenters will be established next year, which will later be scaled up, and cost reduction will also be considered by altering the culture medium composition, such as using corn steep liquor (CSL) as a nitrogen source, and using sophorolipids to induce and improve cellulase production.
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