JP6515810B2 - セルラーゼ生産微生物 - Google Patents
セルラーゼ生産微生物 Download PDFInfo
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- JP6515810B2 JP6515810B2 JP2015553546A JP2015553546A JP6515810B2 JP 6515810 B2 JP6515810 B2 JP 6515810B2 JP 2015553546 A JP2015553546 A JP 2015553546A JP 2015553546 A JP2015553546 A JP 2015553546A JP 6515810 B2 JP6515810 B2 JP 6515810B2
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- 235000013343 vitamin Nutrition 0.000 description 1
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Description
[1]
アクレモニウム・セルロリティカス(Acremonium cellulolyticus)(タラロマイセス・セルロリティカス(Talaromyces cellulolyticus))S6-25株(NITE BP-01685)及びその誘導株から選択される菌株。
[2]
前記誘導株が、配列番号7〜16に記載の塩基配列から選択される1またはそれ以上の塩基配列を染色体上に有する、前記菌株。
[3]
前記誘導株が、配列番号7〜16に記載の塩基配列の全てを染色体上に有する、前記菌株。
[4]
前記誘導株が、creA遺伝子の発現が低下するように、又は、creA遺伝子が破壊されるように改変されている、前記菌株。
[5]
前記菌株を培地で培養し、セルラーゼを該培地中に生成蓄積させること、および
該培地よりセルラーゼを回収すること、
を含む、セルラーゼの製造法。
[6]
植物バイオマスを前処理に供すること、
前記前処理の処理物を、前記方法により得られるセルラーゼを用いた糖化処理に供すること、
前記糖化処理の処理物を含有する培地で目的物質の生産能を有する微生物を培養し、目的物質を該培地中又は該微生物の菌体内に生成蓄積させること、および
該培地又は菌体より目的物質を採取すること、
を含む、目的物質の製造法。
[7]
前記前処理が水熱分解処理である、前記方法。
[8]
前記目的物質がL−アミノ酸である、前記方法。
本発明の微生物は、アクレモニウム・セルロリティカス(Acremonium cellulolyticus)S6-25株及びその誘導株から選択される菌株である。アクレモニウム・セルロリティカスは、タラロマイセス・セルロリティカス(Talaromyces cellulolyticus)とも呼ばれる(FEMS Microbiol. Lett., 2014, 351:32-41)。なお、本発明においては、説明の便宜上、S6-25株及びその誘導株をアクレモニウム・セルロリティカス(タラロマイセス・セルロリティカス)に属するものとして記載するが、将来的に系統分類が変更された場合には、S6-25株及びその誘導株は変更後の種に属するものとして適宜読み替えてよい。
本発明の微生物を利用して、セルラーゼを製造することができる。すなわち、本発明は、本発明の微生物を培地で培養し、セルラーゼを該培地中に生成蓄積させること、および該培地よりセルラーゼを回収することを含む、セルラーゼの製造法を提供する。
本発明のセルラーゼは、セルロースの分解に利用できる。例えば、本発明のセルラーゼを利用して植物バイオマスに含まれるセルロース成分を糖化することにより、グルコースを含有する糖化液が得られる。
(1)Acremonium cellulolyticus S6-25株の取得
Acremonium cellulolyticus TN株(FERM BP-685。以下、TN株と記載)を親株として、以下の手順でAcremonium cellulolyticus S6-25株(NITE BP-01685。以下、S6-25株と記載)を取得した。
上記と同様に寒天培地上に伸長させた菌糸の先端付近をストローで打ち抜いて得られたアガーディスク1個を、20 mL の40 g/L ソルカフロック、24 g/L KH2PO4、5 g/L (NH4)2SO4、2 g/L Urea、1.2 g/L MgSO4・7H2O、0.01 g/L ZnSO4・7H2O、1 g/L MnSO4・5H2O、1 g/L CuSO4・5H2Oを含む液体培地に接種し、30℃、220 rpmで5日間前培養を行なった。次に、5 mLの前培養液を、100 mLの50 g/L ソルカフロック、24 g/L KH2PO4、5 g/L (NH4)2SO4、4 g/L Urea、1.2 g/L MgSO4・7H2O、0.01 g/L ZnSO4・7H2O、1 g/L MnSO4・5H2O、1 g/L CuSO4・5H2Oを含む液体培地に植菌し、通気量1.0 vvm、PL制御8%、培養pH を2N NH4でpH 4.5に制御しながら培養を行った。培養液を適宜分取し、遠心分離(15000 rpmで15分間)して得た上清をセルラーゼ活性の測定試料とした。セルラーゼ活性としては、微結晶セルロース(アビセル)の分解活性とろ紙の分解活性を、それぞれ以下の手順で測定した。
0.4 mgのアビセル(Cellulose microcrystalline, 1.02331.0500, MERCK)を含む20μlの反応液(30 mM CaCl2、50 mM 酢酸緩衝液、pH 5.5)に、適宜希釈した等量の培養上清を加え、50℃で16時間反応を行った。なお、反応を行わないサンプルを用意し、ブランクとした。次に100 μlのDNS溶液(1 % ジニトロサリチル酸、20 % 酒石酸ナトリウム・カリウム、0.05 % 亜硫酸ナトリウム、1 % 水酸化ナトリウム)を加え、95℃で5分間反応後、4℃まで冷却した。反応後の溶液を混合し、MultiScreen-HVプレート(Merck Millipore)に全量を移し、遠心ろ過(2000 rpmで10分間)し、ろ液を回収した。その後、ろ液の540 nmの吸光度を測定し、ブランクの値を差し引き、吸光度の増加量を算出した。次に、段階的に希釈したグルコース濃度と540 nmの吸光度の検量線を用いて、反応溶液中に生成された還元糖量をグルコース換算で算出した。この反応を異なる希釈率の培養上清で行い、培養上清の希釈率とグルコース生成量の検量線を作成し、0.08 mgのグルコースに相当する還元糖を生成するのに必要な培養上清の希釈率を見積もり、希釈前の培養上清のアビセル分解活性(U/mL)を算出した。なお、1分間に1μmolのグルコースに相当する還元糖を生成する酵素活性を「1 U」とした。この結果を図1に示す。
6 mm×10 mmに裁断したろ紙(Whatman No. 1、GE Healthcare)を含む100μlのクエン酸緩衝液(50 mM、pH 5.0)に、50μlの適宜希釈した培養上清を加え、50℃で1時間反応を行った。なお、反応を行わないサンプルを用意し、ブランクとした。次に300 μlのDNS溶液を加え、95℃で5分間反応後、氷上で5分間冷却した。反応後の溶液を混合し、遠心(12000 rpmで5分間)し、100μlの上清を回収した。その後、上清の540 nmの吸光度を測定し、ブランクの値を差し引き、吸光度の増加量を算出した。次に、段階的に希釈したグルコース濃度と540 nmの吸光度の検量線を用いて、反応溶液中に生成された還元糖量をグルコース換算で算出した。この反応を異なる希釈率の培養上清で行い、培養上清の希釈率とグルコース生成量の検量線を作成し、0.2 mgのグルコースに相当する還元糖を生成するのに必要な培養上清の希釈率を見積もり、希釈前の培養上清のろ紙分解活性(FPU/mL)を算出した。なお、1分間に1μmolのグルコースに相当する還元糖を生成する酵素活性を「1 U」とした。培養4日目の結果を図2に示す。
S6-25株を親株として、さらに実施例1の(1)と同様の操作を行い、S6-25株由来の変異株を取得した。
(1)遺伝子組み換え用親株の作成
S6-25株を親株として、以下の手順で遺伝子組み換え用の親株F09株を作成した。
菌糸を回収し、得られた菌糸を0.1 % Tween80、0.05 % MgSO4・7H2O、0.5 % NaClを含む
溶液に懸濁し、懸濁液を遠心(5000 rpmで3分間)する洗浄操作を2回行った。洗浄後の
菌体懸濁液の濁度(OD 660 nm)が1.0になるように適宜希釈し、1 mLをペトリディッシュ(底部径約35 mm)に分注し、15 Wの殺菌灯を用いて紫外線を照射した。照射後の菌体懸
濁液を適宜希釈し、1.2 g/L 5-fluoroorotic acid、1 g/L Uracil、1 g/L Uridineを含む最少培地(10 g/L Glucose、10 mM NH4Cl、10 mM KH2PO4、7 mM KCl、2 mM MgSO4、0.06 mg/L H3BO3、0.26 mg/L (NH4)6Mo7O24・4H2O、1 mg/L FeCl3・6H2O、0.4 mg/L CuSO4・5H2O、0.08 mg/L MnCl2、2 mg/L ZnCl2、20g/L Bacto Agar)に播種し、30℃で培養を行っ
た。5-fluoroorotic acidはUracil生合成経路の中間体のアナログであり、Uracil生合成
経路が正常に機能している株に対して毒性を示す。よって、5-fluoroorotic acidを含む
培地で選択することで、Uracil生合成経路に変異が入り機能しなくなった株を取得することができる。5-fluoroorotic acidを含む培地で生育した変異株を、1 g/L Uracil、1 g/L
Uridineを含む最少培地とこれらを含まない最少培地に植え継ぎ、1 g/L Uracil、1 g/L Uridineを含む最少培地でのみ生育した株をF09株とした。5-fluoroorotic acid耐性を示
す株ではpyrF遺伝子またはpyrG遺伝子に変異が入り機能しなくなっている可能性が高い。そこで、F09株の染色体上のこれら領域について塩基配列の決定を行った。その結果、F09株では、pyrF遺伝子(配列番号17)の塩基が変化しており、この変異によりpyrF遺伝子産物が機能しなくなっていると推察された。
F09株を親株として、以下の手順により、creA遺伝子(配列番号18)を破壊し、セルラーゼ生産が向上する株の取得を試みた。creA遺伝子は、カタボライトリプレッションに関与する転写因子をコードする遺伝子である。creA遺伝子は、糸状菌において、セルラーゼの発現に関与していることが知られている(Mol Gen Genet. 1996 Jun 24;251(4):451-60、Biosci Biotechnol Biochem. 1998 Dec;62(12):2364-70)。
S6-25株とF09株由来creA破壊株について、50 g/L ソルカフロックの代わりに5 g/L ソルカフロックと45 g/L グルコースを用いた点以外は、実施例1の(2)と同様の方法でジャー培養を行い、グルコースを炭素源として含む培養条件でのセルラーゼ生産能を比較した。培養後、実施例1の(4)に記載した方法でアビセル分解活性およびろ紙分解活性を測定した。培養4日目の結果を図4および図5に示す。
S6-25株について、以下の手順で次世代シークエンサーによるゲノム配列解析を実施し、S6-25株特有の変異点を抽出した。
配列番号1〜6:プライマー
配列番号7〜16:Acremonium cellulolyticus S6-25株の変異点を含む塩基配列
配列番号17:Acremonium cellulolyticus S6-25株のpyrF遺伝子の塩基配列
配列番号18:Acremonium cellulolyticus S6-25株のcreA遺伝子の塩基配列
Claims (6)
- アクレモニウム・セルロリティカス(Acremonium cellulolyticus)(タラロマイセス
・セルロリティカス(Talaromyces cellulolyticus))S6-25株(NITE BP-01685)及びその誘導株から選択される菌株であって、
前記誘導株が、配列番号7〜16に記載の塩基配列の全てを染色体上に有する、菌株。 - 前記誘導株が、creA遺伝子の発現が低下するように、又は、creA遺伝子が破壊されるように改変されている、請求項1に記載の菌株。
- 請求項1または2に記載の菌株を培地で培養し、セルラーゼを該培地中に生成蓄積させること、および
該培地よりセルラーゼを回収すること、
を含む、セルラーゼの製造法。 - 請求項3に記載の方法によりセルラーゼを製造すること、
植物バイオマスを前処理に供すること、
前記前処理の処理物を、前記製造したセルラーゼを用いた糖化処理に供すること、
前記糖化処理の処理物を含有する培地で目的物質の生産能を有する微生物を培養し、目的物質を該培地中又は該微生物の菌体内に生成蓄積させること、および
該培地又は菌体より目的物質を採取すること、
を含む、目的物質の製造法。 - 前記前処理が水熱分解処理である、請求項4に記載の方法。
- 前記目的物質がL−アミノ酸である、請求項4または5に記載の方法。
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