TWI619810B - 可用於製備生質酒精的釀酒酵母菌株 - Google Patents
可用於製備生質酒精的釀酒酵母菌株 Download PDFInfo
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- 240000004808 Saccharomyces cerevisiae Species 0.000 title claims abstract description 28
- 238000002360 preparation method Methods 0.000 title claims description 6
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 claims abstract description 27
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 19
- 108010047754 beta-Glucosidase Proteins 0.000 claims description 20
- 102000006995 beta-Glucosidase Human genes 0.000 claims description 17
- 239000002028 Biomass Substances 0.000 claims description 7
- 241000228182 Thermoascus aurantiacus Species 0.000 claims description 4
- 239000000835 fiber Substances 0.000 description 19
- 238000000855 fermentation Methods 0.000 description 16
- 235000000346 sugar Nutrition 0.000 description 14
- GUBGYTABKSRVRQ-CUHNMECISA-N D-Cellobiose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-CUHNMECISA-N 0.000 description 12
- 230000004151 fermentation Effects 0.000 description 12
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- 150000002016 disaccharides Chemical class 0.000 description 10
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 description 8
- 102000004190 Enzymes Human genes 0.000 description 8
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- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 6
- 210000004027 cell Anatomy 0.000 description 6
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- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 4
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 4
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 4
- 229910052799 carbon Inorganic materials 0.000 description 4
- 235000013305 food Nutrition 0.000 description 4
- 238000004128 high performance liquid chromatography Methods 0.000 description 4
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- NGFMICBWJRZIBI-JZRPKSSGSA-N Salicin Natural products O([C@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@H](CO)O1)c1c(CO)cccc1 NGFMICBWJRZIBI-JZRPKSSGSA-N 0.000 description 3
- 238000009825 accumulation Methods 0.000 description 3
- NGFMICBWJRZIBI-UHFFFAOYSA-N alpha-salicin Natural products OC1C(O)C(O)C(CO)OC1OC1=CC=CC=C1CO NGFMICBWJRZIBI-UHFFFAOYSA-N 0.000 description 3
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- NGFMICBWJRZIBI-UJPOAAIJSA-N salicin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=CC=CC=C1CO NGFMICBWJRZIBI-UJPOAAIJSA-N 0.000 description 3
- 229940120668 salicin Drugs 0.000 description 3
- 239000001888 Peptone Substances 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- 108010009736 Protein Hydrolysates Proteins 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
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- 244000005700 microbiome Species 0.000 description 2
- 235000019319 peptone Nutrition 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 150000008163 sugars Chemical class 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- 108010008885 Cellulose 1,4-beta-Cellobiosidase Proteins 0.000 description 1
- 101710112457 Exoglucanase Proteins 0.000 description 1
- 229920002488 Hemicellulose Polymers 0.000 description 1
- 241000235004 Saccharomycopsis fibuligera Species 0.000 description 1
- 238000005903 acid hydrolysis reaction Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000002551 biofuel Substances 0.000 description 1
- 230000025938 carbohydrate utilization Effects 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 101150048033 cbh gene Proteins 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000004821 distillation Methods 0.000 description 1
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- 239000003527 fibrinolytic agent Substances 0.000 description 1
- 230000003480 fibrinolytic effect Effects 0.000 description 1
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- 230000002068 genetic effect Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
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- 239000000203 mixture Substances 0.000 description 1
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
Abstract
本發明提供一種釀酒酵母(
Saccharomyces cerevisiae)菌株,其寄存編號為BCRC 920106,以及其於製備生質酒精之用途
Description
本發明係關於一種釀酒酵母(
Saccharomyces cerevisiae)菌株及其在製備生質酒精之用途。
利用木質纖維素當料源產製第二代纖維酒精,其過程主要有四大關鍵步驟:第一是將生質物進行解聚前處理,第二是利用水解酵素將纖維素分解糖化成單醣,第三是利用微生物進行醣類發酵,第四是將酒精進行蒸餾純化。整個製程量產到商轉工業化主要的挑戰在於第二及第三步驟。
第二步驟之困難點在於生質物解聚後,生質物結構包含纖維素、半纖維素及木質素,其中木質素會與水解酵素作用或與醣類形成複合物(LCC,lignin-carbohydrate complex),進而影響到整體酵素水解效率,此為第二代生質精煉過程中普遍會遭遇之問題。另外,纖維水解酵素組成之β-葡萄糖苷酶(β-glucosidase)比例短缺,會造成後續纖維雙糖的累積,而纖維雙糖對於其他水解酵素-葡萄糖內切酶(endoglucanases; EGs)及葡萄糖外切酶(exoglucanases;又稱為cellobiohydrolase; CBHs)具有強烈抑制作用,纖維雙糖的累積更會造成整體糖化效率大幅下降,故有效降低纖維雙糖的殘留有助於同時糖化共發酵製程(SSCF,Simultaneous Saccharification and Co-Fermentation)之酵素水解效率。
第三步驟之困難點在於微生物的開發,此微生物需具備在酸性、多種抑制物、甚至環境溫度較高的水解液進行生長發酵,並同時有效利用多源醣類產製。目前本領域中的技藝人士正透過不同策略來提高菌株對環境的耐受性、纖維雙糖的發酵效率或葡萄糖、木糖、纖維雙糖等多源糖質的利用情況。
第二代非糧作物量產生質酒精,雖可避免第一代生質燃料與民爭糧、與糧爭地之疑慮,但其經濟效益及製程設備成本是未來產業化之主要考量因素,為了有效降低纖維酒精生產成本,增進發酵效率,仍亟需一種多功能釀酒酵母菌株。
在本領域中,現有許多不同的策略來解決纖維雙糖累積的問題。根據酵素作用位置不同分為胞內、細胞膜及胞外去分解纖維雙糖。胞內分解需建構轉運子系統(transporter),使酵母菌運送纖維雙糖進入體內進行代謝,然而此方法基因建構程序複雜。另外,細胞膜分解則需將β-葡萄糖苷酶固定化於細胞表面,此方法不僅會造成β-葡萄糖苷酶產量會受限於菌體總數及細胞表面積,並且會造成纖維雙糖分解速度變慢等問題。
有文獻揭示利用白麴菌(
Saccharomycopsis fibuligera)的β葡萄糖苷酶基因轉形釀酒酵母菌,使其可外泌β葡萄糖苷酶(Tang
et al., J. Microbiol. Biotechnol 23(11),1577-1585(2013))。
在一方面,本發明提供一種釀酒酵母(
Saccharomyces cerevisiae)菌株,其寄存編號為BCRC 920106。
本發明之釀酒酵母菌株可外泌β葡萄糖苷酶。
另一方面,本發明提供上述釀酒酵母(
Saccharomyces cerevisiae)菌株在製備生質酒精之用途。
本發明之其他目的及優點一部分記載於下述說明中,或可透過本發明的實施例而理解。應了解前文之發明內容及下文之實施方式僅為例示性及闡釋性之說明,而非如申請專利範圍般限定本發明。
除非另有指明,所有在此處使用的技術性和科學性術語具有如同本創作所屬技藝中之通常技術者一般所瞭解的意義。
本文所使用的「一」乙詞,如未特別指明,係指至少一個(一個或一個以上)之數量。
在一方面,本發明提供一種釀酒酵母(
Saccharomyces cerevisiae)菌株。該釀酒酵母菌株於2016年10月5日寄存於食品工業發展研究所生物資源保存及研究中心,寄存編號為BCRC 920106。
根據本發明,該釀酒酵母菌株係穩定表達嗜熱子囊菌(
Thermoascus aurantiacus)的β葡萄糖苷酶,並可外泌β葡萄糖苷酶。
另一方面,本發明提供上述釀酒酵母(
Saccharomyces cerevisiae)菌株在製備生質酒精之用途。
通過以下非限制性實例來進一步說明本發明。
實例
實例
1
:釀酒酵母菌株之製備、培養及檢測法
含有嗜熱子囊菌(
Thermoascus aurantiacus)的β葡萄糖苷酶基因(BGL)之質體建構圖如圖1所示。將質體利用化學法送入Y600衍生酵母菌株,使其具有纖維雙糖分解能力,並透過高效液相層析儀分析檢測。菌液與40%甘油以一比一比例混合,製備成凍管並保存於-80℃冰箱中。
培養基配方如下表1所示,在250 ml搖瓶中依濃度配製,於121℃下高溫高壓滅菌20分鐘後,自凍管取出培養基體積千分之一的釀酒酵母菌株,接種於培養基內並於溫度30℃、轉速150 rpm下培養16-24小時,得到發酵實驗的種菌液(OD600=18-20)。
表1:培養基配方
<TABLE border="1" borderColor="#000000" width="85%"><TBODY><tr><td> YPC培養基 </td><td> % </td></tr><tr><td> 酵母菌萃取物 </td><td> 1 </td></tr><tr><td> 蛋白腖 </td><td> 2 </td></tr><tr><td> 纖維雙糖 </td><td> 2 </td></tr></TBODY></TABLE>
醱酵樣品之數據,係利用高效液相層析儀之Coregel-87H3 (Transgenomics, Co.)管柱,透過流速0.8 mL min
-1之4 mM H
2SO
4沖提液於45℃進行樣品的分離,以折射率偵測器(refractive index detector)偵測發酵液中待測化合物之訊號,並於不同時間點紀錄每種化合物的含量種類資訊。
實例
2
:與傳統釀酒酵母原生株之比較
如圖2所示,本發明之菌株與傳統釀酒酵母原生株(即,Y600衍生酵母菌株)比較,傳統釀酒酵母缺乏β-葡萄糖苷酶以及纖維雙糖轉運子,所以釀酒酵母無法吸收代謝纖維雙糖,本發明之經基因改良之菌株可於24-48小時內有效代謝纖維雙糖。
實例
3
:針對纖維雙糖之分解率及酒精轉化率
如圖3所示,本發明之菌株以纖維雙糖為單一碳源之發酵情形,單純系統主要在驗證發明菌株分解纖維雙糖之能力。本試驗將酵母菌培養在YPC培養基,在30℃、轉速150 rpm條件培養,起始菌量控制在OD600=3-5,經培養取樣,24小時纖維雙糖分解率達96.6%,酒精轉化率達83.3%。
實例
4
:纖維雙糖分解率
如圖4所示,本發明之菌株在多源糖源情況下進行發酵,利用此複雜系統模擬驗證實際應用之情境。本試驗將酵母菌培養在混合糖液中(10 g/L 酵母菌萃取物、20 g/L 蛋白腖、40 g/L葡萄糖、40 g/L木糖、20 g/L纖維雙糖),將菌株以10%(v/v)接種於木片水解液,於溫度30℃、轉速150 rpm條件培養,起始菌量於OD600=3-5,經培養取樣,收取不同時間點的上清液進行HPLC分析,24小時纖維雙糖分解率達90%以上。
實例
5
:於木片水解液之酒精產率
如圖5所示,本發明之菌株在木片水解液中進行發酵,驗證菌株在實際應用於生質酒精相關製程,可於多種抑制物、多源糖類等環境進行發酵。該批木片經由稀酸進行蒸氣爆裂前處理及纖維水解酵素進行酵素水解。木片水解液中,葡萄糖濃度40-50g/L、木糖10-20 g/L、纖維雙糖10-30 g/L及水解抑制物醋酸4-6g/L,將菌株以10%(v/v)接種於木片水解液,於溫度30℃、轉速150 rpm條件培養,經培養取樣,收取不同時間點的上清液進行HPLC分析。本發明菌株之總糖酒精轉化效率可達90%以上,總糖利用率亦可達80%以上,酒精產率可達0.94 g/L/h。
實例
6
:
β
葡萄糖苷酶活性測試
如圖6所示,由於水楊苷(Salicin)結構類似纖維雙糖,利用Salicin當作β葡萄糖苷酶的受質,當β葡萄糖苷酶水解Salicin釋放葡萄糖時,藉由測定還原糖濃度而推估β葡萄糖苷酶活性。實驗結果顯示,本發明之菌株於發酵72小時β葡萄糖苷酶活性會達到最高,酵素活性約0.34 U/mL。
本發明之釀酒酵母菌株至少具有以下不可預期之較佳功效: (1) 相較於文獻(Tang
et al., J. Microbiol. Biotechnol 23(11),1577-1585(2013))以纖維雙糖為單一碳源之酒精產率0.33 g/g,本發明之釀酒酵母菌株以纖維雙糖為單一碳源之酒精產率為0.42 g/g(可由圖3之數據計算,理論產率 x 百分比=0.511 x 0.833=0.42)。 (2) 外泌之β葡萄糖苷酶於pH3-6.5及溫度50-80℃具有活性,可承受環境變動,一旦溫度酸鹼度失控,可避免敗槽的風險。 (3) 可應用於生質酒精不同製程,包括分開水解共發酵SHCF(Separate Hydrolysis and Co-Fermentation)及同時糖化共發酵SSCF(Simultaneous Saccharification and Co-Fermentation),對環境耐受性強。 (4) 可針對葡萄糖、木糖、纖維雙糖同時進行分解發酵,增加纖維酒精產製效率。 (5) 分解纖維雙糖速度快,24小時纖維雙糖分解率達96.6%,可有效縮減生質酒精量產時程。相較於文獻(Ha
et al., PNAS vol 108, no 2 (2011))24小時纖維雙糖分解率大約60-80%。
無
圖1為含有嗜熱子囊菌(
Thermoascus aurantiacus)的β葡萄糖苷酶基因(BGL)之質體建構圖。
圖2 顯示本發明之菌株與酵母菌原生株之差異。
圖3顯示本發明之菌株以纖維雙糖為單一碳源之發酵結果。
圖4顯示本發明之菌株在不同糖類之發酵結果。
圖5為本發明之菌株於木片水解液中發酵生產酒精之曲線圖。
圖6顯示本發明之菌株β-葡萄糖苷酶酵素活性的分析。
食品工業發展研究所生物資源保存及研究中心/BCRC 920106/2016年10月5日
Claims (4)
- 一種釀酒酵母 ( Saccharomyces cerevisiae) 菌株,其寄存編號為BCRC 920106。
- 如請求項1之釀酒酵母菌株,其可外泌β葡萄糖苷酶。
- 如請求項1之釀酒酵母菌株,其穩定表達嗜熱子囊菌 ( Thermoascus aurantiacus) 的β葡萄糖苷酶。
- 一種如請求項1-3項中任一項之釀酒酵母菌株在製備生質酒精之用途。
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