CN107418943B - 从秸秆中提取纤维素酶生产诱导剂的方法及其在秸秆糖化中的应用 - Google Patents
从秸秆中提取纤维素酶生产诱导剂的方法及其在秸秆糖化中的应用 Download PDFInfo
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Abstract
本发明是一种从秸秆中制得纤维素酶的诱导剂的方法及其在秸秆糖化中的应用。属于农作废弃物利用技术领域。步骤包括：将秸秆粉碎至粒径小于1mm；秸秆为水稻、小麦、大麦的任一种；利用水、酸处理上述的秸秆粉粒；添加有机溶剂进行萃取，得到上清液和沉淀物；有机溶剂为甲醇、乙醇或丙酮；萃取温度为70～120°C；冷冻干燥沉淀物，该沉淀物主要为糖类，是制取纤维素酶的诱导剂；由糖类物质诱导剂制取纤维素酶的方法为：将糖类物质添加于培养液中，此培养液为由培养纤维素酶生产菌如里氏木霉、糖单孢菌或芽孢杆菌配制而成的培养液；在培养液中培养时间为3‑8天，温度为20‑40°C；最终制得纤维素酶;然后利用纤维素酶糖化得到的固体残渣，得到秸秆单糖。
Description
技术领域
本发明涉及一种从秸秆中提取纤维素酶生产诱导剂的方法及其在秸秆糖化中的应用，属于农作物废弃物秸秆的综合利用。
背景技术
生物质广义上指通过光合作用而产生的各种有机体，狭义上指植物、微生物以及其加工生产后的产品及废弃物。地球上最广泛存在的生物质为木质纤维素，具有代表性的木质纤维素为树木、秸秆和木材废弃物。有效地转化木质纤维素为能源、材料和化学品,不仅可以减少人类对石油的依赖，还可以减少二氧化碳排放。如果能把丰富的农林废渣等生物质资源应用于生物燃料、生物材料的生产中，不仅不会造成粮食的紧缺，还可以达到振兴农业经济，消减贫困的目的。我国年产农作物秸秆约7亿吨。
近年我国雾霾天气明显增加，污染程度加剧，已经严重影响了人民的日常生活，也给我国带来了国际、国内的负面社会影响。雾霾的形成有多种原因，秸秆的焚烧就是其中之一。焚烧秸秆时，大气中二氧化硫、二氧化氮、可吸入颗粒物等3项污染指数达到高峰值，其中二氧化硫的浓度比平时要高出1倍，二氧化氮、可吸入颗粒物的浓度比平时高出3倍。另外，燃烧秸秆还会导致大气能见度低，严重影响交通。目前，严禁农作物秸秆露天焚烧被列为重污染应急措施之一。可见，缓解雾霾、改善大气环境的当务之急是探寻良好的秸秆等农业废弃物处理方法。
秸秆有纤维素、半纤维素、及木质素构成。通过生物炼制工艺可以把秸秆转化为有用物质。然而，由于秸秆结构复杂，其生物转化需要多重预处理工艺及多种酶的协同作用，导致秸秆的生物转化成本居高不下。因此，如果我们能够发现秸秆的高附加值产品并从秸秆中高效地回收，就可以大大提高秸秆的经济价值，从而解决秸秆的处理难题。同时，利用制备高附加值产品的工艺提高秸秆剩余残渣的糖化效率也可以提高秸秆的预处理效率，得到更多的葡萄糖、木糖等单糖。这些单糖可以作为生物炼制工艺中的主要原料生产生物燃料或材料，如生物乙醇和生物乳酸。
发明内容
本发明的目的在于提高以秸秆生物炼制过程中生产高附加值的纤维素酶生产的诱导剂，并使其制备工艺与秸秆糖化工艺相结合，提高秸秆的糖化效率。
为实现上述目的，本发明所采用的技术方案是：
a，将秸秆粉碎至粒径小于1mm；秸秆为水稻、小麦、大麦、玉米的任一种；
b，利用水、酸、或碱固液处理上述的秸秆粉粒；
c, 添加有机溶剂进行萃取，得到上清液和沉淀物； 有机溶剂为甲醇、或乙醇或丙酮； 萃取温度为70～120°C；
d, 冷冻干燥上述步骤c中的沉淀物，该沉淀物主要成分为糖类物质，将其作为制取纤维素酶生产的诱导剂；
e, 由糖类物质诱导剂制取纤维素酶的方法，其制取方法如下：
将步骤c或d中得到的糖类物质添加于培养液中，此培养液为由培养纤维素酶生产菌如里氏木霉、糖单孢菌或芽孢杆菌配制而成的纤维素酶培养液；在培养液中培养时间为3-8天，温度为20-40°C；最终制得纤维素酶;
f，然后利用上述步骤e中得到的纤维素酶糖化上述步骤c中得到的剩余固体残渣，得到秸秆单糖。
本发明的有益效果在于：
秸秆的生物炼制其主要问题是纤维素酶成本高，如果能够在其转化过程中提取出纤维素酶生产诱导剂这种高附加值产品，提高纤维素酶生产效率，秸秆的生物炼制成本就可以大幅度降低。
附图说明
图1为本发明中不同预处理条件下制备的秸秆纤维素酶生产诱导剂对滤纸酶活的影响图。
图2为本发明中不同预处理条件下制备的秸秆纤维素酶生产诱导剂对β-葡萄糖苷酶活的影响图。
图3为本发明中生产秸秆产纤维素酶诱导剂后的剩余残渣糖化时葡萄糖浓度的变化图。
图4为本发明中生产秸秆产纤维素酶诱导剂后的剩余残渣糖化时木糖浓度的变化图。
具体实施方式
下面结合具体实施例，进一步阐述本发明。
试验例1关于秸秆纤维素酶诱导剂的组分
实验方法：稻秆来自江苏连云港，60℃下烘干，用微型植物试样粉碎机进行粗磨。随后行星式球磨机研磨粗磨后的样品，保持转速45Hz，运转10min，停时间5min，共5-15个循环。得到颗粒均匀、孔径较小的稻秆粉末。利用水为溶媒固液萃取秸秆中低聚糖后，利用70%乙醇沉降低聚糖、旋蒸干燥。该产物即为纤维素酶诱导剂。利用酸、酶水解该纤维素酶诱导剂，测其单糖组分。单糖浓度采用液相色谱仪测定。条件为：RI检测器，色谱柱为AminexHPX-87H column (7.8 mm I.D. ´ 30 cm, Bio-Rad, USA)，柱箱温度65℃，流动相5 mMH2SO4，流速0.6ml/min。
实验结果：如表1所示，球磨次数对纤维素酶诱导剂得率有很大的影响。纤维素酶诱导剂得率随球磨次数的提高而提高。同时，球磨次数对纤维素酶诱导剂组分有一定的影响。随球磨次数的提高纤维素酶诱导剂中木糖和阿拉伯糖的含量略有提高（表2）。但是，不同条件下得到的纤维素酶诱导剂的含量都是以葡萄糖为主。可以认为纤维素酶诱导剂是以低聚纤维素为主体，随球磨次数的增加，增加了少量的低聚半纤维素。
试验例2 利用纤维素酶诱导剂生产纤维素酶，提高纤维素酶活性
实验方法：
a， 纤维素酶生产菌培养：里氏木霉（T. reesei）接种于PDA平板上，于 30 ℃培养3天，菌种立即使用或于 4 ℃保存。每30天新接种一次。活化培养基：1% 蛋白胨, 0.05% 酵母粉, 1% 玉米浆, 2.4% KH2PO4, 0.47% C4H4O6K2, 0.12% MgSO4·7 H2O, 0.5% (NH4)2SO4，0.1% Tween 80. 2%纤维素。pH 4.0.
b， 纤维素酶生产：平板菌种接入20 ml 活化培养基，在 30 ℃、200 r/min的摇床中培养 72 h 进行种母活化。于 100 ml 锥形瓶中加入 20 ml 产酶培养基，灭菌冷却后接入 10 %种母液，并添加适量纤维素酶生产的诱导剂，于30 ℃、 200 r/min 的摇床中培养。取2，4，6，8的发酵液进行酶活测试。发酵培养基：10 g/L 酵母粉, 1.4 g/L (NH4)2SO4, 0.3g/L MgSO4·7H2O, 8 g/L KH2PO4, 0.4 g/L CaCl2·2H2O, 0.0014 g/L ZnSO4·7H2O,0.0016 g/L MnSO4·6H2O, 0.005 g/L FeSO4·7H2O and 0.0002 g/L CoCl2.3%的纤维素或7.5%的生物质作为碳源，纤维素酶生产的诱导剂，pH 4.0。
c， 滤纸酶活的测定：试管中放入卷起折叠好的Whatman No.1滤纸条(1*6cm)和1mL 0.05M柠檬酸缓冲液（pH=5），将滤纸条充分浸没，50℃水浴5min。将待测液适当稀释，试管中加入0.5mL样液，空白管加入0.05mol/L柠檬酸缓冲液0.5mL，50℃继续水浴振荡1h。每管中均加入3mL DNS试剂并混匀，沸水浴5min，0℃冷却5min。2.5mL UP水和200μL样液混合，在540nm下测定样液的吸光值。以葡萄糖为标准作标准曲线。β-葡萄糖苷酶活测定: 试管中加入650μL水、200μL 0.5mol/L醋酸缓冲液、稀释好的酶液50μL(空白管加入50μL 0.05mol/L HAC buffer)。45℃水浴预热5min加入100μL pNPG，水浴10min。立即加入500μL 1mol/LNa2CO3终止反应。紫外分光光度计420nm测定OD值。以对硝基苯酚为标准作标准曲线。
实验结果：
在本实验中，我们利用粉碎机粉碎秸秆至1mm左右粗磨。在此基础上，球磨5-15次。最后，我们把球磨和粗磨的秸秆以1:10的比例加热水中，在60°C、100rpm的条件下进行固液萃取。反应结束后利用离心（8000rpm、5分钟）得到上清液。加95%乙醇至上清液使乙醇浓度达到70%。在此离心得到醇提物，干燥后得到纤维素酶诱导剂。如图1所示，我们通过70%的乙醇沉降粗磨、5-15次球磨的秸秆，得到纤维素酶诱导剂。把纤维素酶诱导剂加入到发酵液中，我们探讨了秸秆纤维素酶诱导剂对纤维素酶生产的影响。如图1所示，纤维素酶诱导剂的添加大幅度提高了纤维素酶活性，特别是对β-葡萄糖苷活性的提高有显著效果。众所周知，里氏木霉由来的纤维素酶β-葡萄糖苷酶活性低。利用里氏木霉的纤维素酶糖化生物质时需要额外添加β-葡萄糖苷酶，导致糖化成本上升。本研究中发现秸秆纤维素酶诱导剂对纤维素酶、特别是β-葡萄糖苷酶活性的提高有促进作用，应用于纤维素酶生产中可以有效提高纤维素酶总体活性。
试验例3 剩余残渣的糖化
实验方法：球磨稻杆（15次）经水洗（60°C ，150 rpm，1 h）后，加入纤维素酶量（10FPU/g水稻秸秆），10% （w/v）体系，调节pH至5，在45℃，200 rpm下糖化，分别于0 h，12 h，24h，36 h，48 h等时间取样。糖类物质的测定采用高效液相色谱仪，此系统配有一个视差检测器，采用糖柱（Aminex HPX-87H column 7.8 mm *30 cm;Bio-Rad, USA）进行分离，各组分分离的流速为1.0 mL/min，温度为80℃，流动相为去离子水，进样体积为10 μL。
实验结果：
在本实验中，我们利用粉碎机粉碎秸秆至1mm左右粗磨后，球磨秸秆15次。最后，我们把球磨的秸秆以1:10的比例加热水中，在60°C、100rpm的条件下进行固液萃取。反应结束后利用离心（8000rpm、5分钟）得到残渣，干燥后利用纤维素酶糖化。作为对照，我们也在相同条件下糖化了没有水洗的秸秆。如图2所示，葡萄糖和木糖浓度随反应时间的增加而提高，在48小时时，残渣中水解出43g/L葡萄糖,6.7g/L木糖。这些值要高于未处理秸秆中水解出的葡萄糖和木糖的浓度，说明提取诱导剂的同时可有效提高秸秆的单糖收率。所以，我们认为耦合诱导剂的提取工艺和糖化工艺是一种有效降低秸秆生物转化成本的方法，可也为及秸秆处理提供一个新的途径。
Claims (1)
1.一种从秸秆中提取纤维素酶生产诱导剂的方法，其特征在于具有如下的过程和步骤：
a，将秸秆粉碎至粒径小于1mm；秸秆为水稻、小麦、大麦、玉米的任一种；
所述粉碎步骤为先将秸秆在60℃下烘干，用微型植物试样粉碎机进行粗磨，随后行星式球磨机研磨粗磨后的样品，保持转速45Hz，运转10min，停时间5min，共5-15个循环，得到颗粒均匀、孔径较小的秸秆粉末；
b，把球磨的秸秆以1:10的比例加热水中，在60℃、100rpm的条件下进行固液萃取；反应结束后利用8000rpm、5分钟的离心操作得到上清液；加95％乙醇至上清液使乙醇浓度达到70％；再次离心得到醇提物，干燥后得到纤维素酶诱导剂；
c，由纤维素酶诱导剂制取纤维素酶的方法，其制取方法如下：
将步骤b中得到的纤维素酶诱导剂添加于培养液中，此培养液为由培养纤维素酶生产菌如里氏木霉、糖单孢菌或芽孢杆菌配制而成的纤维素酶培养液；在培养液中培养时间为3-8天，温度为20-40℃；最终制得纤维素酶；
d，然后利用上述步骤c中得到的纤维素酶糖化上述步骤b中得到的剩余固体残渣，得到秸秆单糖。
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