CN110951628B - 一种用于秸秆降解的高β-葡萄糖苷酶活力里氏木霉工程菌株的构建方法及应用 - Google Patents
一种用于秸秆降解的高β-葡萄糖苷酶活力里氏木霉工程菌株的构建方法及应用 Download PDFInfo
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- CN110951628B CN110951628B CN201911394283.1A CN201911394283A CN110951628B CN 110951628 B CN110951628 B CN 110951628B CN 201911394283 A CN201911394283 A CN 201911394283A CN 110951628 B CN110951628 B CN 110951628B
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Abstract
本发明涉及基因工程技术领域，特别公开了一种用于秸秆降解的高β‑葡萄糖苷酶活力里氏木霉工程菌株的构建方法及应用。该构建方法，其特征在于：所述工程菌株基因组中含有来自黑曲霉β‑葡萄糖苷酶变体编码基因mbglA，其基因型为QM9414OEmbglA，是以里氏木霉QM9414为出发菌，转化含黑曲霉β‑葡萄糖苷酶变体编码基因mbglA的表达盒Pcbh1‑mbglA‑TbglA后筛选、验证获得。本发明还公开了该工程菌株在发酵生产纤维素酶中的应用，证实该工程菌株为目前最高β‑葡萄糖苷酶活力的里氏木霉工程菌株；其发酵生产的纤维素酶系可以高效应用于玉米秸秆降解糖化，具有良好的工业开发和应用前景。
Description
（一）技术领域
本发明涉及基因工程技术领域，特别涉及一种用于秸秆降解的高β-葡萄糖苷酶活力里氏木霉工程菌株的构建方法及应用。
（二）背景技术
将秸秆等生物质酶促水解成可发酵糖，进而转化为沼气或乙醇是发展可再生性能源并克服农作物废弃物环境污染的有效途径。然而过高的纤维素酶生产成本限制了纤维材料能源化的发展。因此，改良纤维素酶系，提高底物糖化效率，对降低酶生产成本、促进纤维素降解转化具有重要意义。丝状真菌里氏木霉（Trichoderma reesei）因其高纤维素酶活力而成为纤维素酶生产的主要工业菌株。因此，对里氏木霉的菌种改良是构建高效纤维素酶系，提高秸秆材料糖化效率的最有效途径。
里氏木霉活力的纤维素降解酶主要有外切葡聚糖苷酶（CBHs）、内切葡聚糖甘酶（EGs）和β-葡萄糖苷酶（BGL）。三者协同将纤维素底物水解成葡萄糖，然而里氏木霉纤维素酶系中BGL的含量不足1%。BGL的不足时常造成纤维二糖的积累，高浓的的纤维二糖进而引起了对外切纤维素酶的反馈抑制。普遍认为里氏木霉降解纤维素的瓶颈是BGL的不足。
向里氏木霉酶系中外源补加BGL能够显著提高水解纤维素作底物时的葡萄糖得率。另外，在里氏木霉中本源和异源表达BGL1可以提高其纤维素酶系降解纤维素底物的能力，但目前纤维素酶系组分中的BGL表达水平仍然不高。提高里氏木霉纤维素降解酶系中的BGL活力是构建纤维素酶高产菌株和提升纤维素酶系整体糖化效率的关键。因此，利用基因工程策略构建高活力的葡萄糖苷酶高活力具有重要工业应用价值和理论指导意义。
（三）发明内容
本发明为了弥补现有技术的不足，提供了一种产量高、生产成本低的用于秸秆降解的高β-葡萄糖苷酶活力里氏木霉工程菌株的构建方法及应用。
本发明是通过如下技术方案实现的：
一种用于秸秆降解的高β-葡萄糖苷酶活力里氏木霉工程菌株的构建方法，其特征在于：所述工程菌株基因组中含有来自黑曲霉β-葡萄糖苷酶变体编码基因mbglA，其基因型为QM9414OEmbglA，是以里氏木霉QM9414（ATCC26921）为出发菌，转化含黑曲霉β-葡萄糖苷酶变体编码基因mbglA的表达盒Pcbh1-mbglA-TbglA后筛选、验证获得。
上述用于秸秆降解的高β-葡萄糖苷酶活力里氏木霉工程菌株的构建方法，其具体为：将来自黑曲霉的β-葡萄苷酶进行定点突变，突变位点是I179S、Q201E和G294W；随后利用融合PCR方法得到含有终止区的融合基因mbglA-TbglA，同样地将来自里氏木霉纤维二糖水解酶1的启动子与mbglA-TbglA融合，得到表达盒Pcbh1-mbglA-TbglA；再将该表达盒转化进出发菌里氏木霉QM9414的基因组，即获得改造的黑曲霉mBGLA的高活力菌株QMB1；其中，所述mbglA基因其上游为纤维二糖水解酶1启动子Pcbh1，下游为黑曲霉β-葡萄糖苷酶编码基因bglA的终止子Tbgl1。所述黑曲霉β-葡萄糖苷酶改造前后的氨基酸序列如SEQ ID NO.1和SEQ ID NO.2所示；β-葡萄糖苷酶改造前后的基因核苷酸序列如SEQ ID NO.3和SEQ IDNO.4所示。
上述构建方法中，定点突变和融合PCR方法包括如下步骤：
（1）利用引物Pcbh1-F/Pcbh1-R扩增1.8kb的cbh1启动子区；
（2）利用引物Bg-1F/Bg-835R（I179S）扩增含有I179S突变的835bp的bglA的部分编码区；
（3）利用引物Bg-2143F（Q201E）/Bg-1146R（G294W）扩增含有Q201E和G294W突变的3415bp的bglA的部分编码区；
（4）利用引物Bg-2170F（G294W）/Bg-529DR扩增包含1823bp含G294W突变的BGL编码区和529bp的BGL终止子区的2352bp的序列；
（5）利用融合PCR的方法以Pcbh1-F/Bg-529DR为引物扩增得到5.3kb的Pcbh1-mbglA-TbglA表达盒。
上述构建方法中，所述黑曲霉β-葡萄糖苷酶基因bglA来自黑曲霉ATCC1015基因组，其NCBI序列号为：XP_026627948.1；并对BGLA进行基因定点突变I179S、Q201E和G294W，即获得mBGLA；所述mbglA基因的启动子Pcbh1来源于里氏木霉QM9414，终止子TbglA来源于黑曲霉ATCC1015。
本发明所述用于秸秆降解的高β-葡萄糖苷酶活力里氏木霉工程菌株在发酵生产纤维素酶中的应用。
上述应用中，发酵生产纤维素酶的方法是：将基因工程菌株里氏木霉（Trichoderma reesei）QMB1接种到装有50mL种子培养基的300mL三角瓶中，于30±2℃、转速为180-220rpm下培养24-36h，得发酵种子液；将得到的种子液先抽滤、收集湿菌丝，再按0.5-1g湿菌丝转接到100mL发酵培养基的比例扩大接种，继续培养120-168h，然后以9000-13000rpm，4℃下离心5-10min，收集上清液，即得到含纤维素酶的酶液。
其中，所述种子培养基的组成成分为：葡萄糖 10g/L，玉米粉 15g/L，(NH4)2SO45g/L，KH2PO4 15g/L，MgSO4 1g/L，CaCl2 0.6g/L。
所述发酵培养基的组成成分为：结晶纤维 40g/L，玉米浆 20g/L，酵母膏 10g/L，(NH4)2SO4 5g/L，KH2PO4 5g/L，MgSO4 1g/L，CaCl2 0.6g/L，CaCO3 2.5g/L，甘油 2.5g/L，Tween-80 2g/L。
本发明所述的用于秸秆降解的高β-葡萄糖苷酶活力里氏木霉工程菌株发酵产生的纤维素酶在水解玉米秸秆中的应用。
本发明公开了一株用于秸秆降解的高β-葡萄糖苷酶活力里氏木霉工程菌及其构建方法与应用。较现有技术中的里氏木霉QM9414比，本发明的里氏木霉工程菌QMB1具有以下突出效果：
本发明所述的里氏木霉菌株QMB1较出发菌株具有高β-葡萄糖苷酶活力，实验证实该工程菌株的葡萄糖苷酶活力110IU/mL，与出发菌株相比提高了55倍，为目前所报道的最高BGL的酶活力水平；另外，利用本发明的里氏木霉工程菌QMB1所生产的纤维素酶系处理玉米秸秆材料时，与出发株相比，葡萄糖得率提高了2.26倍；在水解玉米芯材料96h时，其纤维素转化率能够到达99%，显著高于出发菌株（44%）。
本发明提供的基因工程菌的β-葡萄糖苷酶产量高，生产成本低，并且该酶系可以高效应用于玉米秸秆及其他木质纤维素材料的降解，具有良好的工业开发和应用前景。
（四）附图说明
下面结合附图对本发明作进一步的说明。
图1为本发明黑曲霉Pcbh1-mbglA-TbglA基因表达盒的图谱；
图2为黑曲霉的β-葡萄糖苷酶变体的高活力里氏木霉PCR验证电泳图；
图3为工程菌株β-葡萄糖苷酶活力平板检测图；
图4为工程菌株的β-葡萄糖苷酶活力测定结果对比图；
图5为工程菌株生产纤维素酶的对玉米秸秆的糖化应用检测图。
图中，Y1、Y2和Y3为不同的引物，Y1/Y2和Y2/3为所选用的不同验证引物对，M为1kbDNA marker，QMB1为高活力改造的黑曲霉mBglA的工程菌株，QM9414为出发菌株，为阴性对照；SDC11为一株本源过表达bgl1的工程菌株，为阳性对照。
（五）具体实施方式
下面结合具体实施例对本发明内容进行详细说明。如下所述例子仅是本发明的较佳实施方式而已，并非对本发明作任何形式上的限制，凡是依据本发明的技术实质对实施方式所做的任何简单修改，等同变化与修饰，均属于本发明技术方案的范围内。
一般性说明：
实施例所用出发菌种里氏木霉（Trichoderma reesei）QM9414，购自美国菌种保藏中心，其保藏号为ATCC 26921。黑曲霉（Aspergillius niger）购自美国菌种保藏中心，其保藏号为ATCC 1015。
实施例所用质粒提取试剂盒试剂盒、琼脂糖凝胶DNA回收试剂盒购自美国OMEGABiotek公司，片段扩增所用Taq酶PrimeSTAR和菌株验证所用Taq酶2*Phanta mix购自诺唯赞有限公司；质粒PAN7-1的构建见（Punt et al., Gene. 1987, 56, 117-24.）。
实施例1：黑曲霉的β-葡萄糖苷酶变体编码基因的构建
首先以黑曲霉ATC1015的基因组DNA为模板，以Bg-1F/Bg-835R（I179S）为引物扩增带有I179S突变的835bp的bglA的前端表达元件；接着以引物Bg-2143F（Q201E）/Bg-1146R（G294W）扩增含有Q201E和G294W突变的3415bp的bglA的中端表达元件；然后以Bg-2170F（G294W）/Bg-529DR扩增包含1823bp含G294W突变的BGL末端表达元件和529bp的BglA终止子序列；最后利用融合PCR，将前、中和后表达元件连接，利用引物Bg-1F/Bg-529DR扩增得到3477bp的完整的含终止子区的mbglA编码序列。再以里氏木霉QM9414的基因组DNA为模板，以Pcbh1-F/Pcbh1-R为引物扩增1.8kb的Pcbh1启动子序列，随后利用融合PCR将Pcbh1和含终止子区的mbglA进行融合，以Pcbh1-F/Bg-529DR为引物扩增得到5.3kb的黑曲霉β-葡萄糖苷酶变体表达盒Pcbh1-mbglA-TbglA。表达盒图谱见附图1所示。
上述引物对具体序列如下：
Pcbh1-F：
CTTTTCCCTTCCTCTAGTGTTG；
Pcbh1-R：
CGCCTCGATCAAAGTGAACCTCATGATGCGCAGTCCGCGGTTGACTATTG；
Bg-1F：
CAATAGTCAACCGCGGACTGCGCATCATGAGGTTCACTTTGATCGAGGCG；
Bg-835R（I179S）：
CGTAGGCGATGTAGTGCTTAGCCGTTGCAACCACACCAGCATCCTGGGAACC；
Bg-2143F（Q201E）：
TTGCAACGGCTAAGCACTACATCGCCTACGAGCAGGAGCATTTCCGTGAGGCG；
Bg-1146R（G294W）：
AGACATGTCCAACCACGCCAA；
Bg-2170F（G294W）：
TTGGCGTGGTTGGACATGTCT；
Bg-529DR：
TGTGGGTGGAGGGTGCTGGA。
实施例2：利用表达盒Pcbh1-mbglA-TbglA转化里氏木霉QM9414构建β-葡萄糖苷酶高活力基因工程菌
里氏木霉QM9414的遗传转化是利用PEG/CaCl2介导的原生质体转化方法，潮霉素B抗性基因hph为选择标记。将纯化的10µg Pcbh1-mbglA-TbglA的片段和1µg含有hph抗性基因的质粒PAN7-1 （Punt et al., Gene. 1987, 56, 117-24.）混匀后共转化里氏木霉QM9414的原生质体。
上述PEG/CaCl2介导的原生质体转化的具体方法如下：
（1）里氏木霉QM9414原生质体的制备：
准备新鲜的里氏木霉孢子（1周之内），涂布含有玻璃纸的PDA平板上，30℃培养，待菌丝生长到合适的长度后，将菌丝刮入含有裂解酶的转化液1中，混匀后30℃静置裂解1.5h；将裂解物过滤，收集滤液；2500rpm离心10min，弃上清；用转化液2将轻轻地重新悬浮沉淀，4℃, 2500rpm 再次离心10min后弃上清；加入0.5mL 转化液2重悬浮沉淀，置于冰上以备后续实验。
其中上述PDA培养基组成为：土豆200g，加水煮沸30min，8层纱布过滤，滤液添加葡萄糖20g，定容至1L，2%琼脂粉，115℃灭菌30min；
转化液1：1.2M山梨醇，0.1M KH2PO4，pH 5.6，115℃灭菌30min；
转化液2：1M山梨醇，50mM CaCl2，10mM Tris-HCl，pH 7.5，115℃灭菌30min。
（2）表达盒Pcbh1-mbglA-TbglA转化里氏木霉QM9414的原生质体：
将混匀后的Pcbh1-mbglA-TbglA片段和PAN7-1质粒，加入50μL预冷的转化液3，冰浴20min，后加2mL常温的转化液3，室温放置5min，最后加4mL转化液2，轻轻混匀；然后，将上述混合物加入到含250μg/mL潮霉素B的50mL转化上层培养基中，混匀后倒入预先凝固的转化下层培养基平板，冷却凝固后，30℃培养3-5天，挑选生长良好的转化株，提取染色体，进行分子验证。结果如附图2所示，以Y1/Y3和Y2/Y3为引物分别对mBGLA高活力菌株进行PCR验证，其中转化子能够分别扩增出2.0 和1.0kb的条带，而出发菌株没有条带产生。将验证正确的mBGLA高活力菌株命名为里氏木霉（Trichoderma reesei）QMB1，该菌株基因型为QM9414OEmbglA。
其中上述转化液3培养基为：25% PEG 6000，50mM CaCl2，10mM Tris-HCl，pH7.5。
上层培养基（g/L）：葡萄糖10，SO4•7H2O 1，LKH2PO4 10，(NH4)2SO4 6，C6H5Na3O7 3，山梨醇 182.18，琼脂糖6；
下层培养基（g/L）：葡萄糖10，MgSO4•7H2O 1，KH2PO4 10，(NH4)2SO4 6，C6H5Na3O7 3，琼脂粉20；以上培养基均经115℃灭菌30min后使用。
上述所用引物为：
Y1：CCTTTGGCGTTTCCCTGAITCT；
Y2：GTATTACCCCTCCCCTTGG；
Y3：TCGTAGGCGATGTAGTGC。
实施例3：β-葡萄糖苷酶高活力工程菌株酶活力平板分析
将实施例2获得的工程菌株QMB1和出发菌株QM9414，利用β-葡萄糖苷酶活力平板检测方法对转化株酶活力进行检测，具体方法是：取等量转化株QMB1孢子、出发菌株QM9414孢子以及一株高表达bgl1的菌株SDC11的孢子接种至七叶苷培养基上，培养2d，观察菌落周围的黑色晕圈的形成。如附图3所示，转化株QMB1的菌落周围的黑色晕圈显著大于出发菌株QM9414和对照菌株SDC11菌落周围的黑色晕圈，说明Pcbh1-mbglA-TbglA成功整合到基因组中且β-葡萄糖苷酶基因获得表达。因此，β-葡萄糖苷酶高活力工程菌可用于后继分析应用。
其中，上述七叶苷平板培养基（g/L）为：CMC-Na 10，七叶苷3，柠檬酸铁0.5，(NH4)2SO4 2，MgSO4·7H2O 0.5，K2HPO4 1，酵母膏1，2.5%琼脂粉，115℃灭菌30min。
实施例4：β-葡萄糖苷酶高活力工程菌株酶活力的测定
将实施例2获得的工程菌株QMB1与出发菌株QM9414（108个/mL）分别接种至100mL孢子悬液分别接种到100mL（500mL的三角瓶）种子培养基中，30℃、200rpm培养36h后，取10mL菌液转接到100mL（500mL的三角瓶）发酵培养基中培养，在培养3d、5d和7d时取发酵液，离心，取上清，测定β-葡萄糖苷酶活力。附图4显示工程菌株QMB1的β-葡萄糖苷酶活力为110IU/mL，比出发菌株（2IU/mL）提高了55倍；由此表明，高表达β-葡萄糖苷酶能够显著提高里氏木霉的胞外β-葡萄糖苷酶活力。
其中，上述种子培养基为（g/L）：葡萄糖10g/L，玉米粉 15g/L，(NH4)2SO4 5g/L，KH2PO4 15g/L，MgSO4 1g/L，CaCl2 0.6g/L。
β-葡萄糖苷酶活力（pNPGase）测定方法：
取100μL适当稀释的酶液，加入50μL、10mmol/L对硝基苯-β-D-吡喃葡萄糖苷（pNPG）柠檬酸buffer溶液（pH4.8），混匀，50℃、水浴30min后加入150μl、10% Na2CO3终止反应。420nm测OD值。同时以先10% Na2CO3，后加入酶液作为空白对照。
pNPGase酶活力的定义：在50℃、pH 4.8条件下，每毫升酶液每分钟产生对硝基酚（pNP）的微摩尔数。
实施例5：mBGLA高活力工程菌株产生的纤维素酶系在水解玉米秸秆中的应用
将实施例4获得的工程菌株QMB1和出发菌株QM9414所制备的纤维素酶液应用于实际玉米秸秆的糖化实验中。糖化实验：反应体系为30mL，糖化底物为5%的稀酸处理玉米秸秆，纤维素酶添加量为10FPU/g底物，为了防止染菌加入0.1% NaN3，用0.1M·pH4.8的醋酸缓冲液补齐至30mL，50℃、每分钟150rpm进行糖化，糖化结束后、取糖化液，离心，取上清，利用SBA-40C（BISAS，China）生物传感仪进行葡萄糖的测定。结果如附图5所示，工程菌株QEB1的酶液能够显著提高葡萄糖得率，QMB1的葡萄糖得率（34mg/mL）是出发菌株QM9414的酶液（15.6mg/mL）的2.17倍。工程菌株QEB1的酶液的转化率（99.8%）显著高于QM9414的酶液的转化率（44%）。
其中，上述玉米秸秆的成分是：纤维素65.7%，半纤维素1.8%，木素13.2%，其他成分为19.3%。
综上，本研究实现了里氏木霉QM9414遗传转化体系的改良并成功高水平表达黑曲霉β-葡萄糖苷酶变体mBGLA，构建了具有高活力β-葡萄糖苷酶的改良纤维素酶系，获得了用于玉米秸秆高效降解糖化的工程菌，为下一步实际工业应用打下基础。上述事实证明，利用本发明公开的里氏木霉β-葡萄糖苷酶高活力的纤维素酶系能够显著提高秸秆材料的降解能力，且在实际应用中，能够达到预处理秸秆99%的纤维素转化率。该工程菌株具有较高的理论研究意义和实际应用价值。
SEQUENCE LISTING
<110> 乐陵胜利新能源有限责任公司，山东大学
<120> 一种用于秸秆降解的高β-葡萄糖苷酶活力里氏木霉工程菌株的构建方法及
应用
<130> 3
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 860
<212> PRT
<213> 改造前的黑曲霉β-葡萄糖苷酶（BGLA）
<400> 1
Met Arg Phe Thr Leu Ile Glu Ala Val Ala Leu Thr Ala Val Ser Leu
1 5 10 15
Ala Ser Ala Asp Glu Leu Ala Tyr Ser Pro Pro Tyr Tyr Pro Ser Pro
20 25 30
Trp Ala Asn Gly Gln Gly Asp Trp Ala Glu Ala Tyr Gln Arg Ala Val
35 40 45
Asp Ile Val Ser Gln Met Thr Leu Ala Glu Lys Val Asn Leu Thr Thr
50 55 60
Gly Thr Gly Trp Glu Leu Glu Leu Cys Val Gly Gln Thr Gly Gly Val
65 70 75 80
Pro Arg Leu Gly Ile Pro Gly Met Cys Ala Gln Asp Ser Pro Leu Gly
85 90 95
Val Arg Asp Ser Asp Tyr Asn Ser Ala Phe Pro Ala Gly Val Asn Val
100 105 110
Ala Ala Thr Trp Asp Lys Asn Leu Ala Tyr Leu Arg Gly Gln Ala Met
115 120 125
Gly Gln Glu Phe Ser Asp Lys Gly Ala Asp Ile Gln Leu Gly Pro Ala
130 135 140
Ala Gly Pro Leu Gly Arg Ser Pro Asp Gly Gly Arg Asn Trp Glu Gly
145 150 155 160
Phe Ser Pro Asp Pro Ala Leu Ser Gly Val Leu Phe Ala Glu Thr Ile
165 170 175
Lys Gly Ile Gln Asp Ala Gly Val Val Ala Thr Ala Lys His Tyr Ile
180 185 190
Ala Tyr Glu Gln Glu His Phe Arg Gln Ala Pro Glu Ala Gln Gly Tyr
195 200 205
Gly Phe Asn Ile Thr Glu Ser Gly Ser Ala Asn Leu Asp Asp Lys Thr
210 215 220
Met His Glu Leu Tyr Leu Trp Pro Phe Ala Asp Ala Ile Arg Ala Gly
225 230 235 240
Ala Gly Ala Val Met Cys Ser Tyr Asn Gln Ile Asn Asn Ser Tyr Gly
245 250 255
Cys Gln Asn Ser Tyr Thr Leu Asn Lys Leu Leu Lys Ala Glu Leu Gly
260 265 270
Phe Gln Gly Phe Val Met Ser Asp Trp Ala Ala His His Ala Gly Val
275 280 285
Ser Gly Ala Leu Ala Gly Leu Asp Met Ser Met Pro Gly Asp Val Asp
290 295 300
Tyr Asp Ser Gly Thr Ser Tyr Trp Gly Thr Asn Leu Thr Ile Ser Val
305 310 315 320
Leu Asn Gly Thr Val Pro Gln Trp Arg Val Asp Asp Met Ala Val Arg
325 330 335
Ile Met Ala Ala Tyr Tyr Lys Val Gly Arg Asp Arg Leu Trp Thr Pro
340 345 350
Pro Asn Phe Ser Ser Trp Thr Arg Asp Glu Tyr Gly Phe Lys Tyr Tyr
355 360 365
Tyr Val Ser Glu Gly Pro Tyr Glu Lys Val Asn Gln Phe Val Asn Val
370 375 380
Gln Arg Asn His Ser Glu Leu Ile Arg Arg Ile Gly Ala Asp Ser Thr
385 390 395 400
Val Leu Leu Lys Asn Asp Gly Ala Leu Pro Leu Thr Gly Lys Glu Arg
405 410 415
Leu Val Ala Leu Ile Gly Glu Asp Ala Gly Ser Asn Pro Tyr Gly Ala
420 425 430
Asn Gly Cys Ser Asp Arg Gly Cys Asp Asn Gly Thr Leu Ala Met Gly
435 440 445
Trp Gly Ser Gly Thr Ala Asn Phe Pro Tyr Leu Val Thr Pro Glu Gln
450 455 460
Ala Ile Ser Asn Glu Val Leu Lys Asn Lys Asn Gly Val Phe Thr Ala
465 470 475 480
Thr Asp Asn Trp Ala Ile Asp Gln Ile Glu Ala Leu Ala Lys Thr Ala
485 490 495
Ser Val Ser Leu Val Phe Val Asn Ala Asp Ser Gly Glu Gly Tyr Ile
500 505 510
Asn Val Asp Gly Asn Leu Gly Asp Arg Arg Asn Leu Thr Leu Trp Arg
515 520 525
Asn Gly Asp Asn Val Ile Lys Ala Ala Ala Ser Asn Cys Asn Asn Thr
530 535 540
Ile Val Ile Ile His Ser Val Gly Pro Val Leu Val Asn Glu Trp Tyr
545 550 555 560
Asp Asn Pro Asn Val Thr Ala Ile Leu Trp Gly Gly Leu Pro Gly Gln
565 570 575
Glu Ser Gly Asn Ser Leu Ala Asp Val Leu Tyr Gly Arg Val Asn Pro
580 585 590
Gly Ala Lys Ser Pro Phe Thr Trp Gly Lys Thr Arg Glu Ala Tyr Gln
595 600 605
Asp Tyr Leu Tyr Thr Glu Pro Asn Asn Gly Asn Gly Ala Pro Gln Glu
610 615 620
Asp Phe Val Glu Gly Val Phe Ile Asp Tyr Arg Gly Phe Asp Lys Arg
625 630 635 640
Asn Glu Thr Pro Ile Tyr Glu Phe Gly Tyr Gly Leu Ser Tyr Thr Thr
645 650 655
Phe Asn Tyr Ser Asn Leu Gln Val Glu Val Leu Ser Ala Pro Ala Tyr
660 665 670
Glu Pro Ala Ser Gly Glu Thr Glu Ala Ala Pro Thr Phe Gly Glu Val
675 680 685
Gly Asn Ala Ser Asp Tyr Leu Tyr Pro Asp Gly Leu Gln Arg Ile Thr
690 695 700
Lys Phe Ile Tyr Pro Trp Leu Asn Ser Thr Asp Leu Glu Ala Ser Ser
705 710 715 720
Gly Asp Ala Ser Tyr Gly Gln Asp Ala Ser Asp Tyr Leu Pro Glu Gly
725 730 735
Ala Thr Asp Gly Ser Ala Gln Pro Ile Leu Pro Ala Gly Gly Gly Ala
740 745 750
Gly Gly Asn Pro Arg Leu Tyr Asp Glu Leu Ile Arg Val Ser Val Thr
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Ile Lys Asn Thr Gly Lys Val Ala Gly Asp Glu Val Pro Gln Leu Tyr
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Val Ser Leu Gly Gly Pro Asn Glu Pro Lys Ile Val Leu Arg Gln Phe
785 790 795 800
Glu Arg Ile Thr Leu Gln Pro Ser Glu Glu Thr Gln Trp Ser Thr Thr
805 810 815
Leu Thr Arg Arg Asp Leu Ala Asn Trp Asn Val Glu Thr Gln Asp Trp
820 825 830
Glu Ile Thr Ser Tyr Pro Lys Met Val Phe Val Gly Ser Ser Ser Arg
835 840 845
Lys Leu Pro Leu Arg Ala Ser Leu Pro Thr Val His
850 855 860
<210> 2
<211> 860
<212> PRT
<213> 改造后的黑曲霉β-葡萄糖苷酶（mBGLA）
<400> 2
Met Arg Phe Thr Leu Ile Glu Ala Val Ala Leu Thr Ala Val Ser Leu
1 5 10 15
Ala Ser Ala Asp Glu Leu Ala Tyr Ser Pro Pro Tyr Tyr Pro Ser Pro
20 25 30
Trp Ala Asn Gly Gln Gly Asp Trp Ala Glu Ala Tyr Gln Arg Ala Val
35 40 45
Asp Ile Val Ser Gln Met Thr Leu Ala Glu Lys Val Asn Leu Thr Thr
50 55 60
Gly Thr Gly Trp Glu Leu Glu Leu Cys Val Gly Gln Thr Gly Gly Val
65 70 75 80
Pro Arg Leu Gly Ile Pro Gly Met Cys Ala Gln Asp Ser Pro Leu Gly
85 90 95
Val Arg Asp Ser Asp Tyr Asn Ser Ala Phe Pro Ala Gly Val Asn Val
100 105 110
Ala Ala Thr Trp Asp Lys Asn Leu Ala Tyr Leu Arg Gly Gln Ala Met
115 120 125
Gly Gln Glu Phe Ser Asp Lys Gly Ala Asp Ile Gln Leu Gly Pro Ala
130 135 140
Ala Gly Pro Leu Gly Arg Ser Pro Asp Gly Gly Arg Asn Trp Glu Gly
145 150 155 160
Phe Ser Pro Asp Pro Ala Leu Ser Gly Val Leu Phe Ala Glu Thr Ile
165 170 175
Lys Gly Ser Gln Asp Ala Gly Val Val Ala Thr Ala Lys His Tyr Ile
180 185 190
Ala Tyr Glu Gln Glu His Phe Arg Glu Ala Pro Glu Ala Gln Gly Tyr
195 200 205
Gly Phe Asn Ile Thr Glu Ser Gly Ser Ala Asn Leu Asp Asp Lys Thr
210 215 220
Met His Glu Leu Tyr Leu Trp Pro Phe Ala Asp Ala Ile Arg Ala Gly
225 230 235 240
Ala Gly Ala Val Met Cys Ser Tyr Asn Gln Ile Asn Asn Ser Tyr Gly
245 250 255
Cys Gln Asn Ser Tyr Thr Leu Asn Lys Leu Leu Lys Ala Glu Leu Gly
260 265 270
Phe Gln Gly Phe Val Met Ser Asp Trp Ala Ala His His Ala Gly Val
275 280 285
Ser Gly Ala Leu Ala Trp Leu Asp Met Ser Met Pro Gly Asp Val Asp
290 295 300
Tyr Asp Ser Gly Thr Ser Tyr Trp Gly Thr Asn Leu Thr Ile Ser Val
305 310 315 320
Leu Asn Gly Thr Val Pro Gln Trp Arg Val Asp Asp Met Ala Val Arg
325 330 335
Ile Met Ala Ala Tyr Tyr Lys Val Gly Arg Asp Arg Leu Trp Thr Pro
340 345 350
Pro Asn Phe Ser Ser Trp Thr Arg Asp Glu Tyr Gly Phe Lys Tyr Tyr
355 360 365
Tyr Val Ser Glu Gly Pro Tyr Glu Lys Val Asn Gln Phe Val Asn Val
370 375 380
Gln Arg Asn His Ser Glu Leu Ile Arg Arg Ile Gly Ala Asp Ser Thr
385 390 395 400
Val Leu Leu Lys Asn Asp Gly Ala Leu Pro Leu Thr Gly Lys Glu Arg
405 410 415
Leu Val Ala Leu Ile Gly Glu Asp Ala Gly Ser Asn Pro Tyr Gly Ala
420 425 430
Asn Gly Cys Ser Asp Arg Gly Cys Asp Asn Gly Thr Leu Ala Met Gly
435 440 445
Trp Gly Ser Gly Thr Ala Asn Phe Pro Tyr Leu Val Thr Pro Glu Gln
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Ala Ile Ser Asn Glu Val Leu Lys Asn Lys Asn Gly Val Phe Thr Ala
465 470 475 480
Thr Asp Asn Trp Ala Ile Asp Gln Ile Glu Ala Leu Ala Lys Thr Ala
485 490 495
Ser Val Ser Leu Val Phe Val Asn Ala Asp Ser Gly Glu Gly Tyr Ile
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Asn Val Asp Gly Asn Leu Gly Asp Arg Arg Asn Leu Thr Leu Trp Arg
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Asn Gly Asp Asn Val Ile Lys Ala Ala Ala Ser Asn Cys Asn Asn Thr
530 535 540
Ile Val Ile Ile His Ser Val Gly Pro Val Leu Val Asn Glu Trp Tyr
545 550 555 560
Asp Asn Pro Asn Val Thr Ala Ile Leu Trp Gly Gly Leu Pro Gly Gln
565 570 575
Glu Ser Gly Asn Ser Leu Ala Asp Val Leu Tyr Gly Arg Val Asn Pro
580 585 590
Gly Ala Lys Ser Pro Phe Thr Trp Gly Lys Thr Arg Glu Ala Tyr Gln
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Asp Tyr Leu Tyr Thr Glu Pro Asn Asn Gly Asn Gly Ala Pro Gln Glu
610 615 620
Asp Phe Val Glu Gly Val Phe Ile Asp Tyr Arg Gly Phe Asp Lys Arg
625 630 635 640
Asn Glu Thr Pro Ile Tyr Glu Phe Gly Tyr Gly Leu Ser Tyr Thr Thr
645 650 655
Phe Asn Tyr Ser Asn Leu Gln Val Glu Val Leu Ser Ala Pro Ala Tyr
660 665 670
Glu Pro Ala Ser Gly Glu Thr Glu Ala Ala Pro Thr Phe Gly Glu Val
675 680 685
Gly Asn Ala Ser Asp Tyr Leu Tyr Pro Asp Gly Leu Gln Arg Ile Thr
690 695 700
Lys Phe Ile Tyr Pro Trp Leu Asn Ser Thr Asp Leu Glu Ala Ser Ser
705 710 715 720
Gly Asp Ala Ser Tyr Gly Gln Asp Ala Ser Asp Tyr Leu Pro Glu Gly
725 730 735
Ala Thr Asp Gly Ser Ala Gln Pro Ile Leu Pro Ala Gly Gly Gly Ala
740 745 750
Gly Gly Asn Pro Arg Leu Tyr Asp Glu Leu Ile Arg Val Ser Val Thr
755 760 765
Ile Lys Asn Thr Gly Lys Val Ala Gly Asp Glu Val Pro Gln Leu Tyr
770 775 780
Val Ser Leu Gly Gly Pro Asn Glu Pro Lys Ile Val Leu Arg Gln Phe
785 790 795 800
Glu Arg Ile Thr Leu Gln Pro Ser Glu Glu Thr Gln Trp Ser Thr Thr
805 810 815
Leu Thr Arg Arg Asp Leu Ala Asn Trp Asn Val Glu Thr Gln Asp Trp
820 825 830
Glu Ile Thr Ser Tyr Pro Lys Met Val Phe Val Gly Ser Ser Ser Arg
835 840 845
Lys Leu Pro Leu Arg Ala Ser Leu Pro Thr Val His
850 855 860
<210> 3
<211> 2948
<212> DNA
<213> 改造前的黑曲霉β-葡萄糖苷酶（bglA）
<400> 3
atgaggttca ctttgatcga ggcggtggct ctgactgccg tctcgctggc cagcgctgta 60
cgtgccgtca cttcctttgt cgtgtgaatt gcaattgcgc tcgattggat tcacttcttt 120
gtttcgtcac cactaacaat tgtctattca aaggatgaat tggcctactc cccgccgtat 180
tacccctccc cttgggccaa tggccagggt gactgggcgg aagcatacca gcgcgctgtt 240
gatatcgtct cgcagatgac attggctgag aaggtcaatt tgactacggg aactgggtaa 300
ggctcagtgg cacaaacgat gtgtatgctc ctgctaacga cctccagatg ggaattggaa 360
ttatgtgttg gtcagactgg aggtgttccc cggtaagttt gtgaagattg tcaaaacagg 420
gagcatctac tgatatatgg tgacagattg ggaattccgg gaatgtgtgc acaggatagc 480
cctctgggtg ttcgtgactg taagcaactc ggtgttttta ggctttgatg ctcttgctga 540
cacagcgtag ccgactacaa ctctgcgttc cctgccggtg tcaacgtggc cgcaacctgg 600
gacaagaatc tggcttacct tcgtggccag gctatgggtc aggagtttag tgacaagggt 660
gctgatatcc aattgggtcc agctgccggc cctctcggta gaagtcccga cggcggtcgt 720
aactgggagg gcttctcccc cgacccggcc ctcagtggtg tgctctttgc agagacaatc 780
aagggtattc aggatgctgg tgtggttgca acggctaagc actacatcgc ctacgagcag 840
gagcatttcc gtcaggcgcc tgaagctcaa ggctacggat tcaatattac cgagagtgga 900
agcgcgaacc tcgacgataa gactatgcat gagctgtacc tctggccctt cgcggatgcc 960
atccgtgcag gtgccggtgc tgtgatgtgc tcgtacaacc agatcaacaa cagctatggc 1020
tgccaaaaca gctacactct gaacaagctg ctcaaggctg agctgggttt ccagggcttt 1080
gtcatgagtg attgggcggc tcaccatgcc ggtgtgagtg gtgctttggc gggattggac 1140
atgtctatgc cgggagacgt cgattacgac agtggcacgt cttactgggg taccaacttg 1200
accatcagtg tgctcaacgg gacggtgccc caatggcgtg ttgatgacat ggctgtccgc 1260
atcatggccg cctactacaa ggtcggccgt gaccgtctgt ggactcctcc caacttcagc 1320
tcatggacca gagatgaata cggcttcaag tactactatg tctcggaggg accgtatgag 1380
aaggtcaacc agttcgtgaa cgtgcaacgc aaccatagcg agttgatccg ccgtattgga 1440
gcagacagca cggtgctcct caagaacgat ggcgctcttc ccttgactgg aaaggagcgc 1500
ttggtcgccc ttatcggaga agatgcgggt tccaatcctt atggtgccaa cggctgcagt 1560
gaccgtgggt gcgacaatgg aacattggcg atgggctggg gaagtggcac tgccaacttt 1620
ccctacttgg tgacccccga gcaggccatc tcgaacgagg tgctcaagaa caagaatggc 1680
gtattcactg cgaccgataa ctgggctatt gatcagattg aggcgcttgc taagaccgcc 1740
aggtaagaag atctcgggct ctttcttttt ccttattgtg caatgaatgc tgacaacatg 1800
gtagtgtctc tcttgtcttt gtcaacgccg actctggtga gggttatatc aatgtcgacg 1860
gaaacctggg tgaccgcagg aacctgaccc tgtggaggaa cggcgacaat gtgatcaagg 1920
ctgctgctag caactgcaac aacacgatcg ttattattca ctctgtcggc ccagtcttgg 1980
ttaacgagtg gtacgacaac cccaatgtta ccgctattct ctggggtggt cttcccggtc 2040
aggagtctgg caactccctc gccgacgtgc tctacggccg tgtcaacccc ggtgccaagt 2100
cgcccttcac ctggggcaag actcgtgagg cctaccaaga ttacttgtac accgagccca 2160
acaacggcaa cggagcgccc caggaagact tcgtcgaggg cgtcttcatt gactaccgcg 2220
gatttgacaa gcgcaacgag actcctatct atgagttcgg ctatggtctg agctacacca 2280
ccttcaacta ctcgaacctt caggtggagg ttctgagcgc ccctgcgtac gagcctgctt 2340
cgggcgagac tgaggcagcg ccgactttcg gagaggtcgg aaatgcgtcg gattacctct 2400
accccgatgg actgcagaga atcaccaagt tcatctaccc ctggctcaac agtaccgatc 2460
ttgaggcgtc ttctggggat gctagctatg ggcaggatgc ctcagactat cttcccgagg 2520
gagccaccga tggctctgcg caaccgatcc tgcctgccgg tggtggtgct ggcggcaacc 2580
ctcgcctgta cgacgagctc atccgcgtgt cggtgactat caagaacacc ggcaaggttg 2640
cgggtgatga agttcctcaa ctggtaagta gacagcaaat tcgaacgaag tcggacaaag 2700
ctaataaatc gcagtatgtt tctcttggcg gccctaacga acccaagatc gtgctgcgtc 2760
aattcgagcg tatcacgctg cagccgtcgg aagagacgca gtggagcaca actctgacgc 2820
gccgtgacct tgcgaactgg aatgttgaga cgcaggactg ggagattacg tcgtatccca 2880
agatggtgtt tgtcggaagc tcctcgcgga agctgccgct ccgggcgtct ctgcctactg 2940
ttcactaa 2948
<210> 4
<211> 2948
<212> DNA
<213> 改造后的黑曲霉β-葡萄糖苷酶（mbglA）
<400> 4
atgaggttca ctttgatcga ggcggtggct ctgactgccg tctcgctggc cagcgctgta 60
cgtgccgtca cttcctttgt cgtgtgaatt gcaattgcgc tcgattggat tcacttcttt 120
gtttcgtcac cactaacaat tgtctattca aaggatgaat tggcctactc cccgccgtat 180
tacccctccc cttgggccaa tggccagggt gactgggcgg aagcatacca gcgcgctgtt 240
gatatcgtct cgcagatgac attggctgag aaggtcaatt tgactacggg aactgggtaa 300
ggctcagtgg cacaaacgat gtgtatgctc ctgctaacga cctccagatg ggaattggaa 360
ttatgtgttg gtcagactgg aggtgttccc cggtaagttt gtgaagattg tcaaaacagg 420
gagcatctac tgatatatgg tgacagattg ggaattccgg gaatgtgtgc acaggatagc 480
cctctgggtg ttcgtgactg taagcaactc ggtgttttta ggctttgatg ctcttgctga 540
cacagcgtag ccgactacaa ctctgcgttc cctgccggtg tcaacgtggc cgcaacctgg 600
gacaagaatc tggcttacct tcgtggccag gctatgggtc aggagtttag tgacaagggt 660
gctgatatcc aattgggtcc agctgccggc cctctcggta gaagtcccga cggcggtcgt 720
aactgggagg gcttctcccc cgacccggcc ctcagtggtg tgctctttgc agagacaatc 780
aagggttccc aggatgctgg tgtggttgca acggctaagc actacatcgc ctacgagcag 840
gagcatttcc gtgaggcgcc tgaagctcaa ggctacggat tcaatattac cgagagtgga 900
agcgcgaacc tcgacgataa gactatgcat gagctgtacc tctggccctt cgcggatgcc 960
atccgtgcag gtgccggtgc tgtgatgtgc tcgtacaacc agatcaacaa cagctatggc 1020
tgccaaaaca gctacactct gaacaagctg ctcaaggctg agctgggttt ccagggcttt 1080
gtcatgagtg attgggcggc tcaccatgcc ggtgtgagtg gtgctttggc gtggttggac 1140
atgtctatgc cgggagacgt cgattacgac agtggcacgt cttactgggg taccaacttg 1200
accatcagtg tgctcaacgg gacggtgccc caatggcgtg ttgatgacat ggctgtccgc 1260
atcatggccg cctactacaa ggtcggccgt gaccgtctgt ggactcctcc caacttcagc 1320
tcatggacca gagatgaata cggcttcaag tactactatg tctcggaggg accgtatgag 1380
aaggtcaacc agttcgtgaa cgtgcaacgc aaccatagcg agttgatccg ccgtattgga 1440
gcagacagca cggtgctcct caagaacgat ggcgctcttc ccttgactgg aaaggagcgc 1500
ttggtcgccc ttatcggaga agatgcgggt tccaatcctt atggtgccaa cggctgcagt 1560
gaccgtgggt gcgacaatgg aacattggcg atgggctggg gaagtggcac tgccaacttt 1620
ccctacttgg tgacccccga gcaggccatc tcgaacgagg tgctcaagaa caagaatggc 1680
gtattcactg cgaccgataa ctgggctatt gatcagattg aggcgcttgc taagaccgcc 1740
aggtaagaag atctcgggct ctttcttttt ccttattgtg caatgaatgc tgacaacatg 1800
gtagtgtctc tcttgtcttt gtcaacgccg actctggtga gggttatatc aatgtcgacg 1860
gaaacctggg tgaccgcagg aacctgaccc tgtggaggaa cggcgacaat gtgatcaagg 1920
ctgctgctag caactgcaac aacacgatcg ttattattca ctctgtcggc ccagtcttgg 1980
ttaacgagtg gtacgacaac cccaatgtta ccgctattct ctggggtggt cttcccggtc 2040
aggagtctgg caactccctc gccgacgtgc tctacggccg tgtcaacccc ggtgccaagt 2100
cgcccttcac ctggggcaag actcgtgagg cctaccaaga ttacttgtac accgagccca 2160
acaacggcaa cggagcgccc caggaagact tcgtcgaggg cgtcttcatt gactaccgcg 2220
gatttgacaa gcgcaacgag actcctatct atgagttcgg ctatggtctg agctacacca 2280
ccttcaacta ctcgaacctt caggtggagg ttctgagcgc ccctgcgtac gagcctgctt 2340
cgggcgagac tgaggcagcg ccgactttcg gagaggtcgg aaatgcgtcg gattacctct 2400
accccgatgg actgcagaga atcaccaagt tcatctaccc ctggctcaac agtaccgatc 2460
ttgaggcgtc ttctggggat gctagctatg ggcaggatgc ctcagactat cttcccgagg 2520
gagccaccga tggctctgcg caaccgatcc tgcctgccgg tggtggtgct ggcggcaacc 2580
ctcgcctgta cgacgagctc atccgcgtgt cggtgactat caagaacacc ggcaaggttg 2640
cgggtgatga agttcctcaa ctggtaagta gacagcaaat tcgaacgaag tcggacaaag 2700
ctaataaatc gcagtatgtt tctcttggcg gccctaacga acccaagatc gtgctgcgtc 2760
aattcgagcg tatcacgctg cagccgtcgg aagagacgca gtggagcaca actctgacgc 2820
gccgtgacct tgcgaactgg aatgttgaga cgcaggactg ggagattacg tcgtatccca 2880
agatggtgtt tgtcggaagc tcctcgcgga agctgccgct ccgggcgtct ctgcctactg 2940
ttcactaa 2948
Claims (7)
1.一种用于秸秆降解的高β-葡萄糖苷酶活力里氏木霉工程菌株的构建方法，其特征在于：所述工程菌株基因组中含有来自黑曲霉β-葡萄糖苷酶变体编码基因mbglA，其基因型为QM9414OEmbglA，是以里氏木霉QM9414（ATCC26921）为出发菌，转化含黑曲霉β-葡萄糖苷酶变体编码基因mbglA的表达盒Pcbh1-mbglA-TbglA后筛选、验证获得；
将来自黑曲霉的β-葡萄苷酶进行定点突变，突变位点是I179S、Q201E和G294W；随后利用融合PCR方法得到含有终止区的融合基因mbglA-TbglA，同样地将来自里氏木霉纤维二糖水解酶1的启动子与mbglA-TbglA融合，得到表达盒Pcbh1-mbglA-TbglA；再将该表达盒转化进出发菌里氏木霉QM9414的基因组，即获得改造的黑曲霉mBGLA的高活力菌株QMB1；其中，所述mbglA基因其上游为纤维二糖水解酶1启动子Pcbh1，下游为黑曲霉β-葡萄糖苷酶编码基因bglA的终止子Tbgl1；
所述黑曲霉β-葡萄糖苷酶改造前后的氨基酸序列如SEQ ID NO.1和SEQ ID NO.2所示；β-葡萄糖苷酶改造前后的基因核苷酸序列如SEQ ID NO.3和SEQ ID NO.4所示；
所述黑曲霉β-葡萄糖苷酶基因bglA来自黑曲霉ATCC1015基因组，并对BGLA进行基因定点突变I179S、Q201E和G294W，即获得mBGLA；所述mbglA基因的启动子Pcbh1来源于里氏木霉QM9414，终止子TbglA来源于黑曲霉ATCC1015。
2.根据权利要求1所述的构建方法，其特征为，所述定点突变和融合PCR方法包括如下步骤：（1）利用引物Pcbh1-F/Pcbh1-R扩增1.8kb的cbh1启动子区；（2）利用引物Bg-1F/Bg-835R扩增含有I179S突变的835bp的bglA的部分编码区；（3）利用引物Bg-2143F/Bg-1146R扩增含有Q201E和G294W突变的3415bp的bglA的部分编码区；（4）利用引物Bg-2170F/Bg-529DR扩增包含1823bp含G294W突变的BGL编码区和529bp的BGL终止子区的2352bp的序列；（5）利用融合PCR的方法以Pcbh1-F/Bg-529DR为引物扩增得到5.3kb的Pcbh1-mbglA-TbglA表达盒。
3.根据权利要求1所述的用于秸秆降解的高β-葡萄糖苷酶活力里氏木霉工程菌株在发酵生产纤维素酶中的应用。
4.根据权利要求3所述的应用，其特征在于：将基因工程菌株里氏木霉QMB1接种到装有50mL种子培养基的300mL三角瓶中，于30±2℃、转速为180-220rpm下培养24-36h，得发酵种子液；将得到的种子液先抽滤、收集湿菌丝，再按0.5-1g湿菌丝转接到100mL发酵培养基的比例扩大接种，继续培养120-168h，然后以9000-13000rpm，4℃下离心5-10min，收集上清液，即得到含纤维素酶的酶液。
5.根据权利要求4所述的应用，其特征在于：所述种子培养基的组成成分为：葡萄糖10g/L，玉米粉 15g/L，(NH4)2SO4 5g/L，KH2PO4 15g/L，MgSO4 1g/L，CaCl2 0.6g/L。
6.根据权利要求4所述的应用，其特征在于：所述发酵培养基的组成成分为：结晶纤维40g/L，玉米浆 20g/L，酵母膏 10g/L，(NH4)2SO4 5g/L，KH2PO4 5g/L，MgSO4 1g/L，CaCl20.6g/L，CaCO3 2.5g/L，甘油 2.5g/L，Tween-80 2g/L。
7.根据权利要求3所述的用于秸秆降解的高β-葡萄糖苷酶活力里氏木霉工程菌株发酵产生的纤维素酶在水解玉米秸秆中的应用。
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