CN111944863A - 一种同时降解纤维素及半纤维素的方法 - Google Patents
一种同时降解纤维素及半纤维素的方法 Download PDFInfo
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- CN111944863A CN111944863A CN202010809504.3A CN202010809504A CN111944863A CN 111944863 A CN111944863 A CN 111944863A CN 202010809504 A CN202010809504 A CN 202010809504A CN 111944863 A CN111944863 A CN 111944863A
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- hemicellulose
- cellulase
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- cellulose
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Abstract
本发明涉及农业生物技术领域，具体涉及一种同时降解纤维素及半纤维素的方法。本发明发现一个天然多肽具有双功能内切纤维素酶活性，该酶同时具有降解纤维素与降解半纤维素甘露聚糖的能力，该酶具有酸性、较高的反应温度、能同时降解纤维素及半纤维素成分甘露聚糖，易于发酵生产。所有这些优点都表明着新发明的内切纤维素酶在饲料、食品、医药等行业中具有较大的应用潜力。
Description
技术领域
本发明涉及农业生物技术领域，具体涉及一种同时降解纤维素及半纤维素的方法。
背景技术
非淀粉多糖(non-starch polysaccharide, NSP) 是植物中除了淀粉之外的碳水化合物的总称，包括纤维素、半纤维素和果胶类等。半纤维素主要分为三类，即聚木糖类、聚葡萄甘露糖类和聚半乳糖葡萄甘露糖类。
非淀粉多糖是饲料纤维的主要成分，由于这些纤维将饲料中的营养物质包围在细胞内部，在一定程度上抑制了动物的对营养物质的降解与吸收。自然界中广泛存在着可以降解非淀粉多糖的酶类，包括纤维素酶、甘露聚糖酶、木聚糖酶、葡聚糖酶等，这些酶可以有效降解饲料中的NSP，提高饲料营养价值并改善动物生长性能。
多糖酶解产生寡糖可以进一步刺激和增强动物的免疫反应，调控动物胃肠道微生态环境。饲料中非淀粉多糖的降解往往需要多种酶的复合作用，比如纤维素酶(CMCase)、半纤维素酶(β-1,3-1,4-葡聚糖酶、甘露聚糖酶、木聚糖酶)共同作用。但是，复合酶的添加增加了饲料使用的成本，成为限制其广泛应用的一个重要因素。多功能酶（一种酶同时具有两种或以上功能）的研究与应用无疑是简化饲料加工工艺、降低饲料成本的有效途径。获得单催化域酶催化两种甚至多种底物的多功能酶，或者利用分子改良拓宽酶高效作用底物的范围，获得功能多样性的高效NSP酶意义重大。获得性质优良的纤维素酶基因资源是降低使用纤维素酶的成本的有效途径之一，尤其是具有较高酶活力的多功能纤维素酶。截至目前，研究人员利用各种策略尝试获得多功能酶。
发明内容
本发明的目的是提供一种同时降解纤维素及半纤维素的方法。
本发明的再一目的是提供氨基酸序列如SEQ ID NO:1所示的多肽作为内切纤维素酶的应用。
根据本发明的同时降解纤维素及半纤维素方法，所述方法包括使用氨基酸序列如SEQ ID NO:1所示的多肽降解纤维素及半纤维素的步骤。
本发明还提供了氨基酸序列如SEQ ID NO:1所示的多肽作为内切纤维素酶的应用。
根据本发明的同时降解纤维素及半纤维素方法，其中，所述多肽的作用pH为pH3.5-pH 6.0，优选pH5.0。
根据本发明的同时降解纤维素及半纤维素方法，其中，所述多肽的作用温度为55℃-75℃。
根据本发明的同时降解纤维素及半纤维素方法，其中，所述多肽的编码基因的核苷酸序列如SEQ ID NO:3所示。
本发明发现氨基酸序列如SEQ ID NO:1所示的多肽具有双功能内切纤维素酶功能，该酶具有酸性、较高的反应温度、能同时降解纤维素及半纤维素成分甘露聚糖，易于发酵生产。所有这些优点都表明着新发明的内切纤维素酶在饲料、食品、医药等行业中具有较大的应用潜力。
根据本发明的具体实施方式，对氨基酸序列如SEQ ID NO:1的多肽具有双功能内切纤维素酶活性，该酶全长342个氨基酸，去除信号肽后的全长为325个氨基酸。
DNA全序列分析结果表明，该酶的结构基因全长为1364bp，共有4个内含子，其序列如SEQ ID NO:2所示。
根据本发明具体实施方式，该酶编码基因经密码子优化后的核酸序列如SEQ IDNO.3所示：
本发明还提供了一种制备酶切纤维素酶的方法，包括以下步骤：
以包含核苷酸序列如SEQ ID NO:3所示基因的重组载体转化宿主细胞，获得重组菌株；
将重组菌株进行培养，诱导重组内切纤维素酶的表达；以及
回收并纯化所表达的内切纤维素酶。本发明发现一个天然多肽具有双功能内切纤维素酶活性，该酶同时具有降解纤维素与降解半纤维素甘露聚糖的能力，该酶具有酸性、较高的反应温度、能同时降解纤维素及半纤维素成分甘露聚糖，易于发酵生产。所有这些优点都表明着新发明的内切纤维素酶在饲料、食品、医药等行业中具有较大的应用潜力。
附图说明
图1本发明重组内切纤维素酶RMX的最适pH值。
图2本发明内切纤维素酶RMX的pH稳定性。
图3本发明内切纤维素酶RMX最适反应温度。
图4本发明内切纤维素酶RMX热稳定性。
具体实施方式
试验材料和试剂
1、菌株及载体：毕赤酵母(Pichia pastoris GS115)；毕赤酵母表达载体pPIC9及菌株GS115购自于Invitrogen公司。
2、酶类及其它生化试剂：内切酶购自Thermo公司，连接酶购自Invitrogen公司，其它都为国产试剂（均可从普通生化试剂公司购买得到）。
3、培养基：
(1) 产酶培养基：30 g/L 麦麸，30 g/L玉米芯粉，30 g/L 豆粕，5 g/L 大麦葡聚糖，5g/L (NH4)SO4，1 g/L KH2PO4，0.5 g/L MgSO4·7H2O，0.01 g/L FeSO4·7H2O，0.2 g/L CaCl2于1 L去离子水中，121°C，15磅条件下灭菌处理20 min
(2)大肠杆菌培养基LB (1%蛋白胨、0.5%酵母提取物、1% NaCL，pH7.2)。
(3) BMGY培养基；1%酵母提取物，2%蛋白胨，1.34% YNB，0.000049< Biotin，1% 甘油(v/v)。
(4) BMMY培养基：除以0.5%甲醇代替甘油，其余成份均与BMGY相同。
说明：以下实施例中未作具体说明的分子生物学实验方法，均参照《分子克隆实验指南》（第三版）J．萨姆布鲁克一书中所列的具体方法进行，或者按照试剂盒和产品说明书进行。
实施例1 获取内切纤维素酶编码基因RMX及构建表达载体
根据Genbank中公布的蛋白序列（序列号为：RMX75592.1），对其进行密码子优化，化学合成获得优化后的基因，编号为RMX。
将基因RMX连接在pUC57载体上通过酶切连接的方法实现目的基因与表达载体pPIC9r的连接，完成表达载体的构建。
利用限制性内切酶EcoRI、NotI对表达载体pPIC9进行酶切，随后1%的琼脂糖凝胶电泳并回收PCR产物和酶切产物。回收后的表达载体pPIC9r和目的片段通过T4 DNA连接酶进行连接。在20℃条件下连接1 h，反应结束后置于冰上冷却数秒，连接产物直接用于转化或保存于-20℃。
连接产物转化入E. coli Trans-T1感受态中，转化菌液涂布在LB 平板(含100 μg/mL 氨苄青霉素) 上37 ℃培养箱过夜培养，挑取克隆子用菌落PCR方法鉴定并测序，测序正确的重组质粒命名为pPIC9r-RMX。
实施例2内切纤维素酶RMX工程菌株的构建
（1）表达载体的构建及在酵母的表达
测序正确的pPIC9r-RMX阳性转化子用于大量制备重组质粒。重组质粒用限制性内切酶Bgl II线性化酶切后，电击转化至酵母GS115感受态细胞，30°C培养48 h，挑取转化子进行验证，具体操作请参考毕赤酵母表达操作手册。
（2）高活性内切纤维素酶转化子的筛选
用灭过菌的牙签从长有转化子的MD板上挑取单菌落，按照编号先点到MD平板上，将 MD平板置于30°C培养箱中培养1~2天，至菌落长出。按编号从MD平板上挑取转化子接种于装有3 mL BMGY培养基的离心管中，30°C、220 rpm摇床培养48h；将摇床培养48 h的菌液4,000×g离心5 min，去上清，离心管中再加入1mL含有0.5%甲醇的BMMY培养基，继续在30°C、220rpm条件下诱导培养；48 h后离心去除菌体，取上清用于酶活性检测，筛选出高内切纤维素酶活性的转化子，具体操作请参考毕赤酵母表达操作手册。
实施例3重组内切纤维素酶的制备
（1）内切纤维素酶基因RMX利用毕赤酵母在摇瓶水平的大量表达
筛选出酶活较高的转化子，接种于300 mL BMGY液体培养基中，30°C，220 rpm摇床振荡培养48 h；5,000 rpm离心5 min，弃上清，再向菌体加入200 mL含有0.5%甲醇的BMMY液体培养基，30°C，220 rpm诱导培养48 h。诱导培养期间，间隔12 h补加一次甲醇溶液，使甲醇浓度保持在0.5%左右；(3) 12,000×g离心20 min，收集上清发酵液，检测酶活性并进行SDS-PAGE蛋白电泳分析。
（2）重组内切纤维素酶的纯化
收集摇瓶表达的重组内切纤维素酶上清液，利用10 kDa膜包（Vivascience，Hannover，Germany）进行浓缩，并且利用透析的方式进行脱盐处理，将其置换到buffer A(pH6.5，10mM磷酸氢二钠-柠檬酸缓冲液）中。利用离子交换柱（HiTrap Q HP）对粗酶液进行纯化，收集具有酶活力的组份，利用SDS-PAGE验证其蛋白的纯度及分子量大小，并进行液相色谱-电喷雾离子化-质谱联用（liquid chromatography-electrospray ionization-tandem massspectrometry，LC-ESI-MS）鉴定。
实施例4重组内切纤维素酶基本酶学性质分析
采用二硝基水杨酸(DNS) 法测定重组内切纤维素酶的基本酶学性质。具体方法如下：在pH 5.0，75°C条件下，1 mL的反应体系包括100 µL适当的稀释酶液，900 µL底物，反应10min，加入1.5 mL DNS终止反应；沸水浴煮5 min后冷却至室温，在540 nm波长下测定OD值。纤维素酶活性单位定义：在一定条件下，每分钟分解底物生成1 μmoL 葡萄糖所需要的酶量为1个活性单位(U)。酶学性质研究所用酶液均需达到电泳纯。甘露聚糖酶活测定方法同纤维素酶。甘露聚糖酶活性单位定义：在一定条件下，每分钟分解底物生成1 μmoL 甘露单糖所需要的酶量为1个活性单位(U)。
（1）内切纤维素酶的最适pH及pH稳定性
经纯化的(实施例3)表达的内切纤维素酶RMX在不同的pH下进行酶促反应以测定其最适pH。所用缓冲液为pH 1.0-3.0 甘氨酸-盐酸缓冲体系，pH3.0-8.0的柠檬酸一磷酸氢二钠系列缓冲体系，pH 8.0-l0.0是Tris-HCl缓冲体系。纯化的RMX在不同pH的缓冲体系．65°C下测定其最适pH结果（图1）表明：以羧甲基纤维素钠为底物时，RMX的最适pH为5.0，在pH 3.5-pH 6.0范围内，该酶能够维持其60%以上的相对酶活。整体来看，RMX属于酸性的内切纤维素酶，在pH接近中性的条件下，相对酶活仅剩余50%左右。
将纯化后的酶液用最适pH对应的缓冲体系稀释到50μg/mL，在不同pH值的缓冲液中于37°C下处理60 min，适当稀释后，在最适温度及pH下测定剩余酶活。结果表明（图2），分析结果表明pH2.0-pH10.0之间能够维持30%以上的酶活力，且在pH4.0-pH6.0范围内可以保持90%以上的酶活力，说明该酶具有极好的耐酸性能。
（2）内切纤维素酶反应的最适温度及热稳定性
纯化的内切纤维素酶在pH 5.0条件下，测定不同温度（30-80℃）下的酶活性，以确定重组酶最适温度。实验结果表明，该酶的最适反应温度为75℃，在80℃时依然具有60%以上的酶活力（图3）。
将纯化后的酶液用最适pH对应的缓冲体系稀释到100μg/mL，分别在55℃、65℃、75℃下进行热处理，处理时间分别为2min、5min、10min、30min和60min，处理后的样品置于冰上以终止酶的热处理，冷却后稀释适当倍数，在最适反应条件下测定剩余酶活，以未处理酶液所测的活性为100%。
结果如图4所示，RMX的热稳定性在中低温段表现良好，在55℃条件下处理1h几乎没有酶活损失；处理温度达到65℃时，处理30 min后，损失30%左右的酶活，处理1 h后可以剩余50%左右的酶活；但是当处理温度提高至75℃时，处理2 min就会损失90%的酶活。
（3）比活分析比较
纯化后的纤维素酶野生型RMX在pH5.0，75℃和pH5.0，70℃件下以羧甲基纤维素钠及角豆胶进行酶促反应以测定其酶活性。比活测定结果显示，在以羧甲基纤维素钠为底物时，野生型RMX的纤维素比活为1214 U/mg；在以角豆胶为底物时，野生型RMX的甘露聚糖比活为251 U/mg，其降解半纤维素角豆胶的活力是降解纤维素底物的20%，表明该酶同时具有降解纤维素与降解半纤维素甘露聚糖的能力。
序列表
<110> 中国农业科学院北京畜牧兽医研究所
<120> 一种同时降解纤维素及半纤维素的方法
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gcaccggagt cgtccctcga caaacgtgca ggaaacttca agttctttgg cgtcaatgag 120
gccgggcccg agtttggaaa ccagaacttg cctggtaagg atattcaaga ctgcagattg 180
aagactactt actgaacagc atcgctcttg aaggagtcta caacaaggac tacgtcttcc 240
caaccctcag taccgtaaga agaaccagca tccctcacag atcctcagaa caagatcagg 300
gattaataca atcgaacagt atgacacgtt catctctaag gggttcaaca cgttccgtct 360
gaacatccag atggaacgcc ttgccccgaa cgccatcaac ggcaacttag acactaccta 420
cctcaacatg atcaaggagc aggtcaacta cgtcactggt aaaggcgctt acatgatgat 480
caacccacac aactacggcc ggtactatgg tcagatctat cgtgatacgc aatccttcgg 540
taagtctctt tcaaaagcca gaagacttgc ttcttaggat gcagagagag agtggctgat 600
gcatagctat ccacaggtca attctgggcc cacctagccc aagaattcaa gtccaactcg 660
cgcgtcatct tcgacacgga caacgagttt cacgacgagc caggccagct cgtggcggat 720
ctcaaccagg cagccatcaa cgccatccgc gctacaggcg ctacgaacca gtacatcgcc 780
gtggagggaa atgcatggac gggagcctgg acttggacga ctgccaaggg aaccgatggt 840
ctgacgaatg cccagacgat gggcaatttg aaagacccga gcaataagat tctgtacgag 900
gtaagcggcg cacgtttcac actcacctgt gaacctcaac caggcaatgc tgactacctt 960
gtccagatgc accagtacct cgactcggac ggctccggca cctcgacgac ctgcgtcagc 1020
tccacgatcg gctctgagcg gctcaaagcc gcgacgcaat ggctgcgggc taacggcaag 1080
aaagggctcc taggcgaata tgctggcgct gtgaacccga catgtcaggc cgccgtcaag 1140
gacatgctga gctacatggt caagaacaag gatgtctggg agggcgctgt atggtgggcg 1200
gcgggtccat ggtggggaga gtacgtatct tcgctttgaa tttacttgtc ggttcccgtg 1260
tgtgtggtgt ttgctgactt gagcgtagct acatgttctc cattgagcct acgaatggcc 1320
cggcctacaa cacctacgtg cctctcatca ctcagtacgc ttag 1364
<210> 3
<211> 975
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
gctgctccag ctccagaatc ttctttggat aagagagccg gcaactttaa gtttttcggc 60
gtcaacgaag ctggtccaga atttggtaac caaaacttgc caggtgttta caacaaggat 120
tacgttttcc caaccttgtc tacttacgac acctttatct ctaagggctt caacaccttt 180
cgtttgaaca tccagatgga aagattggct ccaaacgcta ttaacggtaa cttggacact 240
acttacttga acatgattaa ggagcaggtc aactacgtta ctggtaaggg tgcttacatg 300
atgattaacc cacacaacta cggtagatac tacggtcaaa tctacagaga tactcagtcc 360
tttggtcagt tttgggctca tttggcccaa gaatttaagt ctaactcccg tgtcattttt 420
gacactgaca acgagtttca tgatgaacca ggtcaattgg ttgctgattt gaaccaagct 480
gccattaacg ctattagagc tactggtgct actaaccaat acattgctgt tgaaggtaac 540
gcttggactg gtgcttggac ttggactact gctaagggta ctgatggttt gactaacgct 600
caaactatgg gtaacttgaa ggatccatcc aacaagatat tgtacgagat gcaccaatac 660
ttggattctg atggttctgg tacttctact acttgtgtct cctctactat cggttctgaa 720
agattgaagg ctgctactca atggttgaga gctaacggta agaagggttt gttgggtgaa 780
tacgctggtg ctgttaaccc aacttgtcaa gctgctgtta aggatatgtt gtcctacatg 840
gtcaagaaca aggatgtttg ggaaggtgct gtttggtggg ctgctggtcc atggtggggt 900
gattacatgt tctccattga accaactaac ggtccagctt acaacactta cgttccattg 960
atcactcaat acgcc 975
Claims (8)
1.一种同时降解纤维素及半纤维素方法，其特征在于，所述方法包括使用氨基酸序列如SEQ ID NO:1所示的多肽降解纤维素及半纤维素的步骤。
2.根据权利要求1所述的同时降解纤维素及半纤维素方法，其特征在于，所述多肽的作用pH为pH 3.5-pH 6.0。
3.根据权利要求1所述的同时降解纤维素及半纤维素方法，其特征在于，所述多肽的作用 pH5.0。
4.根据权利要求1所述的同时降解纤维素及半纤维素方法，其特征在于，所述多肽的作用温度为55℃-75℃。
5.根据权利要求1所述的同时降解纤维素及半纤维素方法，其特征在于，所述多肽的作用温度为55℃。
6.根据权利要求1所述的同时降解纤维素及半纤维素方法，其特征在于，所述多肽的编码基因的核苷酸序列如SEQ ID NO:3所示。
7.氨基酸序列如SEQ ID NO:1所示的多肽作为内切纤维素酶的应用。
8.一种制备内切纤维素酶的方法，其特征在于，所述方法包括以下步骤：
以包含核苷酸序列如SEQ ID NO:3所示基因的重组载体转化宿主细胞，获得重组菌株；
将重组菌株进行培养，诱导重组内切纤维素酶的表达；以及
回收并纯化所表达的内切纤维素酶。
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