CN114410596A - 一种裂解性多糖单加氧酶及其应用 - Google Patents
一种裂解性多糖单加氧酶及其应用 Download PDFInfo
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- CN114410596A CN114410596A CN202210092989.8A CN202210092989A CN114410596A CN 114410596 A CN114410596 A CN 114410596A CN 202210092989 A CN202210092989 A CN 202210092989A CN 114410596 A CN114410596 A CN 114410596A
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- substrate
- plpmocb
- chitin
- pllpmocb3
- xylan
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Abstract
本发明属于生物工程技术领域，尤其是涉及一种细菌来源的裂解性多糖单加氧酶及其应用。所述PlLPMOCB3的氨基酸序列，如序列表SEQIDNO.1所示。PlLPMOCB3不仅自身具有降解纤维素、木聚糖、几丁质等生物质多糖的能力，而且还可与商品纤维素酶、木聚糖酶或几丁质酶协同作用提高纤维素、木质纤维素、木聚糖和几丁质底物的降解效率。其中，PlLPMOCB3与商品纤维素酶共同作用时可使纤维素和玉米芯底物时协同度分别达到1.43和1.22；和木聚糖酶共同作用时协同度达到协同度为4.57；和几丁质酶共同作用时协同度达到1.70。为丰富裂解性多糖单加氧酶库，以及生物质降解高效复合酶系的人工复配提供新的途径。
Description
技术领域：
本发明属于生物工程技术领域，尤其是涉及一种细菌来源的裂解性多糖单加氧酶及其应用。
背景技术：
纤维素和几丁质是地球上最丰富的天然多糖，是未来替代化石资源的主要生产材料和可再生能源。作为天然高分子聚合物，它们需被降解成低聚物或单体物质才能得到有效利用。目前，主要是利用糖苷水解酶系来完成生物质多糖的催化降解。但是，它们顽固性的结晶结构使其难以被高效水解。虽然可采用化学或物理等方法进行预处理降低底物结晶度，但高能耗、环境不友好等限制了其工业大规模应用。为提高生物质多糖酶解效率，一些高效的辅助蛋白被报道可有效提高生物质多糖的降解效率。其中，裂解性多糖单加氧酶LPMOs的发现揭示了一种新型多糖降解酶的催化机制，改变了人们对传统酶法降解结晶多糖的认识，已在生物技术和生物无机化学领域引起了科研工作者的极大兴趣，是一类非常具有应用前景的一种生物质多糖降解辅助酶。
LPMOs广泛存在于真菌、细菌、古菌、海洋生物甚至病毒中，目前共发现约70种LPMOs，主要作用于几丁质、淀粉和纤维素，可协助几丁质酶、淀粉酶或纤维素酶降解几丁质、淀粉和纤维素。目前，国内对该类蛋白的研究仍处于起步阶段，上海交通大学冯雁课题组克隆了来源于Actinosynnema mirum和Thermobifida fusca中的3个LPMO发现它们可有效促进几丁质酶水解几丁质；山东农业大学李多川课题组和华中科技大学张晓昱课题组研究表明嗜热毛壳菌LPMO和白腐菌LPMO均可有效提高木质纤维素底物还原糖的释放；本课题组研究发现来自于黑曲霉的AnLPMO15g与纤维素酶共同作用于纤维素和稻草粉时还原糖产量分别提高了96％和131％(ZL201810265764.1)。
目前，LPMOs的相关研究还非常有限，因此，为丰富LPMOs资源库及实现天然多糖高效降解，急需挖掘更多不同来源的LPMOs并对其结构域组成及催化机理进行研究，为生物质的高效降解提供可靠的理论依据，对生物质高效转化具有重大意义。
发明内容：
本发明的目的是筛选可高效降解生物质多糖的酶或辅助酶，丰富裂解性多糖单加氧酶库，为生物质降解高效复合酶系的人工复配提供候选因子，最终为降低多糖类生物质转化利用成本并最终实现其高效降解提供理论依据。
本发明提供的技术方案之一，是一种细菌来源的裂解性多糖单加氧酶PlLPMOCB3在酶解纤维素底物、木质纤维素底物、木聚糖底物、几丁质底物中的应用；
进一步地，所述PlLPMOCB3的氨基酸序列，如序列表SEQIDNO.1所示；
进一步地，所述PlLPMOCB3的核苷酸序列，如序列表SEQIDNO.2所示；
进一步地，所述PlLPMOCB3来源于乳酸类芽孢杆菌(Paenibacillus lactis)154。
进一步地，采用PlLPMOCB3酶解纤维素底物、木质纤维素底物、木聚糖底物或几丁质底物的方法具体如下：
每1g底物添加0.005-0.8g的PlLPMOCB3，在pH 4-6，35-60℃条件下进行酶解反应；
进一步地，底物浓度为0.5-2％(w/v)、PlLPMOCB3的用量为0.1-4mg/mL，抗坏血酸添加量为1-5mM，pH 4-6，35-60℃反应30-50h。
本发明提供的技术方案之二，是上述裂解性多糖单加氧酶PlLPMOCB3在与纤维素酶协同降解纤维素底物中的应用；
进一步地，采用PlLPMOCB3在与纤维素酶协同降解纤维素底物的方法具体如下：
每个单位酶活(FPU)纤维素酶添加0.04-6mg的PlLPMOCB3，协同降解纤维素底物；
进一步地，协同降解纤维素的体系中包括：0.10～3.20mg/mL PlLPMOCB3、0.55～2.20FPU/mL纤维素酶，0.5-2％(w/v)的纤维素底物和1-5mM抗坏血酸，pH 4-6，35-60℃反应30-50h。
本发明提供的技术方案之三，是上述裂解性多糖单加氧酶PlLPMOCB3在与木聚糖酶协同降解木聚糖底物中的应用；
进一步地，采用PlLPMOCB3在与木聚糖酶协同降解木聚糖底物的方法具体如下：
每个单位酶活(U)木聚糖酶添加0.04-6mg的PlLPMOCB3，协同降解木聚糖底物；
进一步地，协同降解木聚糖底物的体系中包括：0.10-3.20mg/mL PlLPMOCB3、0.55-2.20U/mL木聚糖酶、0.5-2％(w/v)木聚糖底物和1-5mM抗坏血酸，pH 4-6，35-60℃反应30-50h。
本发明提供的技术方案之四，是上述裂解性多糖单加氧酶PlLPMOCB3在与几丁质酶协同降解几丁质底物中的应用；
进一步地，采用PlLPMOCB3在与几丁质酶协同降解几丁质底物的方法具体如下：
每mg几丁质酶添加0.1-320mg的PlLPMOCB3，协同降解几丁质底物；
进一步地，协同降解几丁质的体系中包括：0.10-3.20mg/mL PlLPMOCB3、0.01-1mg/ml几丁质酶、0.5-2％(w/v)几丁质底物和1-5mM抗坏血酸，pH 4-6，35-60℃反应30-50h。
进一步地，本发明所述纤维素底物包括但不限于：微晶纤维素、酸溶胀纤维素、羧甲基纤维素钠等；
进一步地，本发明所述木质纤维素底物包括但不限于：玉米芯、滤纸、小麦秸秆、水稻秸秆等；
进一步地，本发明所述木聚糖底物包括但不限于：桦木木聚糖、燕麦木聚糖、小麦阿拉伯木聚糖、榉木木聚糖等；
进一步地，本发明所述几丁质底物包括但不限于：α-几丁质、壳聚糖、胶体几丁质等。
有益效果：
根据NCBI数据库中对核苷酸序列SEQIDNO.2编码的蛋白功能注释为几丁质结合蛋白，但是，本发明经对该蛋白表达纯化后对其催化性质研究表明，该蛋白具有氧化断裂纤维素和几丁质糖苷键并产生还原糖的功能，属于裂解性多糖单加氧酶，对其命名为PlLPMOCB3。
本发明发现PlLPMOCB3不仅自身具有降解纤维素、木聚糖、几丁质等生物质多糖的能力，而且还可与商品纤维素酶、木聚糖酶或几丁质酶协同作用提高纤维素、木质纤维素、木聚糖和几丁质底物的降解效率。其中，PlLPMOCB3与商品纤维素酶共同作用时可使纤维素和玉米芯底物还原糖产率分别提高43％和23％；和木聚糖酶共同作用时可使木聚糖还原糖产率提高516.7％；和几丁质酶共同作用时可使几丁质还原糖产率提高70％。
附图说明：
图1为Rosetta-CB3菌液PCR结果验证
其中，泳道M为marker；泳道1是以Rosetta-CB3为模板，pET-Up、T7-Term为引物对PCR扩增得到的结果；
图2为重组蛋白的SDS-PAGE分析
其中，泳道M为marker；泳道1是未上柱之前的破碎上清液；泳道2是破碎上清液上柱后，目的蛋白挂柱上，其余流出柱子的液体；泳道3是用20mM咪唑对挂柱的目的蛋白进行洗脱后的洗脱液。
图3为PlLPMOCB3作用于不同底物时的还原糖产量。
图4为PlLPMOCB3酶解产物的MALDI-TOF/TOF分析
其中，a为PlLPMOCB3作用于微晶纤维素；b为PlLPMOCB3作用于胶体几丁质。
图5PlLPMOCB3作用的最适温度及pH
其中，a为底物为微晶纤维素，温度对PlLPMOCB3反应的影响；b为底物为胶体几丁质，温度对PlLPMOCB3反应的影响；c为底物为微晶纤维素，pH对PlLPMOCB3反应的影响；d为底物为胶体几丁质，pH对PlLPMOCB3反应的影响。
具体实施方式：
为了使本专利的目的、技术方案及优点更加清楚明白，以下结合具体实施例，对本专利进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本专利，并不用于限定本发明。
本发明通过测定还原糖的含量来计算和表征PlLPMOCB3的酶活水平，以及PlLPMOCB3与不同纤维素酶、木聚糖酶或几丁质酶协同作用的水平。
还原糖测定方法采用PAHBAH比色法，具体如下：
标准曲线的绘制：用去离子水分别配制0、0.1、0.2、0.3、0.4、0.5mg/mL的葡萄糖溶液和GlcNAc溶液。将300μL葡萄糖溶液(或GlcNAc溶液)与900μL 1％(w/v)PAHBAH工作液混匀，于沸水中加热5min，冷却得反应液，取一定量的反应液，测定410nm处的吸光值，根据吸光值和葡萄糖(或GlcNAc)浓度绘制标准曲线。
样品还原糖浓度的测定：取1.5mL离心管若干，进行编号，每管加入300μL样品(酶与底物反应后的样品)与900μL PAHBAH工作液，于沸水中加热5min，冷却后得反应液，取700μL反应液，测定410nm处的吸光值。作用于纤维素类、木质纤维素类、木聚糖类底物时根据葡萄糖标准曲线计算出还原糖浓度，作用于几丁质类底物时根据GlcNAc标准曲线计算出还原糖的浓度。
PAHBAH储备液：用0.5M的盐酸溶液配制5％(w/v)的PAHBAH，于4℃避光保存。
PAHBAH工作液：用0.5M的NaOH溶液稀释PAHBAH储备液至1％(w/v)，现配现用。
本发明所述的纤维素酶的单位酶活FPU，是指采用国际理论与应用化学协会(IUPAC)推荐的标准测定方法测定得到的酶活，即以葡萄糖作标准曲线，FPA 酶活力酶活力单位定义：在pH 4.8、50℃的条件下，1mL酶液每分钟水解50mg Waterman No.1滤纸条产生1μmol葡萄糖当量的还原糖的酶用量为1个酶活力单位，用FPU/mL表示。
本发明所述的木聚糖酶的单位酶活U，是指1mL酶液每分钟水解木聚糖底物生成相当于1μmol木糖(还原糖)所需要的酶量为1个酶活性单位(U)，用U/mL表示酶液活性。
以下通过具体实施例对本发明作进一步地解释说明。
实施例1：裂解性多糖单加氧酶PlLPMOCB3目的基因的获取
(1)目的基因由金唯智公司按照SEQIDNO.2合成，合成时添加5'(EcoRI)和3'(EcoRI)酶切位点，将基因通过5'EcoRI和3'EcoRI克隆至载体pET-28a(+)构建重组质粒。上游引物和下游引物的核苷酸序列如下所示:
上游引物：
5’-ATGGGTCGCGGATCCATGATTCAAAACGTTCACGTTCAA-3’；
下游引物:
5’-TTGTCGACGGAGCTCGAATTCTTAATGGTGGTGGTGATGATG-3’；
将质粒pET-28a(+)线性化，通过DNA Extraction Mini Kit纯化线性化质粒。
(2)用T4DNA连接酶将酶切后的载体和目的基片段连接在一起，连接体系如下:5μL目的基因，3μL线性化载体，1μL T4DNA连接酶，1μLBuffer。在16℃过夜连接。
(3)用连接后的质粒转化大肠杆菌感受态。取10μL上一步的连接产物加入到100μl的大肠杆菌DH5α感受态中，轻柔混匀后置干冰上静置30min。将上一步的混合物在42℃水浴锅中热击90秒立即置于冰上放置3-5min。向离心管中加入1mL不含氨苄的LB培养基。在37℃恒温摇床中120rpm震荡培养1h取100μL涂布到含有氨苄(1％)的LB平板上，在37℃培养箱中倒置培养过夜，挑取阳性克隆，用通用引物pET-Up和T7-Term进行PCR验证，将验证条带大小正确的克隆抽提送去金唯智公司进行测序。测序结果正确，即成功构建了重组pET-28a(+)质粒。用索莱宝质粒小提试剂盒提取。
实施例2:重组大肠杆菌Rosetta-CB3的构建及诱导表达
(1)转化大肠杆菌。取10μL实施例1构建的重组pET-28a(+)质粒加入80μLE.coliRosetta感受态细胞，轻柔混匀后于冰上放置30min，在水浴锅中42℃热激90s，立即冰浴2-5min。加入1mL不含抗性的LB培养基，220rpm、37℃恢复培养1h，离心取100～200μL菌液涂布在含卡那霉素和氯霉素抗性的LB固体培养基上，37℃倒置过夜培养。挑取LB平板中的单菌落至5mL含卡那霉素和氯霉素抗性的LB液体培养基中，37℃、220rpm培养12h，以菌液为模板，PCR后进行1％(w/v)的琼脂糖凝胶电泳验证结果，筛选阳性克隆验证，验证得到1700bp条带，与预期一致，电泳图如图1所示。选取阳性克隆的菌液送至金唯智公司进行测序，测序正确的菌株命名为重组大肠杆菌Rosetta-CB3，并将菌液菌液保存至甘油管中，放在-20℃冰箱以备下一步使用。
(2)将上一步菌液接种到5mL含卡那霉素和氯霉素抗性的液体LB培养基，37℃、220rpm震荡过夜培养。按1％的接种量，将种子液接种至液体LB培养基中，37℃、220rpm震荡培养约3～4h，生长至大肠杆菌对数生长期，此时OD 600约0.7～1.0；此时向菌液中加入IPTG至终浓度为0.5mM，在16℃、220rpm诱导约20h。4℃、6000rpm离心收集菌体，用PBS溶液清洗菌体两次，以去除菌体上附着的培养基。将收集的菌体用4℃预冷的、约1/10菌液体积的PBS溶液重悬，于冰上超声破碎20min。将超声破碎液体，于4℃离心，收集上清。对上清液进行Ni柱纯化并用20mM咪唑对目的蛋白进行洗脱。将收集到的蛋白转移至截留分子量30kda的超滤管中，然后用50mM柠檬酸钠缓冲溶液(pH 5.0)进行超滤，以置换目的蛋白的缓冲液。
采用BCA蛋白浓度测定试剂盒对纯化后的蛋白进行测定，最终收集的溶液中的目的蛋白PlLPMOCB3的浓度达到1.26g/L。
将20mM咪唑洗脱的蛋白进行SDS-PAGE分析验证，得到47.9kda条带，结果正确，如图2所示。
实施例3：PlLPMOCB3的底物特异性分析
取1.5mL离心管若干，向其中分别加入终浓度1％(w/v)的胶体几丁质，壳聚糖，微晶纤维素，CMC，玉米芯粉末(经过粉碎、干燥处理)，滤纸，榉木木聚糖，加入纯化后的PlLPMOCB3使其终浓度为1mg/mL，加入抗坏血酸使其终浓度为1mM，用50mM，pH 5.0的柠檬酸钠缓冲溶液将体积精准补至1mL，用封口膜封住，50℃，220rpm震荡反应36h，于沸水中加热5min以终止反应，12000rpm离心1min收集上清液，通过PAHBAH法测还原糖的产量。不加酶的反应体系作为空白对照，所有样品每个做3个平行。
结果如图3所示，PlLPMOCB3可作用于胶体几丁质、壳聚糖、微晶纤维素、CMC、玉米芯、滤纸和榉木木聚糖，并产生一定量的还原糖。其中，当PlLPMOCB3作用于微晶纤维素时还原糖产量最高；其次为壳聚糖，玉米芯，榉木木聚糖和CMC；作用于滤纸和胶体几丁质时还原糖产量较低。作用于不同底物产生的还原糖产量不同，可能是由于不同的底物结构与组分组成对多糖单加氧酶的活性影响不同。
实施例4:PlLPMOCB3水解微晶纤维素和胶体几丁质的产物的MALDI-TOF/TOF分析
分别以实施例3获得的微晶纤维素水解产物、胶体几丁质水解产物为样品，先用酚氯仿沉淀去除杂蛋白，再用AMBERLITE IR-120强酸型阳离子交换树脂处理去除离子进行样品纯化。用2，5-二羟基苯甲酸(DHB)做基质，20mg/mL的DHB溶于TA 30(0.1％的三氟醋酸(TFA)乙腈溶液与超纯水以3:7的体积比配制而成的溶液)配制而成。取1μL纯化后的样品与2μL DHB基质混匀，取1μL的混合物垂直悬空滴加在一个MTP 384剖光精钢靶板上，在空气中自然干燥。将靶板按步骤装入Ultraflextreme MALDI-TOF/TOF(基体辅助激光解吸电离飞行时间)光谱仪上进行检测，该系统通过(Bruker Daltonics,Germany)Flex Control3.0软件进行控制。MALDI-TOF/TOF仪器在正反射模式下检测并且收集500-2600m/z范围内的产物波谱。每个样品收集多个平行波谱，数据用Flex Analysis 3.0软件进行分析，分析结果如图4。
如图4中a所示，PlLPMOCB3水解微晶纤维素的产物中含有纤维寡糖钠加合物，对应的1,5-δ-内酯和糖醛酸及糖醛酸的钠加合物及其双钠加合物，由此推断PlLPMOCB3可作用于纤维素糖苷链的C1位。如图4中b所示，PlLPMOCB3水解胶体几丁质的产物，出现了代表几丁寡糖的糖醛酸钠加合物及其双钠加合物的质谱峰，由此确定，PlLPMOCB3作用于几丁质糖苷链的C1位。
实施例5：不同酶加载量对协同降解底物的影响
1、对于PlLPMOCB3与纤维素酶的协同作用研究，需要3种不同的反应体系，分别是PlLPMOCB3体系、纤维素酶体系和添加了PlLPMOCB3的纤维素酶体系：
①PlLPMOCB3体系中含有：1％(w/v)底物(微晶纤维素或玉米芯)，PlLPMOCB3的浓度分别为0，0.10，0.20，0.40，0.80，1.60，3.20mg/mL；
②纤维素酶体系中含有：1％(w/v)底物(微晶纤维素或玉米芯)，商品纤维素酶Ctec2的浓度分别为0，0.55，1.10，2.20FPU/mL；
③添加PlLPMOCB3的纤维素酶体系中含有：1％(w/v)底物(微晶纤维素或玉米芯)，PlLPMOCB3的浓度分别为0，0.10，0.20，0.40，0.80，1.60，3.20mg/mL，商品纤维素酶Ctec2的浓度分别为0，0.55，1.10，2.20FPU/mL。
以上三个体系分别加入终浓度为1mM的抗坏血酸，并用50mM，pH 5.0的柠檬酸钠缓冲溶液将体积总量补至1mL。以不加酶的反应体系作为空白对照。每个样品做3个平行，50℃，220rpm条件下震荡36h，反应结束后沸水浴终止反应，12000rpm离心1min，取上清，用PAHBAH法测定还原糖产量。分析PlLPMOCB3与纤维素酶的加载量对协同作用的影响。
本发明协同度计算公式为：PlLPMOCB3和商品酶共同作用的还原糖产量/两者单独作用时的还原糖产量之和。
PlLPMOCB3与纤维素酶共同作用于微晶纤维素结果如表1所示，协同度计算结果如表2。当1.60mg/mL PlLPMOCB3与1.10FPU/mL纤维素酶共同作用于微晶纤维素时，与纤维素酶单独作用相比，PlLPMOCB3使相对还原糖产量提高42.66％，协同度达到1.43。PlLPMOCB3与纤维素酶共同作用于玉米芯底物结果如表3所示，协同度计算结果如表4。当0.40mg/mLPlLPMOCB3与2.20FPU/mL纤维素酶共同作用于玉米芯时，与纤维素酶单独作用相比，PlLPMOCB3使相对还原糖产量提高23.00％，协同度为1.22。
2、对于PlLPMOCB3与木聚糖酶的协同作用研究，需要3种不同的反应体系，分别是PlLPMOCB3体系、木聚糖酶体系和添加了PlLPMOCB3的木聚糖酶体系：
①PlLPMOCB3体系中含有：1％(w/v)榉木木聚糖底物、PlLPMOCB3的浓度分别为0，0.10，0.20，0.40，0.80，1.60，3.20mg/mL；
②木聚糖酶体系中含有：1％(w/v)榉木木聚糖底物，商品木聚糖酶的浓度分别为0，0.55，1.10，2.20U/mL；
③添加PlLPMOCB3木聚糖酶体系中含有：1％(w/v)榉木木聚糖底物，PlLPMOCB3的浓度分别为0，0.10，0.20，0.40，0.80，1.60，3.20mg/mL，木聚糖酶的浓度分别为0，0.55，1.10，2.20U/mL。
以上三个体系分别加入终浓度为1mM的抗坏血酸，并用50mM，pH 5.0的柠檬酸钠缓冲溶液将体积总量补至1mL。以不加酶的反应体系作为空白对照。每个样品做3个平行，50℃，220rpm条件下震荡36h，反应结束后沸水浴终止反应，12000rpm离心1min，取上清，用PAHBAH法测定还原糖产量。分析PlLPMOCB3与木聚糖酶的加载量对协同作用的影响。
本发明协同度计算公式为：PlLPMOCB3和商品酶共同作用的还原糖产量/两者单独作用时的还原糖产量之和。
PlLPMOCB3与木聚糖酶共同作用于榉木木聚糖结果如表5所示，协同度计算结果如表6。当0.20mg/mLPlLPMOCB3与0.55U/mL木聚糖酶共同作用于榉木木聚糖时，与木聚糖酶单独作用相比，PlLPMOCB3的加入使相对还原糖产量最高提高516.70％，协同度为4.57。
表1 PlLPMOCB3与纤维素酶协同降解微晶纤维素的还原糖产量
表2 PlLPMOCB3与纤维素酶协同降解微晶纤维素的协同度
表3 PlLPMOCB3与纤维素酶协同降解玉米芯的还原糖产量
表4 PlLPMOCB3与纤维素酶协同降解玉米芯的协同度
表5 PlLPMOCB3与木聚糖酶协同降解榉木木聚糖的还原糖产量
表6 PlLPMOCB3与木聚糖酶协同降解榉木木聚糖的协同度
实施例6：不同酶加载量对协同降解几丁质的影响
PlLPMOCB3与几丁质酶的加载量对协同作用的研究同样需要3种不同的反应体系，分别是PlLPMOCB3体系、几丁质酶体系和添加了PlLPMOCB3的几丁质酶体系：
①PlLPMOCB3体系中含有：1％(w/v)胶体几丁质底物、PlLPMOCB3的浓度分别为0、0.04，0.40mg/mL；
②几丁质酶体系中含有：1％(w/v)胶体几丁质底物，商品几丁质酶的浓度分别为0、0.05，0.50mg/mL；
③添加PlLPMOCB3的几丁质酶体系中含有：1％(w/v)胶体几丁质底物，PlLPMOCB3的浓度分别为0、0.04，0.40mg/mL，商品几丁质酶的浓度分别为0、0.05，0.50mg/mL；
以上三个体系分别加入终浓度为1mM的抗坏血酸，并用50mM，pH 5.0的柠檬酸钠缓冲溶液将体积总量补至1mL。以不加酶的反应体系作为空白对照。每个样品做3个平行。45℃，220rpm条件下震荡反应36h，反应结束后沸水浴终止反应，12000rpm离心1min，取上清，用PAHBAH法测定还原糖产量。分析PlLPMOCB3与几丁质酶的协同作用。PlLPMOCB3与几丁质酶降解几丁质的还原糖产量如表7所示，协同度计算结果如表8。
当PlLPMOCB3为0.40mg/mL与几丁质酶为0.05mg/mL在反应温度为45℃时，协同度最高，可达1.70。当酶的加载量变成PlLPMOCB3为0.04mg/mL与几丁质酶为0.50mg/mL时，协同度在1.04。
表7 PlLPMOCB3与几丁质酶协同降解几丁质的还原糖产量
表8 PlLPMOCB3与几丁质酶协同降解胶体几丁质的协同度
实施例7最适温度及pH分析
反应体系为：底物(微晶纤维素或几丁质)浓度为1％(w/v)、PlLPMOCB3的用量为0.8mg/mL，抗坏血酸添加量为1mM，pH 5.0，反应36h，分别测定反应温度为30℃、40℃、50℃、60℃时的还原糖产量，进而计算相对酶活。
反应体系为：底物(微晶纤维素或几丁质)浓度为1％(w/v)、PlLPMOCB3的用量为0.8mg/mL，抗坏血酸添加量为1mM。反应温度为50℃，反应36h，分别测定不同pH(1.0-10.0)反应时的还原糖产量，进而计算相对酶活。
结果如图5所示。将反应的最高还原糖产量定义为100％。PlLPMOCB3作用微晶纤维素和几丁质两种底物的最适温度均为50℃，最适pH是5.0。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本专利构思的前提下，上述各实施方式还可以做出若干变形、组合和改进，这些都属于本专利的保护范围。因此，本专利的保护范围应以权利要求为准。
SEQUENCE LISTING
<110> 天津科技大学
<120> 一种裂解性多糖单加氧酶及其应用
<130> 1
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 453
<212> PRT
<213> 乳酸类芽孢杆菌（Paenibacillus lactis）154
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<213> 乳酸类芽孢杆菌（Paenibacillus lactis）154
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Claims (10)
1.一种裂解性多糖单加氧酶PlLPMOCB3的应用，其特征在于，具体为PlLPMOCB3在酶解纤维素底物、木质纤维素底物、木聚糖底物、几丁质底物中的应用；
所述PlLPMOCB3的氨基酸序列，如序列表SEQ ID NO.1所示。
2.如权利要求1所述的应用，其特征在于，采用PlLPMOCB3酶解纤维素底物、木质纤维素底物、木聚糖底物或几丁质底物的方法具体如下：
每1g底物添加0.005-0.8g的PlLPMOCB3，在pH 4-6，35-60℃条件下进行酶解。
3.如权利要求2所述的应用，其特征在于，酶解体系如下：底物浓度为0.5-2％(w/v)、PlLPMOCB3的用量为0.1-4mg/mL，抗坏血酸添加量为1-5mM，pH 4-6，35-60℃反应30-50h。
4.裂解性多糖单加氧酶PlLPMOCB3在与纤维素酶协同降解纤维素底物中的应用，其特征在于，每个单位酶活(FPU)纤维素酶添加0.04-6mg的PlLPMOCB3，协同降解纤维素底物；
所述PlLPMOCB3的氨基酸序列，如序列表SEQ ID NO.1所示。
5.如权利要求4所述的应用，其特征在于，协同降解纤维素的体系中包括：0.10～3.20mg/mL PlLPMOCB3、0.55～2.20FPU/mL纤维素酶，0.5-2％(w/v)的纤维素底物和1-5mM抗坏血酸，pH 4-6，35-60℃反应30-50h。
6.裂解性多糖单加氧酶PlLPMOCB3在与木聚糖酶协同降解木聚糖底物中的应用，其特征在于，每个单位酶活(U)木聚糖酶添加0.04-6mg的PlLPMOCB3，协同降解木聚糖底物；
所述PlLPMOCB3的氨基酸序列，如序列表SEQ ID NO.1所示。
7.如权利要求6所述的应用，其特征在于，协同降解木聚糖底物的体系中包括：0.10-3.20mg/mL PlLPMOCB3、0.55-2.20U/mL木聚糖酶、0.5-2％(w/v)木聚糖底物和1-5mM抗坏血酸，pH 4-6，35-60℃反应30-50h。
8.裂解性多糖单加氧酶PlLPMOCB3在与几丁质酶协同降解几丁质底物中的应用，其特征在于，每mg几丁质酶添加0.1-320mg的PlLPMOCB3，协同降解几丁质底物；
所述PlLPMOCB3的氨基酸序列，如序列表SEQ ID NO.1所示。
9.如权利要求8所述的应用，其特征在于，协同降解几丁质的体系中包括：0.10-3.20mg/mL PlLPMOCB3、0.01-1mg/ml几丁质酶、0.5-2％(w/v)几丁质底物和1-5mM抗坏血酸，pH 4-6，35-60℃反应30-50h。
10.如权利要求1-9任意一项所述的应用，其特征在于，
所述纤维素底物包括但不限于：微晶纤维素、酸溶胀纤维素、羧甲基纤维素钠；
所述木质纤维素底物包括但不限于：玉米芯、滤纸、小麦秸秆、水稻秸秆；
所述木聚糖底物包括但不限于：桦木木聚糖、燕麦木聚糖、小麦阿拉伯木聚糖、榉木木聚糖；
所述几丁质底物包括但不限于：α-几丁质、壳聚糖、胶体几丁质；
所述PlLPMOCB3的核苷酸序列，如序列表SEQ ID NO.2所示。
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| CN116790696A (zh) * | 2023-08-28 | 2023-09-22 | 中国海洋大学 | 利用裂解性多糖单加氧酶OsLPMO10A制备N-乙酰化壳二糖的方法 |
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| CN116790696A (zh) * | 2023-08-28 | 2023-09-22 | 中国海洋大学 | 利用裂解性多糖单加氧酶OsLPMO10A制备N-乙酰化壳二糖的方法 |
| CN116790696B (zh) * | 2023-08-28 | 2023-11-03 | 中国海洋大学 | 利用裂解性多糖单加氧酶OsLPMO10A制备N-乙酰化壳二糖的方法 |
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