CN112852845B - 一种新型多功能酶基因hg32及应用 - Google Patents
一种新型多功能酶基因hg32及应用 Download PDFInfo
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Abstract
本发明公开了一种新型多功能酶基因HG32及应用，该酶来源于中华竹鼠肠道微生物，能够兼容真核生物细胞表达系统，表达高生物活性的纤维素酶，其可在畜禽饲料酶制剂、转基因动物制备、食品和药品行业等领域应用，以解决植物纤维素难降解、饲料营养利用率低等问题，减轻动物排泄物中氮和、有机物等污染问题。本发明通过对竹鼠肠道微生物宏转录组测序，序列拼接，与NCBI上已有的糖苷水解酶家族进行同源性对比，筛选高表达的、同源性高于50％的疑似纤维素酶的基因，预测其信号肽，并用猪腮腺分泌蛋白信号肽替换原有的信号肽，按猪密码子偏好优化后克隆到真核表达载体上，通过脂质体转染到猪肾上皮细胞中表达，收集细胞培养上清液进行酶活分析比较后获得。
Description
技术领域
本发明涉及基因工程领域，特别涉及一种新型多功能酶基因HG32及应用。
背景技术
纤维素是自然界中丰富的绿色碳源，是组成植物细胞壁的主要成分。适量的纤维素有利于动物肠道蠕动，促进粪便排出等。但纤维素也是植物细胞壁的主成分，大多数畜禽是单胃动物如猪、鸡等，其胃和小肠缺少纤维素酶，难以破坏植物的细胞壁，阻碍了细胞内营养物质的充分吸收，这些未消化的营养物质，随着排泄物进入自然环境，造成环境污染。天然常常以纤维素超分子聚集态结构存在，难以被降解，解决这个问题的方法就是寻找优良的纤维素降解酶。最早科学家们是通过培养皿筛选能产生纤维素的菌株(Liu and Qinet al.,2013)，由于大多数微生物难于人工培养，采用细菌营养液培养筛选得到的微生物只是群落里的冰山一角(Amann and Binder et al.,1990)。高重复率的分离方式使后续发现具有生物活性的新成员越来越困难(Chang and Brady,2013)。1995年，科学家们首先在厌氧消化池中通过宏基因组学筛选首次发现了纤维素酶基因(Healy and Ray et al.,1995)，至此一种全新的筛选基因诞生。宏基因组学又称为元基因组学，是绕过微生物分离培养，直接从环境样本中分离基因组DNA为基础的一种文库构建方法，在获得宏基因组学数据后，利用生物信息学分析的方法获得该微生物环境群落结构，物种分类，系统进阶过程，基因功能注释以及代谢通路网等，主要用于识别特定或挖掘未被发现微生物群落中生物活性分子和酶(Segata and Izard et al.,2011)，避免复杂生物环境污染、实验周期短、信息量全。因此在挖掘新的基因资源中有更大的优势。
发明内容
本公开通过宏转录组测序后拼接的核酸序列与NCBI上已有的糖苷水解酶家族进行同源性对比，将同源性高于50％的疑似纤维素酶的基因挑出，用猪腮腺分泌蛋白信号肽替换原有的信号肽，并进行猪密码子优化得到一个能在真核载体上表达的酶基因。转染到猪肾上皮细胞中表达，收集细胞培养上清液后进行酶活分析。从而，获得了一种纤维素酶基因HG32，其可在畜禽饲料酶制剂、转基因动物制备，食品和药品行业等领域应用，以解决纤维素难降解吸收和排泄物污染环境的问题。
根据本发明的第一个方面，提供了一种新型多功能酶基因HG32，其中，该新型多功能酶基因HG32的核苷酸序列包括如SEQ ID NO:3所示的核苷酸序列。
根据本发明的第二个方面，提供了一种新型多功能酶基因HG32，其中，该新型多功能酶基因HG32的核苷酸序列包括与SEQ ID NO:3具有80％同源性的序列。
根据本发明的第三个方面，提供了一种新型多功能酶基因HG32，其中，该新型多功能酶基因HG32包含猪腮腺分泌蛋白信号肽序列，其核苷酸序列包括如SEQ ID NO:5所示的核苷酸序列。
根据本发明的第四个方面，提供了一种新型多功能酶基因HG32，其中，该新型多功能酶基因HG32的氨基酸序列包括如SEQ ID NO:4所示的氨基酸序列。
根据本发明的第五个方面，提供了一种新型多功能酶基因HG32，其中，该新型多功能酶基因HG32的氨基酸序列包括与SEQ ID NO:4具有85％同源性的氨基酸序列。
根据本发明的第六个方面，提供了一种新型多功能酶基因HG32，其中，该新型多功能酶基因HG32包含猪腮腺分泌蛋白信号肽序列，其氨基酸序列包括如SEQ ID NO:6所示的氨基酸序列。
根据本发明的第七个方面，提供了一种多功能纤维素酶，其中，该多功能纤维素酶含有如SEQ ID NO:4所示的氨基酸序列；或含有与SEQ ID NO:4具有85％同源性的氨基酸序列；或含有如SEQ ID NO:6所示的氨基酸序列。
根据本发明的第八个方面，提供了核苷酸序列包括如SEQ ID NO:3所示的核苷酸序列的新型多功能酶基因HG32在制备畜禽饲料酶制剂上的应用；或核苷酸序列包括与SEQID NO:3具有80％同源性的序列的新型多功能酶基因HG32在制备畜禽饲料酶制剂上的应用；或核苷酸序列包括如SEQ ID NO:5所示的核苷酸序列的新型多功能酶基因HG32在制备畜禽饲料酶制剂上的应用；或氨基酸序列包括如SEQ ID NO:4所示的氨基酸序列的新型多功能酶基因HG32在制备畜禽饲料酶制剂上的应用；或氨基酸序列包括与SEQ ID NO:4具有85％同源性的序列的新型多功能酶基因HG32在制备畜禽饲料酶制剂上的应用；或氨基酸序列包括如SEQ ID NO:6所示的氨基酸序列的新型多功能酶基因HG32在制备畜禽饲料酶制剂上的应用；优选的，在制备纤维素酶、微晶纤维素酶、低粘度羧乙基纤维素酶或葡聚糖酶任一项或多项酶制剂上的应用；优选的，在制备具有强胰蛋白和糜蛋白酶耐受性的纤维素酶、微晶纤维素酶、低粘度羧乙基纤维素酶或葡聚糖酶任一项或多项酶制剂上的应用。
根据本发明的第九个方面，提供了核苷酸序列包括如SEQ ID NO:3所示的核苷酸序列的新型多功能酶基因HG32在制备转基因动物上的应用；或核苷酸序列包括与SEQ IDNO:3具有80％同源性的序列的新型多功能酶基因HG32在制备转基因动物上的应用；或核苷酸序列包括如SEQ ID NO:5所示的核苷酸序列的新型多功能酶基因HG32在制备转基因动物上的应用；或氨基酸序列包括如SEQ ID NO:4所示的氨基酸序列的新型多功能酶基因HG32在制备转基因动物上的应用；或氨基酸序列包括与SEQ ID NO:4具有85％同源性的序列的新型多功能酶基因HG32在制备转基因动物上的应用；或氨基酸序列包括如SEQ ID NO:6所示的氨基酸序列的新型多功能酶基因HG32在制备转基因动物上的应用。
根据本发明的第十个方面，提供了核苷酸序列包括如SEQ ID NO:3所示的核苷酸序列的新型多功能酶基因HG32在改善胃肠道内环境、药品、保健品、食品上的应用；或核苷酸序列包括与SEQ ID NO:3具有80％同源性的序列的新型多功能酶基因HG32在改善胃肠道内环境、药品、保健品、食品上的应用；或核苷酸序列包括如SEQ ID NO:5所示的核苷酸序列的新型多功能酶基因HG32在改善胃肠道内环境、药品、保健品、食品上的应用；或氨基酸序列包括如SEQ ID NO:4所示的氨基酸序列的新型多功能酶基因HG32在改善胃肠道内环境、药品、保健品、食品上的应用；或氨基酸序列包括与SEQ ID NO:4具有85％同源性的序列的新型多功能酶基因HG32在改善胃肠道内环境、药品、保健品、食品上的应用；或氨基酸序列包括如SEQ ID NO:6所示的氨基酸序列的新型多功能酶基因HG32在改善胃肠道内环境、药品、保健品、食品上的应用。
根据本发明的第十一个方面，提供了核苷酸序列含有如SEQ ID NO:3所示的核苷酸序列的新型多功能酶基因HG32的重组载体；或核苷酸序列含有与SEQ ID NO:3具有80％同源性序列的新型多功能酶基因HG32的重组载体；或核苷酸序列含有如SEQ ID NO:5所示的核苷酸序列的新型多功能酶基因HG32的重组载体。
在某些实施方式中，该新型多功能酶基因HG32重组载体在制备畜禽饲料酶制剂上的应用；优选的，在制备纤维素酶、微晶纤维素酶、低粘度羧乙基纤维素酶或葡聚糖酶任一项或多项酶制剂上的应用；优选的，在制备具有强胰蛋白和糜蛋白酶耐受性的纤维素酶、微晶纤维素酶、低粘度羧乙基纤维素酶或葡聚糖酶任一项或多项酶制剂上的应用。
在某些实施方式中，该新型多功能酶基因HG32重组载体在制备转基因动物上的应用。
在某些实施方式中，该新型多功能酶基因HG32重组载体在在改善胃肠道内环境、药品、保健品、食品上的应用。
根据本发明的第十二个方面，提供了核苷酸序列含有如SEQ ID NO:3所示的核苷酸序列的新型多功能酶基因HG32的重组菌株；或核苷酸序列含有与SEQ ID NO:3具有80％同源性序列的新型多功能酶基因HG32的重组菌株；或核苷酸序列含有如SEQ ID NO:5所示的核苷酸序列的新型多功能酶基因HG32的重组菌株；或氨基酸序列含有如SEQ ID NO:4所示的氨基酸序列的新型多功能酶基因HG32的重组菌株；或氨基酸序列含有与SEQ ID NO:4具有85％同源性序列的新型多功能酶基因HG32的重组菌株；或氨基酸序列含有如SEQ IDNO:6所示的氨基酸序列的新型多功能酶基因HG32的重组菌株。
在某些实施方式中，该新型多功能酶基因HG32重组菌株在制备畜禽饲料酶制剂上的应用；优选的，在制备纤维素酶、微晶纤维素酶、低粘度羧乙基纤维素酶或葡聚糖酶任一项或多项酶制剂上的应用；优选的，在制备具有强胰蛋白和糜蛋白酶耐受性的纤维素酶、微晶纤维素酶、低粘度羧乙基纤维素酶或葡聚糖酶任一项或多项酶制剂上的应用。
在某些实施方式中，该新型多功能酶基因HG32重组菌株在在改善胃肠道内环境、药品、保健品、食品上的应用。
本发明的有益效果：
1、从宏转录组学数据提取更多有价值的糖苷水解酶，使整个过程更加高效简便。同时利用基因克隆技术克隆微生物源中的酶基因，用猪腮腺分泌信号肽进行替换，经过对其成熟肽区猪密码子优化后，使其能在动物细胞上表达。该方法获得了在动物细胞上表达更强的纤维素酶、微晶纤维素酶活性、低粘度羧乙基纤维素酶活性和葡聚糖酶活性且具有较强的胰蛋白和糜蛋白酶耐受性的酶基因HG32。
2、本发明经过对微生物源基因进行信号肽替换和密码子优化，筛选到在动物细胞中表达更强的纤维素酶、微晶纤维素酶活性、低粘度羧乙基纤维素酶活性和葡聚糖酶活性且具有较强的胰蛋白和糜蛋白酶耐受性的酶基因HG32，该基因非常适合动物肠道环境，可用于畜禽饲料酶制剂制备和开发、转基因动物制备、改善胃肠道内环境、药品、保健品、食品等领域。
附图说明
图1为中华竹鼠肠道微生物的RNA提取后电泳结果图；
图2为HG32真核表达载体pcDNA3.1-HG32图谱；
图3为不同pH缓冲液下HG32的纤维素酶活性检测结果图；
图4为不同pH缓冲液下HG32的葡聚糖酶活性检测结果图；
图5为HG32的纤维素酶的蛋白耐受能力检测结果图；
图6为HG32的葡聚糖酶的蛋白耐受能力结果图；
图7为不同基因的纤维素酶活最适pH比较结果图；
图8为不同基因的葡聚糖酶活最适pH比较结果图；
图9为pH 4.3胃蛋白酶的纤维素酶耐受比较结果图；
图10为pH 4.3胃蛋白酶的葡聚糖酶耐受比较结果图；
图11为pH 6.5胰蛋白酶的纤维素酶耐受比较结果图；
图12为pH 6.5胰蛋白酶的葡聚糖酶耐受比较结果图；
图13为pH 8.0胰蛋白酶的纤维素酶耐受比较结果图；
图14为pH 8.0胰蛋白酶的葡聚糖酶耐受比较结果图；
图15为pH 6.5糜蛋白酶的纤维素酶耐受比较结果图；
图16为pH 6.5糜蛋白酶的葡聚糖酶耐受比较结果图；
图17为pH 8.0糜蛋白酶的纤维素酶耐受比较结果图；
图18为pH 8.0糜蛋白酶的葡聚糖酶耐受比较结果图；
图19为pH 6.0对不同底物的酶促反应活性测定结果图。
具体实施方式
下面结合附图和实施例对发明作进一步详细的说明。
一、动物喂养试验
用高纤维饲料如竹子、苞米皮和玉米芯等喂养中华竹鼠一段时间，进一步诱导竹鼠肠道消化纤维素的菌群高效增殖。在饲养三周后，屠宰，在超净工作台上采用双结扎法取盲肠和结肠微生物，放入液氮速冻保存备用。
二、竹鼠肠道微生物的RNA提取及宏转录组测序和序列分析
2.1、用QIAGE公司的ZR Soil/Fecal RNA MicroPrep(R2040)提取中华竹鼠盲肠和结肠中肠道微生物RNA，电泳结果如图1所示。
2.2、NEB普通建库，上机测序，质控分析，转录本拼接(均由北京诺禾致源科技股份有限公司完成)
2.3、初次筛选：把测序后获得转录本拼接序列，与现有的糖苷水解酶库(http://www.cazy.org)里面的序列进行比对，将同源性在50％以上的序列筛选出来。
2.4、第二次筛选：用国家生物技术信息中心的ORF查找器(NCBI；www.ncbi.nlm.nih.gov/orffinder/)识别搜索序列中的开放阅读框(ORF)。对片段长度大于等于500nt的序列用BLASTp程序(http://www.ncbi.nlm.nih.gov/BLAST/)和ClustalW程序比对氨基酸序列，筛选出一大批候选基因，去除其信号肽，拼接猪腮腺蛋白信号肽，构建真核表达载体，转染猪肾pK15细胞，测定其表达酶活力进行验证，其中包含HG32的基因序列，HG32基因核苷酸序列如SEQ ID NO:1所示，氨基酸序列如SEQ ID NO:2所示；将HG32基因去除原始信号肽后的核苷酸序列如SEQ ID NO:3所示，氨基酸序列如SEQ ID NO:4所示；用猪信号肽替换原始信号肽后的HG32基因核苷酸序列如SEQ ID NO:5所示，氨基酸序列如SEQID NO:6所示。将含有猪信号肽的HG32基因核苷酸序列通过金斯瑞公司进行人工合成。
2.5、HG32真核表达载体构建：将人工合成的HG32基因序列连接至pcDNA3.1(+)质粒，构建HG32真核表达载体pcDNA3.1-HG32。
三、猪肾细胞水平酶活验证
3.1 细胞复苏与传代
把液氮冻存的PK15细胞放在37℃水浴锅中轻轻摇晃复苏，吸取细胞液到1.5mL离心管，1000rpm离心5min，吸去上清液，加入1mL含8％FBS，1/100双抗的高糖DMEM培养基(完全培养基)进行重悬，铺板到10cm细胞板中，补充9mL完全培养基，并放到37℃、5％CO2培养箱中培养。细胞长满了后，吸去完全培养基，用2mL PBS清洗贴壁细胞后，加入2mL0.05％胰蛋白酶-EDTA消化酶，在37℃、5％CO2培养箱中消化4min左右，显微镜下看细胞变圆变亮后，吸去消化酶，并加入3mL完全培养基终止消化，并用枪轻轻吹打贴壁细胞，加1/3细胞到新的10cm板中继续培养。像这样连续传代培养了2代后，消化并均匀铺6孔细胞板中，待细胞密度有80％后准备转染。
3.2 酶活检测用细胞上清的制备。
以6孔板为例，参照
LTX Reagent(#15338030)进行转染，其中pcDNA3.1空载体用量为2.5μg/孔，根据摩尔比换算得出待测质粒用量，pcDNA3.1-HG32为3.82μg/孔，每个质粒设置3个重复。室温孵育5min后，沿培养皿壁缓慢加入到细胞培养基中，37℃、5％CO2培养6h后换液，72h后收集细胞液，90xg，离心3min取上清液，获得细胞上清粗酶液，放-20℃保存备用，用于后续酶活检测试验。
四、酶活测定
4.1 试验材料
羧甲基纤维素钠、β-葡聚糖、葡萄糖、3,5-二硝基水杨酸、四水酒石酸钾钠、苯酚、无水亚硫酸钠、磷酸氢二钠均购自Sigma(美国)。
4.2 主要溶液配制
DNS试剂：称取3,5-二硝基水杨酸3.15g，加水500mL，搅拌5s，水浴至45℃。然后，逐步加入100mL氢氧化钠溶液，同时不断搅拌，直到溶液清澈透明(注：在加入氢氧化钠过程中，溶液温度不要超过48℃)。再逐步加入四水酒石酸钾钠91.0g、苯酚2.5g和无水亚硫酸钠2.5g。继续45℃水浴加热，同时补加水300mL，不断搅拌，直到加入的物质完全溶解。停止加热，冷却至室温后，用水定容至1000mL。用0.22μm过滤器过滤，避光保存棕色瓶。室温下放7天后可以使用，有效期6个月。
4.3 缓冲液配制
0.05M甘氨酸-盐酸缓冲液配制：0.2M甘氨酸和0.2M盐酸混合，pH调至1.5～3.5(共5个梯度)，定容到200mL，放置在室温备用。0.2M乙酸-乙酸钠缓冲液配制：先配制0.2M乙酸溶液1L(定容)，0.2M乙酸钠溶液1L(定容)，两者混匀，把pH调至4.0～6.0和4.3，放置在室温备用。0.1M磷酸氢二钠-柠檬酸缓冲液配制：把0.2M的磷酸氢二钠和0.1M柠檬酸缓冲液混合，把pH调至6.5～7.5，放置在室温备用。0.1M Tris-HCl：配置50mL 0.1M三羟甲基氨基甲烷与0.1M盐酸混合，把pH调至8.0～9.0，放置在室温备用。
4.4 最适pH酶活测定底物溶液配制
1％(W/V)羧甲基纤维素钠(CMC-Na)溶液：称取粉末状CMC-Na 0.1g，分别加入10mLpH1.5～9.0的缓冲液，缓慢加热并不断搅拌，直至CMC-Na完全溶解，4℃保存备用。1％(W/V)β-葡聚糖溶液：称取β-葡聚糖0.08g，分别加入10mL pH1.5～9.0的缓冲液，缓慢加热并不断搅拌，直至β-葡聚糖完全溶解，4℃保存备用。
4.5 葡聚糖/纤维素标准曲线制备
用10mg/mL的葡聚糖/纤维素溶液和pH4.5的乙酸乙酸钠溶液混合，制备浓度梯度为0.1～0.7mg/mL标准液。具体操作步骤如下：
处理组：50μl标准液+150μl pH4.5乙酸乙酸钠缓冲液+200μl DNS，煮沸5min；
空白组：200μl pH4.5乙酸乙酸钠缓冲液+200μl DNS，煮沸5min；
冷却后加600μl水，混匀离心，96孔板每孔200μl混合液，540nm检测吸光值。
以吸光值为x轴，浓度mg/mL为y轴绘制标曲(R2＞0.995)，可得出以下公式：
葡聚糖/纤维素酶活力＝D×((A1-A0)×3.9273+0.1409)/(180.2×30)×1000，180.2g/mol为葡萄糖相对分子质量，单位U/ml，D为稀释浓度，A1为标准液OD值。
4.6 HG32纤维素酶最适PH测定
处理组：50μl细胞上清粗酶液(试验3.2中获得的细胞上清粗酶液)+150μl pH1.5～9.0 1％(W/V)羧甲基纤维素钠(CMC-Na)溶液，39.5℃孵育30min后加入200μl DNS，煮沸5min；
灭活组：200μl DNS+50μl细胞上清粗酶液(试验3.2中获得的细胞上清粗酶液)+150μl pH 1.5～9.0 1％(W/V)羧甲基纤维素钠(CMC-Na)溶液，39.5℃孵育30min，煮沸5min；
冷却后加600μl水，混匀离心，96孔板每孔200μl混合液，540nm检测吸光值，根据上面得出公式计算出酶活。HG32基因在pH 1.5-4.0几乎没有纤维素酶活性，pH4.0后开始近垂直式上升，在pH5.0时达到最高点，pH5.0-8.0曲折下降，pH8.0后直线下降，结果如图3所示。
4.7 HG32葡聚糖最适PH值测定
处理组：50μl细胞上清粗酶液(试验3.2中获得的细胞上清粗酶液)+150μl pH 1.5～9.0 1％(W/V)β-葡聚糖溶液，39.5℃孵育30min+200μl DNS，煮沸5min
灭活组：200μl DNS+50μl细胞上清粗酶液(试验3.2中获得的细胞上清粗酶液)+150μl pH 1.5～9.0 1％(W/V)β-葡聚糖溶液，39.5℃孵育30min，煮沸5min
冷却后加600μl水，混匀离心，96孔板每孔200μl混合液，540吸光值检测，根据上面得出公式计算出酶活。
HG32基因在pH 1.5-4.0时几乎没有葡聚糖酶活性，pH4.0后开始垂直上升，在pH5.0时到达顶峰，pH5.0-9.0在曲折下降，结果如图4所示。
4.8 纤维素酶蛋白耐受测定
处理液配制：10mL pH2.0甘氨酸-盐酸缓冲液和10mL pH4.3乙酸乙酸钠缓冲液分别加入0.01g胃蛋白酶，10mL pH6.5磷酸氢二钠-柠檬酸缓冲液和10mL pH8.0 Tris-HCl+EDTA分别加入0.01g胰蛋白酶，10mL pH6.5磷酸氢二钠-柠檬酸缓冲液和10mL pH8.0 Tris-HCl+EDTA分别加入0.01g糜蛋白酶，混匀后放置室温备用。把10mL处理液分别加入0.1g羧甲基纤维素钠(CMC-Na)作底物。具体操作步骤如下：
处理组：50μl细胞上清粗酶液(试验3.2中获得的细胞上清粗酶液)+200μl胃蛋白酶/胰蛋白酶/糜蛋白缓冲液，39.5℃孵育2h，再加入150μl底物，39.5℃孵育30min后加入200μl DNS，煮沸5min；
空白组：50μl细胞上清粗酶液(试验3.2中获得的细胞上清粗酶液)+200μl胃蛋白酶/胰蛋白酶/糜蛋白缓冲液，39.5℃孵育2h，加入200μl DNS和150μl底物，39.5℃孵育30min，煮沸5min。
冷却后加400μl水，混匀离心，96孔板每孔200μl混合液，540nm检测吸光值，根据上面得出公式计算出酶活。
HG32基因用pH2.0和pH4.3的胃蛋白酶处理前后纤维素酶活性可忽略不计；分别用pH6.5、pH8.0胰蛋白酶和糜蛋白酶处理前后，纤维素酶活性差异不大，且均高于0.3U/mL，说明HG32对胰蛋白酶和糜蛋白酶有很好的耐受性，结果如图5所示。
4.9 葡聚糖酶蛋白耐受测定
处理液配制：10mL pH2.0甘氨酸-盐酸缓冲液和10mL pH4.3乙酸乙酸钠缓冲液分别加入0.01g胃蛋白酶，10mL pH 6.5磷酸氢二钠-柠檬酸缓冲液和10mL pH8.0 Tris-HCl+EDTA分别加入0.01g胰蛋白酶，10mL pH6.5磷酸氢二钠-柠檬酸缓冲液和10mL pH8.0 Tris-HCl+EDTA分别加入0.01g糜蛋白酶，混匀后放置室温备用。把10mL处理液分别加入0.1g羧甲基纤维素钠(CMC-Na)作底物。具体操作步骤如下：
处理组：50μl细胞上清粗酶液(试验3.2中获得的细胞上清粗酶液)+200μl胃蛋白酶/胰蛋白酶/糜蛋白缓冲液，39.5℃孵育2h后加入150μl底物，再39.5℃孵育30min，加入200μl DNS，煮沸5min；
空白组：50μl细胞上清粗酶液(试验3.2中获得的细胞上清粗酶液)+200μl胃蛋白酶/胰蛋白酶/糜蛋白缓冲液，39.5℃孵育2h后加入200μl DNS和150μl底物，39.5℃孵育30min，煮沸5min。
冷却后加400μl水，混匀离心，96孔板每孔200μl混合液，540nm检测吸光值，根据上面得出公式计算出酶活。
HG32基因用pH2.0和pH4.3的胃蛋白酶处理前后葡聚糖酶活几乎没有；用pH6.5、pH8.0胰蛋白酶和糜蛋白酶处理前后，葡聚糖酶活均高于1U/mL，且差异不大。说明HG32在猪胃肠道均能很好地发挥作用，对胰蛋白酶和糜蛋白酶有很好的耐受性，结果如图6所示。
4.10 与其他酶基因进行最适pH比较，发现HG32在pH 4.5-9.0有更好的纤维素酶活性，结果如图7所示；在pH 5.0-9.0有更高的葡聚糖酶活性，结果如图8所示。bg17虽在pH1.5-4.5有较好的葡聚糖酶活性，但纤维素酶活性较低；eg1314在pH 5.0-8.5有较稳定的葡聚糖酶活性，但几乎没有纤维素酶活性；egII在pH 3.0-6.0之间有较好的纤维素酶和葡聚糖酶，仅此于HG32。
4.11 用pH 4.3的胃蛋白酶处理bg17、TeEG1、egII前后纤维素酶剩余活性差异显著，但与HG32纤维素酶剩余活性差异不显著，结果如图9所示；用pH 4.3的胃蛋白酶处理HG32前后，其葡聚糖酶剩余酶活仅此于eg1314的葡聚糖酶剩余活性，且差异不大，结果如图10所示。综上所述，HG32对pH 4.3的胃蛋白酶有较好的耐受性。
4.12 用pH 6.5胰蛋白酶处理egII前后纤维素酶剩余活性显著降低20％，而HG32处理前后的纤维素酶剩余活性最高且始终保持在0.4U/mL以上，结果如图11所示，四个基因处理前后葡聚糖酶活性差异显著，其中HG32的葡聚糖酶活性处理前后最稳定，从处理后结果看，HG32的葡聚糖酶剩余活性最高，结果如图12所示。说明HG32对pH 6.5胰蛋白酶有较好的耐受性。
4.13 用pH 8.0胰蛋白酶处理bg17和egII前后纤维素酶剩余活性差异显著，其中egII在处理后迅猛下降60％左右，HG32活性最稳定，且处理后HG32的活性最高，结果如图13所示；用pH 8.0胰蛋白酶处理四个基因前后葡聚糖酶剩余活性差异均显著，其中bg17、eg1314、egII大幅度下降70％-90％，而HG32仅下降10％左右，具有更好的稳定性，且从处理后的结果看，HG32有最高的葡聚糖酶活，结果如图14所示。综上所述，在HG32对pH 8.0的胰蛋白酶耐受性最好，最稳定。
4.14 用pH 6.5糜蛋白酶处理TeEG1和egII前后纤维素酶剩余活性差异显著，HG32处理前后的纤维素酶活性都是最好的，egII次之，结果如图15所示。用pH 6.5糜蛋白酶处理eg1314前后葡聚糖酶活性差异显著，HG32在处理前后前后不大，活性保持在1.2U/mL以上，且在处理后活性最高，结果如图16所示。综上所述，HG32对pH 6.5糜蛋白酶酶有较好的耐受性。
4.15 用pH 8.0糜蛋白酶处理bg17和egII前后纤维素酶剩余活性显著降低，HG32处理前后的纤维素酶剩余活性都是最高的，在0.3U/mL以上，结果如图17所示；用pH 8.0糜蛋白酶处理bg17、eg1314和egII前后葡聚糖酶剩余活性差异显著，bg17和egII处理后的葡聚糖酶剩余活性几乎为零，HG32处理前的葡聚糖酶活仅次于bg17，处理后的葡聚糖酶活性最高，结果如图18所示。综上所述，HG32对pH 8.0糜蛋白酶有较好的耐受性。
4.16 HG32对不同底物的酶促反应活性测定
酶活测定底物溶液配制：分别用1％(W/V)微晶纤维素溶液，1％(W/V)低粘度羟乙基纤维素溶液，1％(W/V)中粘度甲基纤维素溶液，1％(W/V)羧甲基纤维素钠溶液，0.8％(W/V)β-葡聚糖溶液，1％(W/V)木聚糖，1％(W/V)多聚半乳糖醛酸溶液，0.5％(W/V)植酸钠(缓冲液均为0.2M pH 6.0的乙酸乙酸钠溶液)作为底物。
1.微晶纤维素酶活测定：
处理组：50μl细胞上清粗酶液(试验3.2中获得的细胞上清粗酶液)+150μl 1％(W/V)微晶纤维素溶液，39.5℃孵育11h后再加200μl DNS，煮沸5min；
灭活组：200μl DNS+50μl细胞上清粗酶液(试验3.2中获得的细胞上清粗酶液)+150μl 1％(W/V)微晶纤维素溶液，39.5℃孵育11h，煮沸5min；
冷却后加400μl水，混匀离心，96孔板每孔200μl混合液，540nm吸光值检测，根据葡聚糖/纤维素标曲得出的公式计算出酶活。
2.低粘度羟乙基纤维素/中粘度甲基纤维素测定：
处理组：50μl细胞上清粗酶液(试验3.2中获得的细胞上清粗酶液)+150μl 1％(W/V)低粘度羟乙基纤维素溶液/1％(W/V)中粘度甲基纤维素溶液，39.5℃孵育30min后再加200μl DNS，煮沸5min；
灭活组：200μl DNS+50μl细胞上清粗酶液(试验3.2中获得的细胞上清粗酶液)+150μl 1％(W/V)低粘度羟乙基纤维素溶液/1％(W/V)中粘度甲基纤维素溶液，39.5℃孵育30min，煮沸5min；
冷却后加400μl水，混匀离心，96孔板每孔200μl混合液，540nm吸光值检测，根据葡聚糖/纤维素标曲得出的公式计算出酶活。
纤维素酶活和葡聚糖酶活测定步骤参照4.6和4.7实验方法。
3.木聚糖活性测定：
D-木糖标准曲线：用10mg/mL的D-木糖溶液和0.2M pH 6.0的乙酸乙酸钠溶液混合，制备浓度梯度0.1～0.7mg/mL的标准液。具体操作步骤如下：
处理组：50μl标准液+150μl 0.2M pH 6.0乙酸乙酸钠缓冲液+200μl DNS，煮沸5min
空白组：200μl 0.2M pH 6.0乙酸乙酸钠缓冲液+200μl DNS，煮沸5min
冷却后加600μl水，混匀离心，96孔板每孔200μl混合液，540nm检测吸光值
以吸光值为x轴，浓度mg/mL为y轴绘制标曲(R2＞0.995)，可得出以下公式：
木聚糖酶活力＝D×((A1-A0)×3.3185+0.2044)/(150.2×30)×1000，150.2g/mol为D-木糖相对分子质量，单位U/ml，D为稀释浓度，A1为标准液OD值。
HG32木聚糖酶活测定步骤如下：
处理组：50μl细胞上清粗酶液(试验3.2中获得的细胞上清粗酶液)+150μl 1％(W/V)木聚糖溶液，39.5℃孵育30min后再加200ul DNS，煮沸5min；
灭活组：200μl DNS+50ul细胞上清粗酶液(试验3.2中获得的细胞上清粗酶液)+150μl 1％(W/V)木聚糖溶液，39.5℃孵育30min，煮沸5min；
冷却后加600μl水，混匀离心，96孔板每孔200μl混合液，540nm吸光值检测，根据木聚糖标曲得出的公式计算出酶活。
4.果胶酶活性测定：
半乳糖醛酸标准曲线：用10mg/mL的半乳糖醛酸溶液和0.2M pH6.0的乙酸乙酸钠溶液混合，制备浓度梯度0.1～0.7mg/mL的标准液。具体操作步骤如下：
处理组：50μl标准液+100μl 0.2M pH6.0乙酸乙酸钠缓冲液+450μl DNS，煮沸5min；
空白组：150μl 0.2M pH6.0乙酸乙酸钠缓冲液+450μl DNS，煮沸5min；
冷却后加400μl水，混匀离心，96孔板每孔200μl混合液，540nm检测吸光值
以吸光值为x轴，浓度mg/mL为y轴绘制标曲(R2＞0.995)，可得出以下公式：
果胶酶活力＝D×((A1-A0)×2.8701+0.0486)/(216.12×30)×1000，216.12g/mol为半乳糖醛酸相对分子质量，单位U/ml，D为稀释浓度，A1为标准液OD值。
HG32果胶酶活测定步骤如下：
处理组：50μl细胞上清粗酶液(试验3.2中获得的细胞上清粗酶液)+100μl 1％(W/V)多聚半乳糖醛酸溶液，39.5℃孵育30min后再加450μlDNS，煮沸5min；
灭活组：450μl DNS+50μl细胞上清粗酶液(试验3.2中获得的细胞上清粗酶液)+100μl 1％(W/V)多聚半乳糖醛酸溶液，39.5℃孵育30min，煮沸5min；
冷却后加400μl水，混匀离心，96孔板每孔200μl混合液，540nm吸光值检测，根据果糖标曲得出的公式计算出酶活。
5.植酸酶活性测定：
100g/L钼酸铵溶液配制：取10g于90mL水中，加热溶解后，再加入1mL0.25％氨水，定容至100mL。
偏钒酸铵溶液配制：取0.235g偏钒酸铵，加2mL硝酸溶液溶解后，定容至100mL。
硝酸溶液：以1:2水的比例进行配制。
植酸显色液：1(100g/L钼酸铵)+1(偏钒酸铵)+2(硝酸溶液)，现配现用。
磷酸二氢钾标曲。用500μl 50mmol/L磷酸二氢钾溶液分别与0.25M pH5.5乙酸乙酸钠缓冲液混合配制成终浓度为25-1.5625mmol/L的标准液，用具体操作步骤如下：
处理组：50μl标准液+150μl 0.25M pH5.5的乙酸乙酸钠缓冲液+400μl底物0.5％(W/V)植酸钠，39.5℃孵育30min，加450ul植酸显色液；
空白组：150μl 0.25M pH5.5的乙酸乙酸钠缓冲液+400μl植酸显色液，39.5℃孵育30min；
冷却后混匀离心，96孔板每孔200μl混合液，415nm检测吸光值；
以吸光值为x轴，浓度mg/mL为y轴绘制标曲(R2＞0.995)，可得出以下公式：
植酸酶活力＝(D×[(A1-A0)×1.0505+0.0023])/(0.05×30)，1.0505g/mol为磷酸二氢钾相对分子质量，单位U/ml，D为稀释浓度，A1为标准液OD值。HG32植酸酶活测定步骤如下：
处理组：50μl细胞上清粗酶液(试验3.2中获得的细胞上清粗酶液)+550μl 0.5％(W/V)植酸钠，39.5℃孵育30min后再加400μl植酸显色液；
灭活组：400μl植酸显色液+50μl细胞上清粗酶液(试验3.2中获得的细胞上清粗酶液)+550μl 0.5％(W/V)植酸钠，39.5℃孵育30min；
冷却后混匀离心，96孔板每孔200μl混合液，415nm吸光值检测，根据磷酸二氢钾标曲得出的公式计算出酶活。
从图19检测结果可见，HG32有较高的纤维素酶、微晶纤维素酶、低粘度羧乙基纤维素酶和葡聚糖酶活性。
序列表
<110> 温氏食品集团股份有限公司
华南农业大学
<120> 一种新型多功能酶基因HG32及应用
<130> 20210203
<160> 6
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aaagcggtgg gagagatgga gaggaccggc ctgctgaacg gctatccgga cggcagcttc 240
cgcccggatg agccgataac ggcggctcag tttgtggcgg tgacagcccg gtgtgccggg 300
ctgtcaccag tcccggggca gacaaaccac tgggccgccg gccggctcca ggcggccctc 360
caggccggct ggtacgactg ggacgagatc ccgcccaccg gggaggggtt tgaccagccc 420
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gactacagca cggagttcca gaagatgcgg gactttgccc ggctggacgg ccggtactac 540
gaggcggtgc tggccgccta cgccgccggc gtggtcaagg gggacgacat gggcaatttc 600
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cagtggtacg gccagtacgc caacagccgg tccattcgca atacggcgga ctggggggcc 900
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Glu Ile Pro Pro Thr Gly Glu Gly Phe Asp Gln Pro Ile Ala Arg Gln
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Arg Cys Ala Gly Leu Ser Pro Val Pro Gly Gln Thr Asn His Trp Ala
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Asp Tyr Ser Thr Glu Phe Gln Lys Met Arg Asp Phe Ala Arg Leu Asp
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Gly Glu Asp Leu Pro Val Pro Glu Asp Pro Gln Pro Glu Thr Pro Gln
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Pro Ala Val Thr Val Arg Gly Gly Val Ser Glu Asn Gly Trp Leu Gln
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Val Lys Gly Thr Arg Leu Cys Asn Glu Lys Gly Glu Ala Val Val Leu
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Ser Arg Ser Ile Arg Asn Thr Ala Asp Trp Gly Ala Asn Leu Phe Arg
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Val Ala Met Tyr Thr Gly Glu Gly Gly Tyr Leu Ser Ser Pro Asp Ser
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Leu Tyr Val Ile Ile Asp Trp His Ile Leu Ser Asp Gly Asn Pro Met
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Ser His Val Glu Glu Ala Lys Ala Phe Phe Gly Glu Met Ala Ala Arg
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Tyr Gly Asp Asn Pro Ala Val Leu Tyr Glu Ile Cys Asn Glu Pro Asn
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atgttccagc tgtggaagct ggtgttcctg tgcggactgc tgatcggcac ctccgccagc 60
gccttcccag acgtgcgccc agaggcctgg tacgctaagg ctgtgggaga gatggagagg 120
accggactgc tgaacggcta cccagacggc agcttcagac ccgacgagcc aatcaccgcc 180
gcccagttcg tggccgtgac cgctaggtgc gctggactgt ccccagtgcc aggccagacc 240
aaccactggg ctgctggccg gctgcaggcc gccctgcagg ccggctggta cgactgggac 300
gagatcccac caaccggaga gggattcgac cagccaatcg ctaggcagct ggctgtgaag 360
atcctgatga gggccctgct gccacaggcc agaggcgact acagcaccga gttccagaag 420
atgagagact tcgctaggct ggacggccgg tactacgagg ccgtgctggc tgcttacgct 480
gctggcgtgg tgaagggcga cgacatgggc aacttcaacc caaggaaggg actgagcaga 540
gccgaggcct gcgtgctgtt ccagaaggct atgaagctgt ccggagctgg agaggacctg 600
ccagtgccag aggacccaca gccagagacc ccacagccag ctgtgaccgt gaggggaggc 660
gtgagcgaga acggatggct gcaggtgaag ggaaccaggc tgtgcaacga gaagggagag 720
gctgtggtgc tgaggggaat gtccacccac ggactgcagt ggtacggcca gtacgccaac 780
tcccggagca tcaggaacac cgccgactgg ggcgccaacc tgttcagagt ggccatgtac 840
accggagagg gaggatacct gtccagccca gactccatga agaagaaggt gaccgacgct 900
gtggacgctg ctatcgagaa cgacctgtac gtgatcatcg actggcacat cctgagcgac 960
ggcaacccca tgtcccacgt ggaggaggcc aaggccttct tcggagagat ggctgctaga 1020
tacggcgaca acccagccgt gctgtacgag atctgcaacg agccaaacgg aggagtgacc 1080
tgggagggcg acgtgaagcc atacgcccgg gccgtgatcc cagtgatcag agagagggct 1140
ccaaagagcg tgatcctggt gggctcccca acctggagcc aggacatcca cctggctgct 1200
cagtccccac tggagggcga gaacctgatg tacaccctgc acttctacgc tggaacccac 1260
ggagctgacc tgcgccggag gatcgacgac gccctggccg ccggcgcccc cgtgttcgtg 1320
tccgagtggg gcaccagccg cgccgacgga tccggaggcg tgttcctgaa ggaggctgga 1380
gagtggctgg acttcctgga ccagaggggc atcagctggg ccaactggtc cctgtgcgac 1440
aagaacgaga ccagcgccgc cctgagacca ggagcttccc cagacggagc ttggggagag 1500
gccgacctgt ccgagagcgg ccgcttcgtg gccagccggc tggccgactg a 1551
<210> 6
<211> 516
<212> PRT
<213> Artificial
<400> 6
Met Phe Gln Leu Trp Lys Leu Val Phe Leu Cys Gly Leu Leu Ile Gly
1 5 10 15
Thr Ser Ala Ser Ala Phe Pro Asp Val Arg Pro Glu Ala Trp Tyr Ala
20 25 30
Lys Ala Val Gly Glu Met Glu Arg Thr Gly Leu Leu Asn Gly Tyr Pro
35 40 45
Asp Gly Ser Phe Arg Pro Asp Glu Pro Ile Thr Ala Ala Gln Phe Val
50 55 60
Ala Val Thr Ala Arg Cys Ala Gly Leu Ser Pro Val Pro Gly Gln Thr
65 70 75 80
Asn His Trp Ala Ala Gly Arg Leu Gln Ala Ala Leu Gln Ala Gly Trp
85 90 95
Tyr Asp Trp Asp Glu Ile Pro Pro Thr Gly Glu Gly Phe Asp Gln Pro
100 105 110
Ile Ala Arg Gln Leu Ala Val Lys Ile Leu Met Arg Ala Leu Leu Pro
115 120 125
Gln Ala Arg Gly Asp Tyr Ser Thr Glu Phe Gln Lys Met Arg Asp Phe
130 135 140
Ala Arg Leu Asp Gly Arg Tyr Tyr Glu Ala Val Leu Ala Ala Tyr Ala
145 150 155 160
Ala Gly Val Val Lys Gly Asp Asp Met Gly Asn Phe Asn Pro Arg Lys
165 170 175
Gly Leu Ser Arg Ala Glu Ala Cys Val Leu Phe Gln Lys Ala Met Lys
180 185 190
Leu Ser Gly Ala Gly Glu Asp Leu Pro Val Pro Glu Asp Pro Gln Pro
195 200 205
Glu Thr Pro Gln Pro Ala Val Thr Val Arg Gly Gly Val Ser Glu Asn
210 215 220
Gly Trp Leu Gln Val Lys Gly Thr Arg Leu Cys Asn Glu Lys Gly Glu
225 230 235 240
Ala Val Val Leu Arg Gly Met Ser Thr His Gly Leu Gln Trp Tyr Gly
245 250 255
Gln Tyr Ala Asn Ser Arg Ser Ile Arg Asn Thr Ala Asp Trp Gly Ala
260 265 270
Asn Leu Phe Arg Val Ala Met Tyr Thr Gly Glu Gly Gly Tyr Leu Ser
275 280 285
Ser Pro Asp Ser Met Lys Lys Lys Val Thr Asp Ala Val Asp Ala Ala
290 295 300
Ile Glu Asn Asp Leu Tyr Val Ile Ile Asp Trp His Ile Leu Ser Asp
305 310 315 320
Gly Asn Pro Met Ser His Val Glu Glu Ala Lys Ala Phe Phe Gly Glu
325 330 335
Met Ala Ala Arg Tyr Gly Asp Asn Pro Ala Val Leu Tyr Glu Ile Cys
340 345 350
Asn Glu Pro Asn Gly Gly Val Thr Trp Glu Gly Asp Val Lys Pro Tyr
355 360 365
Ala Arg Ala Val Ile Pro Val Ile Arg Glu Arg Ala Pro Lys Ser Val
370 375 380
Ile Leu Val Gly Ser Pro Thr Trp Ser Gln Asp Ile His Leu Ala Ala
385 390 395 400
Gln Ser Pro Leu Glu Gly Glu Asn Leu Met Tyr Thr Leu His Phe Tyr
405 410 415
Ala Gly Thr His Gly Ala Asp Leu Arg Arg Arg Ile Asp Asp Ala Leu
420 425 430
Ala Ala Gly Ala Pro Val Phe Val Ser Glu Trp Gly Thr Ser Arg Ala
435 440 445
Asp Gly Ser Gly Gly Val Phe Leu Lys Glu Ala Gly Glu Trp Leu Asp
450 455 460
Phe Leu Asp Gln Arg Gly Ile Ser Trp Ala Asn Trp Ser Leu Cys Asp
465 470 475 480
Lys Asn Glu Thr Ser Ala Ala Leu Arg Pro Gly Ala Ser Pro Asp Gly
485 490 495
Ala Trp Gly Glu Ala Asp Leu Ser Glu Ser Gly Arg Phe Val Ala Ser
500 505 510
Arg Leu Ala Asp
515
Claims (18)
1.一种多功能酶，其中，所述多功能酶的表达基因的核苷酸序列如SEQ ID NO:3所示。
2.根据权利要求1所述的多功能酶，其中，所述多功能酶的表达基因还包含表达猪腮腺分泌蛋白信号肽的核苷酸序列，其核苷酸序列如SEQ ID NO:5所示。
3.一种多功能酶，其中，所述酶的氨基酸序列如SEQ ID NO:4所示。
4.根据权利要求3所述的多功能酶，其中，所述多功能酶还包含猪腮腺分泌蛋白信号肽序列，其氨基酸序列如SEQ ID NO:6所示。
5.权利要求1-4任一项所述的多功能酶在制备畜禽饲料酶制剂上的应用。
6.根据权利要求5所述的应用，其中，所述的应用为在制备纤维素酶、微晶纤维素酶、低粘度羧乙基纤维素酶或葡聚糖酶任一项或多项酶制剂上的应用。
7.根据权利要求6所述的应用，其中，所述的应用为在制备具有强胰蛋白和糜蛋白酶耐受性的纤维素酶、微晶纤维素酶、低粘度羧乙基纤维素酶或葡聚糖酶任一项或多项酶制剂上的应用。
8.权利要求1-4任一项所述的多功能酶在制备改善胃肠道内环境的食品保健品上的应用。
9.含有可表达如权利要求1-4任一项所述的多功能酶的重组载体。
10.权利要求9所述的重组载体在制备畜禽饲料酶制剂上的应用。
11.根据权利要求10所述的应用，其中，所述的应用为在制备纤维素酶、微晶纤维素酶、低粘度羧乙基纤维素酶或葡聚糖酶任一项或多项酶制剂上的应用。
12.根据权利要求11所述的应用，其中，所述的应用为在制备具有强胰蛋白和糜蛋白酶耐受性的纤维素酶、微晶纤维素酶、低粘度羧乙基纤维素酶或葡聚糖酶任一项或多项酶制剂上的应用。
13.权利要求9所述的重组载体在制备改善胃肠道内环境的食品保健品上的应用。
14.含有可表达如权利要求1-4任一项所述的多功能酶的重组菌株。
15.权利要求14所述的重组菌株在制备畜禽饲料酶制剂上的应用。
16.根据权利要求15所述的应用，其中，所述的应用为在制备纤维素酶、微晶纤维素酶、低粘度羧乙基纤维素酶或葡聚糖酶任一项或多项酶制剂上的应用。
17.根据权利要求16所述的应用，其中，所述的应用为在制备具有强胰蛋白和糜蛋白酶耐受性的纤维素酶、微晶纤维素酶、低粘度羧乙基纤维素酶或葡聚糖酶任一项或多项酶制剂上的应用。
18.权利要求14所述的重组菌株在制备改善胃肠道内环境的食品保健品上的应用。
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