CN116042581A - 一种共表达突变酶重组菌及其制剂和在制备水产动物的饲料添加剂中的应用 - Google Patents
一种共表达突变酶重组菌及其制剂和在制备水产动物的饲料添加剂中的应用 Download PDFInfo
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- CN116042581A CN116042581A CN202211721671.8A CN202211721671A CN116042581A CN 116042581 A CN116042581 A CN 116042581A CN 202211721671 A CN202211721671 A CN 202211721671A CN 116042581 A CN116042581 A CN 116042581A
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Abstract
本发明公开了一种共表达突变酶重组菌及其制剂和在制备水产动物的饲料添加剂中的应用。本发明通过筛选获得酶活力提高的木聚糖酶突变体xynM和甘露聚糖酶突变体AnManM;再将xynM和AnManM克隆入载体,构建共表达体系,再转入毕赤酵母中,获取到共表达突变酶重组菌,将菌株发酵液与胆汁酸复合的制剂,经过验证,可以明显提高对虾的特定生长率和消化率,降低饵料系数,从而提高饲料的利用率,降低饲养成本,且效果优于单独添加木聚糖酶或甘露聚糖酶的制剂,因此在饲料酶制剂等领域有良好的应用潜力。
Description
技术领域
本发明属于酶工程领域,具体涉及一种共表达突变酶重组菌及其制剂和在制备水产动物的饲料添加剂中的应用。
背景技术
木聚糖是一种五碳糖,是半纤维素的重要组分,它是自然界中含量仅次于纤维素的第二丰富的多聚糖,几乎占地球可更新有机碳含量的三分之一。木聚糖广泛存在于饲料原料中,如玉米、麦麸、米糠、秸秆、豆粕等,属于饲料中的非淀粉多糖是重要的抗营养因子,在动物消化系统中不能被有效的降解,还会阻碍营养物质,尤其是脂肪和蛋白质的消化吸收,降低饲料的利用率。甘露聚糖是自然界中含量仅次于木聚糖的半纤维素,以β-1,4糖苷键链接形成主链,由半乳糖、葡萄糖等参与形成侧链。广泛存在于自然界中,它是多种植物、微生物细胞壁的主要组成成分,在植物性饲料原料中也和木聚糖一起被视为抗营养因子,难以被消化。
在养殖行业中,通过在饲料中添加木聚糖酶和甘露聚糖酶,以降解抗营养因子木聚糖、甘露聚糖对提高饲料利用率非常经济有效,同时通过酶解获得的甘露寡糖,可以起到保护动物肠道,提高免疫力的作用。
随着市场需求量的增加,提高木聚糖酶和甘露聚糖酶的表达量,改善其在饲料中的使用效果是急需解决的问题。借助于基因工程和酶的体外定向进化手段,从酶的基因和结构入手,改良酶学特性,筛选得到优良性质的酶制剂,同时利用合适的表达系统,进行高效的表达生产,对降低目前的饲用生产成本,提高其利用效率,以及对产品的开发和应用具有重要的意义。
发明内容
本发明提供了一种共表达突变酶重组菌及其制剂和在制备水产动物的饲料添加剂中的应用。本发明筛选获得酶活力提高的木聚糖酶突变体xynM和甘露聚糖酶突变体AnManM,并利用两者构建共表达体系,与胆汁酸共同制备的制剂可以提高饲料的利用转化效率。
为了实现上述发明目的,本发明采用以下技术方案予以实现:
本发明提供了一种木聚糖酶突变体xynM,其氨基酸序列如SEQ ID NO:3所示。
进一步的:所述木聚糖酶突变体xynM是由氨基酸序列为SEQ ID NO:1的木聚糖酶xyn的第156位的天冬氨酸变为谷氨酸和第176位的天冬氨酸变为天冬酰胺得到的。
本发明提供了所述的木聚糖酶突变体xynM的编码基因,其核苷酸序列如SEQ IDNO:4所示。
本发明提供了一种含有所述的编码基因的共表达突变酶重组菌,其中还同时含有甘露聚糖酶的编码基因。
进一步的:所述甘露聚糖酶为甘露聚糖酶突变体AnManM,其氨基酸序列如SEQ IDNO:7所示,其编码基因的核苷酸序列如SEQ ID NO:8所示。
进一步的:所述甘露聚糖酶突变体AnManM是由氨基酸序列为SEQ ID NO:5的甘露聚糖酶AnMan的第76位的缬氨酸变为丙氨酸、第99位的苏氨酸变为谷氨酸和第172位的丝氨酸变为脯氨酸得到的。
本发明提供了一种共表达突变酶复合制剂,其中包含权利要求4所述的共表达突变酶重组菌的发酵液和胆汁酸。
进一步的:所述发酵液和胆汁酸的体积比为5~10:3。
本发明提供了所述的共表达突变酶复合制剂在制备水产动物的饲料添加剂中的应用。
进一步的:所述共表达突变酶复合制剂的用量为水产动物基础饲料重量的0.1%-0.5%。
与现有技术相比,本发明的优点和技术效果是:
本发明分别将来源于瘤胃真菌Neocallimastix patriciarum的木聚糖酶xyn以及黑曲霉Aspergillus niger的甘露聚糖酶AnMan为基础进行突变改良,筛选获得突变位点为D156E/D176N的木聚糖酶突变体xynM和突变位点为V76A/T99E/S172P的AnManM;两者与原始基因相比,酶活力均得到明显提升。
本发明将xynM和AnManM克隆入载体,构建共表达体系,再转入毕赤酵母中,获取到共表达突变酶重组菌,将菌株发酵液与胆汁酸复合的制剂,经过验证,可以明显提高对虾的特定生长率和消化率,降低饵料系数,从而提高饲料的利用率,降低饲养成本,且效果优于单独添加木聚糖酶或甘露聚糖酶的制剂,因此在饲料酶制剂等领域有良好的应用潜力。
附图说明
图1是所述木聚糖酶基因的扩增结果。
图2是所述甘露聚糖酶基因扩增结果。
图3是所述共表达体系和单独表达木聚糖酶或甘露聚糖酶对比。
图4是所述共表达木聚糖酶-甘露聚糖酶在30L发酵罐中的发酵数据。
具体实施方式
为了便于理解本发明,以下将结合附图及实施例对本文发明做更全面、详细地说明,但本发明的保护范围并不限于以下具体实施例。
以下实施例中未作具体说明的分子生物学实验方法,可以参照《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。具体实施例中使用的试剂和生物材料如无特殊说明均可从商业途径获得。
本发明所用培养基配方如下:
LB培养基:1%胰蛋白胨,0.5%酵母提取物,1%NaCl;
MD培养基:1.34%YNB,0.4mg/L生物素,2%葡萄糖;
YPD培养基:1%酵母提取物,2%蛋白胨,2%葡萄糖;
BMGY培养基:1%酵母提取物,2%蛋白胨,100mmol/L磷酸钾缓冲液(pH6.0),1.34%YNB,0.4mg/L生物素,1%甘油;
BSM培养基:26.7mL 85%的磷酸,0.93g二水硫酸钙,14.9g二水硫酸镁,4.13g氢氧化钾,18.2g硫酸钾,40g甘油,4.0mL PMT1。
以上培养基为固体时加入2%的琼脂粉。
本发明中酶活力测定:《GB/T 36861-2018饲料添加剂β-甘露聚糖酶活力的测定分光光度法》中的测定方法。
毕赤酵母GS115(X33)转化方法:将活化的毕赤酵母GS115接种到含有20mL YPD液体培养基中,30℃摇瓶培养至OD600为1.2~1.5后低温离心收集菌体,依次用20mL冰冷无菌水和5mL浓度为1mol/L的冰冷山梨醇溶液清洗菌体,最后用1mL山梨醇溶液重悬菌体制得酵母感受态悬液。将100μL酵母感受态与10μL线性化载体混匀后转移到预冷的电转杯中电击转化,电转化的条件为1.5kV,6msec。电击后加入1mL山梨醇溶液,转移至1.5mL离心管中30℃温育1h。5000rpm离心5min,收集菌体涂布于MD筛选平板上倒置培养,直至长出阳性单克隆。
实施例1:木聚糖酶突变体基因构建和筛选
参考瘤胃真菌Neocallimastix patriciarum的木聚糖酶xyn(GenBank:AKN90969.1)的氨基酸序列,翻译成对应的核苷酸序列,并经过基因序列优化后,由人工合成,得到的氨基酸序列如SEQ ID NO:1所示,核苷酸序列如SEQ ID NO:2所示。设计如下引物,在5’端设计EcoR I限制性酶切位点,3’端设计Not I限制性酶切位点。
xyn-F:CCGGAATTCCAAAGTTTCTGTAGTTCAGC(SEQ ID NO:9);
xyn-R:ATAAGCGGCCGCCTAATCACCAATGTAAACCT(SEQ ID NO:10)。
用GeneMorph II随机突变PCR试剂盒,以人工合成的基因为模版,使用上述引物进行随机突变,PCR扩增结果如图1所示。
将扩增好的随机突变PCR产物用EcoR I和Not I双酶切,纯化回收后连接到pET-21a(+)载体上,转化大肠杆菌BL21-DE3,氨苄青霉素抗性LB平板筛选阳性克隆,得到pET-xynMx。同样方法将合成的原始基因连接到pET-21a(+)载体上并转化大肠杆菌BL21-DE3,得到pET-xyn0。
将筛选的单菌落接种到96孔深孔板。每个平板接入2个表达xyn0的单菌落为对照。每孔装入300uL LB液体培养基(含有100μg/mL氨苄青霉素),37℃、200rpm震荡培养4小时后,转移50uL菌液到新的96孔平板保种,在平板剩余菌液中添加200uL含有IPTG的LB-Amp培养基,使IPTG终浓度为1mM,氨苄青霉素终浓度为100μg/mL,37℃、200rpm摇床培养10h诱导表达木聚糖酶。
将诱导完成的菌液反复冻融进行破碎,将破碎后的细胞液离心取上清,然后检测木聚糖酶的酶活性。将酶活高于对照的突变体基因进行测序。经过测序后获得木聚糖酶突变体xynM,其氨基酸序列如SEQ ID NO:3所示,核苷酸序列如SEQ ID NO:4所示,突变位点为D156E/D176N。
实施例2:甘露聚糖酶突变体基因构建和筛选
参考黑曲霉Aspergillus niger的甘露聚糖酶AnMan(GenBank:ADK88903.1)的氨基酸序列,翻译成对应的核苷酸序列,并经过基因序列优化后,由人工合成,得到的氨基酸序列如SEQ ID NO:5所示,核苷酸序列如SEQ ID NO:6所示。设计如下引物,在5’端设计EcoRI限制性酶切位点,3’端设计Not I限制性酶切位点。
AnMan-F:CCGGAATTCCTGCCGAAAGCCTCCCCTGC(SEQ ID NO:11);
AnMan-R:ATAAGCGGCCGCTTAGGCGCTACCAATAGCAG(SEQ ID NO:12)。
用GeneMorph II随机突变PCR试剂盒,以人工合成的基因为模版,使用上述引物进行随机突变,PCR扩增结果如图2所示。
将扩增好的随机突变PCR产物用EcoR I和Not I双酶切,纯化回收后连接到pET-21a(+)载体上,转化大肠杆菌BL21-DE3,氨苄青霉素抗性LB平板筛选阳性克隆,得到pET-AnManx。同样方法将合成的原始基因连接到pET-21a(+)载体上并转化大肠杆菌BL21-DE3,得到pET-AnMan0。
将筛选的单菌落接种到96孔深孔板。每个平板接入2个表达AnMan0的单菌落为对照。每孔装入300uL LB液体培养基(含有100μg/mL氨苄青霉素),37℃、200rpm震荡培养4小时后,转移50uL菌液到新的96孔平板保种,在平板剩余菌液中添加200uL含有IPTG的LB-Amp培养基,使IPTG终浓度为1mM,氨苄青霉素终浓度为100μg/mL,37℃、200rpm摇床培养10h诱导表达甘露聚糖酶。
将诱导完成的菌液反复冻融进行破碎,将破碎后的细胞液离心取上清,然后检测甘露聚糖酶的酶活性。将酶活高于对照的突变体基因进行测序。经过测序后获得甘露聚糖酶突变体AnManM,氨基酸序列如SEQ ID NO:7所示,核苷酸序列如SEQ ID NO:8所示。
实施例3:共表达载体构建及转化毕赤酵母X33
分别将实施例1和2的木聚糖酶突变体基因和甘露聚糖酶突变体基因用EcoR I和Not I双酶切位点连接到pPICZα质粒上并转化至大肠杆菌DH5α中,获得重组表达载体,测序验证。
(1)将木聚糖酶突变体重组载体用Bgl II酶切;
(2)将甘露聚糖酶突变体重组载体用Bgl II和BamH I双酶切;
(3)将甘露聚糖酶突变体双酶切片段连接到步骤(1)的木聚糖酶突变体的Bgl II位点上,得到共表达重组载体;
(4)将共表达重组载体转化大肠杆菌DH5α中,测序验证。
将共表达重组载体用Sal I酶切线性化后电转入毕赤酵母X33中,Zeocin抗性平板筛选得到转化子并转接到Zeocin抗性的YPD平板中活化(一般挑24-48个转化子)。活化后的转化子接于摇瓶进行发酵(每瓶含20mL BMGY培养基),30℃振荡培养18h后,加1%甲醇诱导,继续振荡培养,此后每24h添加培养体积1%的甲醇。诱导表达96h后,将培养液离心获得上清,测定发酵液上清的平均酶活性。
按照国标中木聚糖酶和甘露聚糖酶测定的方法,取发酵液离心后的上清液测定酶活力,并和单独发酵的酶活力比较。
结果如图3所示,单独表达木聚糖酶酶活力平均是5000U/mL;单独表达甘露聚糖酶平均是220U/mL;共表达体系中,木聚糖酶酶活力是3600U/mL,甘露聚糖酶是110U/mL。所以共表达经过一次发酵可以获得两种酶,且经过测定两种酶活力之和高于单独表达之和的1/2,表明该共表达体系在饲料酶制剂等领域有良好的应用潜力。
实施例4:木聚糖酶和甘露聚糖酶共表达体系在30L发酵罐中发酵和制备
将木聚糖酶-甘露聚糖酶共表达的基因工程菌分别划线于YPD平板上,30℃培养3天长出单菌落,挑取长势良好的单菌落继续在YPD平板上划线培养,如此活化三代得到的毕赤酵母单菌落接种于20mL BMGY培养基中,30℃、200rpm培养24h。以2%的接种量接种到300mL BMGY培养基中,30℃、200rpm培养至OD600为5,用作种子液接种发酵罐。发酵生产工艺:BSM培养基,pH4.8、温度30℃、搅拌速率500rpm、通风量1.5(v/v),溶氧控制在20%以上发酵过程分为三个阶段:(1)菌体培养阶段:按8%比例接入种子液,30℃培养20~24h,使发酵液中甘油耗尽;(2)饥饿阶段:当碳源甘油耗尽后,暂不补加任何碳源,待溶氧上升至80%饥饿阶段结束;(3)诱导表达阶段:用氨水或磷酸调节pH至所需值,流加甲醇诱导,并且保持溶氧在20%以上,诱导时间为160~200h;待发酵结束后,发酵液用于应用测试。
发酵过程曲线如图4所示:每隔8h取样,测定产酶水平,发酵168h后酶活力水平达到最高点。
实施例5:共表达突变酶液在对虾养殖中的应用实验
将实施例4的双酶共表达菌株发酵液与胆汁酸以与胆汁酸(有效成分组成包括猪胆酸、猪去氧胆酸和鹅去氧胆酸,其中猪胆酸和猪去氧胆酸之和的重量百分比为78.0%,鹅去氧胆酸的重量百分比为20.0%,其余为水分和灰分)以7:3的体积比混合均匀(发酵液和胆汁酸的单位均为ml),得到一种共表达突变酶复合制剂。
作为对照,分别将木聚糖酶和甘露聚糖酶依照上述方法转入毕赤酵母X33中,并划线培养发酵,得到的发酵液与胆汁酸以7:3的体积比混合均匀,制取单酶制剂。
选取体重在9.0±0.1g之间的健康的南美白对虾120尾,随机分为4组,每组30尾,每组做3个重复,每重复10尾,饲喂周期为40d。试验设计如下表1所示。养殖温度在20~24℃,每天投喂2次,日投喂量为体重的5%(根据对虾摄食情况调节投喂量)。每天收集残饵2次,时间为投饵后一小时,即8:00和18:00。饲喂5d后,每天下午投饵料之前,开始收集粪便1次。用虹吸方法将残饵和粪便收集于小烧杯中,用适量淡水清洗去盐份,70℃烘干,共收集35d。
表1实验设计
实验结束前24h停止投喂,将对虾从水中捞出,用干滤纸轻轻吸取表面水分,称量体重,计算特定生长率、消化率和饵料系数,试验结果如表2所示。
特定生长率SGR=100*(lnW末-lnW初)/t(养殖天数);
饵料系数=摄食量/增重量=(W1-W2)/W末-W初);
消化率(%)=(摄食量-粪便量)/摄食量=[(W1-W2)-W3]/W1-W2;
注:t表示试验天数,W1、W2、W3分别表示投饵量、残饵量、粪便量。
表2饵料中添加木聚糖酶、甘露聚糖酶对对虾养殖的影响
从表2可以看出,添加木聚糖酶或者甘露聚糖酶的制剂,可以在一定程度上提高对虾的特定生长率,实验组1-3分别提高了13.58%、8.64%、24.69%,消化率方面实验组略有提高,同时降低了降饵料系数。特别是,共表达突变酶复合制剂的各项指标与对照组相比有明显改善。实验结果表明,共表达突变酶复合制剂的添加可以明显提高对虾的特定生长率和消化率,降低饵料系数,从而提高饲料的利用率,降低饲养成本。
以上实施例仅用以说明本发明的技术方案,而非对其进行限制;尽管参照前述实施例对本发明进行了详细的说明,对于本领域的普通技术人员来说,依然可以对前述实施例所记载的技术方案进行修改,或者对其中部分技术特征进行等同替换;而这些修改或替换,并不使相应技术方案的本质脱离本发明所要求保护的技术方案的精神和范围。
Claims (10)
1.一种木聚糖酶突变体xynM,其特征在于,所述木聚糖酶突变体xynM的氨基酸序列如SEQ ID NO:3所示。
2.根据权利要求1所述的木聚糖酶突变体xynM,其特征在于,所述木聚糖酶突变体xynM是由氨基酸序列为SEQ ID NO:1的木聚糖酶xyn的第156位的天冬氨酸变为谷氨酸和第176位的天冬氨酸变为天冬酰胺得到的。
3.权利要求1所述的木聚糖酶突变体xynM的编码基因,其特征在于,所述编码基因的核苷酸序列如SEQ ID NO:4所示。
4.一种含有权利要求3所述的编码基因的共表达突变酶重组菌,其特征在于,所述共表达突变酶重组菌中还同时含有甘露聚糖酶的编码基因。
5.根据权利要求4所述的共表达突变酶重组菌,其特征在于,所述甘露聚糖酶为甘露聚糖酶突变体AnManM,其氨基酸序列如SEQ ID NO:7所示,其编码基因的核苷酸序列如SEQ IDNO:8所示。
6.根据权利要求5所述的共表达突变酶重组菌,其特征在于,所述甘露聚糖酶突变体AnManM是由氨基酸序列为SEQ ID NO:5的甘露聚糖酶AnMan的第76位的缬氨酸变为丙氨酸、第99位的苏氨酸变为谷氨酸和第172位的丝氨酸变为脯氨酸得到的。
7.一种共表达突变酶复合制剂,其特征在于,所述共表达突变酶复合制剂中包含权利要求4所述的共表达突变酶重组菌的发酵液和胆汁酸。
8.根据权利要求7所述的共表达突变酶复合制剂,其特征在于,所述发酵液和胆汁酸的体积比为5~10:3。
9.权利要求7所述的共表达突变酶复合制剂在制备水产动物的饲料添加剂中的应用。
10.根据权利要求9所述的应用,其特征在于,所述共表达突变酶复合制剂的用量为水产动物基础饲料重量的0.1%-0.5%。
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