CN116064479B - 一种含木聚糖酶突变体和胆汁酸的复合制剂及其应用 - Google Patents
一种含木聚糖酶突变体和胆汁酸的复合制剂及其应用 Download PDFInfo
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2477—Hemicellulases not provided in a preceding group
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- C12N9/2482—Endo-1,4-beta-xylanase (3.2.1.8)
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Abstract
本发明公开了一种含木聚糖酶突变体和胆汁酸的复合制剂及其应用。本发明是将来源于瘤胃真菌Neocallimastix patriciarum的木聚糖酶xyn进行突变，筛选获得氨基酸序列如SEQ ID No.3、SEQ ID No.5、或者SEQ ID No.7所示的木聚糖酶突变体xynM1、xynM2和xynM3。和原始基因相比，所述的木聚糖酶突变体经过85℃处理3分钟后的热稳定性由52％分别提高至58％、65％、69％。利用木聚糖酶突变体与胆汁酸、杜仲叶提取物复合制备得到全新的制剂，能够明显提高肉鸭的出栏重并降低料重比，进而提高肉鸭养殖中的饲料转化率和生产性能，有利于其在饲料领域的开发和应用。
Description
技术领域
本发明属于酶工程领域，具体涉及一种含木聚糖酶突变体和胆汁酸的复合制剂及其应用。
背景技术
木聚糖是一种五碳糖，是半纤维素的重要组分，也是自然界中含量仅次于纤维素的多聚糖。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，其在动物消化系统中不能被有效的降解。木聚糖本身难以被单胃动物消化，同时结合大量的水，使采食动物消化道中食糜的体积增大、粘度增加、养分与消化道内源酶的作用降低，从而阻碍营养物质，尤其是脂肪和蛋白质的消化吸收，降低饲料的利用率。在养殖行业中，通过在饲料中添加木聚糖酶降解抗营养因子木聚糖对提高饲料利用率非常经济有效。
木聚糖酶是可将木聚糖降解成低聚糖或木糖的一类酶的总称，主要包括内切β-1,4木聚糖酶、木糖苷酶、阿拉伯糖苷酶等，其中内切β-1,4木聚糖酶在这一类酶中发挥主要作用。目前市场上大多数木聚糖酶适宜温度在40～60℃，pH在5.0-7.0。来源于瘤胃真菌的内切木聚糖酶由于其良好的酶解特性，使其在饲料、造纸等行业具有很大的应用潜力。为了使瘤胃真菌内切木聚糖酶广泛应用到众多工业领域中，提高其酶活力、耐热性、耐酸性等性能，降低生产成本是急需解决的问题。
发明内容
本发明提供了一种含木聚糖酶突变体和胆汁酸的复合制剂及其应用。本发明通过大量筛选得到耐热性提高的木聚糖酶突变体xynM1、xynM2和xynM3，并与胆汁酸、杜仲叶提取物复合成新的制剂，用来提高饲料利用率。
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，所述木聚糖酶突变体的氨基酸序列如SEQ IDNo.3所示、或者如SEQ ID No.5所示、或者如SEQ ID No.7所示。
进一步的：所述木聚糖酶突变体具体为：由氨基酸序列为SEQ ID No.1所示的木聚糖酶的第40位的丙氨酸变为天冬酰胺得到的木聚糖酶突变体xynM1；由氨基酸序列为SEQID No.1所示的木聚糖酶的第40位的丙氨酸变为天冬酰胺和第70位的赖氨酸变为谷氨酸得到的木聚糖酶突变体xynM2；由氨基酸序列为SEQ ID No.1所示的木聚糖酶的第40位的丙氨酸变为天冬酰胺和第191位的甲硫氨酸变为苯丙胺酸得到的木聚糖酶突变体xynM3。
本发明还提供了一种编码基因，所述编码基因为所述的木聚糖酶突变体的编码基因，其核苷酸序列如SEQ ID No.4所示、或者如SEQ ID No.6所示、或者如SEQ ID No.8所示。
本发明还提供了一种重组工程菌，其包含所述的编码基因。
本发明还提供了一种复合制剂，所述复合制剂中包含权利要求1所述的木聚糖酶突变体、胆汁酸和杜仲叶提取物。
进一步的：所述复合制剂中，所述木聚糖酶突变体的重组工程菌发酵液与胆汁酸、杜仲叶提取物的质量比为1:8～12:2～5。
进一步的：所述复合制剂中，所述木聚糖酶突变体的酶活力不少于10万U/g。
本发明还提供了所述的木聚糖酶突变体在酶解玉米皮中应用。
进一步的：所述木聚糖酶突变体的用量为100U/g～500U/g。
本发明还提供了所述的木聚糖酶突变体或者所述的复合制剂在制备提高饲料利用率的饲料添加剂中的应用。
进一步的：所述木聚糖酶突变体或者复合菌剂的用量为饲料重量的0.01％～0.05％。
与现有技术相比，本发明的优点和技术效果是：
1、本发明以瘤胃真菌Neocallimastix patriciarum的木聚糖酶基因为基础进行突变改良，筛选获得了木聚糖酶突变体xynM1、xynM2和xynM3，和原始基因相比，本发明得到的木聚糖酶突变体经过85℃处理3分钟后的热稳定性由52％最高提高69％。
2、本发明利用包含木聚糖酶突变体编码基因的重组工程菌发酵得到的发酵液，与胆汁酸、杜仲叶提取物复合制备得到全新的制剂，能够明显提高肉鸭的出栏重并降低料重比，进而提高肉鸭养殖中的饲料转化率和生产性能，有利于其在饲料领域的开发和应用。
附图说明
图1是木聚糖酶基因的扩增电泳结果图。
图2是木聚糖酶突变体热稳定性比较。
图3是木聚糖酶突变体在15L发酵罐中的发酵曲线。
图4是木聚糖酶突变体酶解玉米皮的应用试验。
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。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。
酶活力测定：参照《GBT 23874-2009饲料添加剂木聚糖酶活力的测定分光光度法》中的测定方法。
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人工合成，得到的氨基酸序列如SEQ ID No.1所示，对应核苷酸序列如SEQ ID No.2所示。根据序列设计引物如下，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，以及上述设计的引物序列进行随机突变，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个表达原始木聚糖酶的单菌落xyn0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活性以及其耐热性。将耐热性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xynM1，其氨基酸序列如SEQ ID No.3所示，其编码的核苷酸序列如SEQ ID No.4所示。
实施例2：以木聚糖酶突变体xynM1基因为模板进行随机突变
以实施例1中筛选到的突变体xynM1为模版，使用与实施例1相同的随机突变方法，进行第二轮突变和筛选，筛选时以xynM1为对照，检测木聚糖酶的活性，将耐热性高于xynM1的突变体基因进行测序。
经过大量筛选测定后获得耐热性较好的两株突变体xynM2和xynM3：
xynM2突变方式为A40N/K70E，其氨基酸序列如SEQ ID No.5所示，核苷酸序列如SEQ ID No.6所示；
xynM3的突变方式为A40N/M191F，其氨基酸序列如SEQ ID No.7所示，核苷酸序列如SEQ ID No.8所示。
实施例3：木聚糖酶突变体转化毕赤酵母GS115
将上述的木聚糖酶突变体基因用EcoR I和Not I双酶切位点连接到pPIC9K质粒上并转化至大肠杆菌DH5α中，获得表达载体，测序验证。将表达载体用Sal I酶切线性化后电转入毕赤酵母GS115中，MD平板筛选得到转化子并转接到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，以及经过85℃处理3分钟后的热稳定性。
结果如图2所示，木聚糖酶突变体xynM1、xynM2、xynM3在85℃处理后的酶活力由对照的52％分别提高至58％、65％、69％。
实施例4：木聚糖酶突变体xynM3在15L发酵罐中发酵和制备
将表达木聚糖酶突变体xynM3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300mLBMGY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，pH 4.8、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，发酵液通过板框过滤机处理后经喷塔喷干成粉制剂，用于应用测试。
发酵曲线如图3所示：每隔8h取样，测定产酶水平，发酵162h后酶活力水平达到最高点。
实施例5：木聚糖酶突变体酶解玉米皮的应用试验
以2％的玉米皮为底物，用缓冲液配制成悬浮液。对照组：不添加酶制剂组；实验组分为4组：即xyn0处理组、xynM1处理组、xynM2处理组和xynM3处理组，每组分别添加200U/g木聚糖酶xyn0、xynM1、xynM2和xynM3；每组3个重复。
模拟体外酶解玉米皮过程包括以下步骤：
(1)用缓冲液调节pH至5.2，40℃，120rpm恒温振荡消化1h；(2)用缓冲液调节pH至2.5，40℃，120rpm恒温振荡消化1h；(3)用缓冲液调节pH至6.8，40℃，120rpm恒温振荡消化4h。消化完成之后，测定消化前后食糜干重和还原糖含量，并计算干物质消失率、还原糖增长率。
结果如图4所示，表明xyn0以及突变体xynM1、xynM2和xynM3消化后的玉米皮干物质消失率分别为20.7％、21.2％、19.3％、20.5％；而还原糖增长率分别25.8％、26.1％、25.9％、26.0％。这说明突变后的木聚糖酶xynM1、xynM2和xynM3在模拟消化道环境中降解玉米皮原料效率和xyn0相当，在提高木聚糖酶耐热性的基础上，没有影响其对玉米皮原料的酶解效率。
实施例6：木聚糖酶突变体制剂在肉鸭养殖中的应用实验
将实施例4制备的木聚糖酶突变体发酵液与胆汁酸、杜仲叶提取物以1:10:3的体积比混合均匀，获得复合制剂。
其中，木聚糖酶突变体的酶活力为10万U/g。
胆汁酸的有效成分组成包括猪胆酸、猪去氧胆酸和鹅去氧胆酸，所述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(重量百分比总和以100％计算)。
杜仲叶提取物的制备方法为：取杜仲叶用水提取，提取温度55-65℃，提取时间2-4h，然后进行过滤，取滤液在60℃下真空浓缩至比重1.2，经喷雾干燥塔喷干制得杜仲叶提取物。
选择体重接近、健康无病的雏鸭，分为4组，每组500只，每组设置2个重复，每个重复250只鸭，饲养期41天。饲养41天出栏后，检测各组的生长指标。其中，含木聚糖酶突变体xynM1、xynM2和xynM3的制剂试验设计如表1所示。
表1试验分组设计
试验统计结果如表2所示，试验组和对照组在成活率方面差别不大，但添加复合制剂后，肉鸭的出栏重明显提高，并且料重比相比于对照组也明显降低。由此说明，添加了含木聚糖酶突变体的复合制剂后，肉鸭在出栏重、料重比方面都优于对照组，复合制剂添加能够提高肉鸭养殖中的饲料转化率，提高生产性能。
表2不同分组的肉鸭生产指标对比
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 (9)
1.一种木聚糖酶突变体，其特征在于，所述木聚糖酶突变体的氨基酸序列如SEQ IDNo.3所示、或者如SEQ ID No.5所示、或者如SEQ ID No.7所示。
2.一种编码基因，其特征在于，所述编码基因为权利要求1所述的木聚糖酶突变体的编码基因，其核苷酸序列如SEQ ID No.4所示、或者如SEQ ID No.6所示、或者如SEQ ID No.8所示。
3.一种重组工程菌，其特征在于，其包含权利要求2所述的编码基因。
4.一种复合制剂，其特征在于，所述复合制剂中包含权利要求3所述的重组工程菌的发酵液、胆汁酸和杜仲叶提取物。
5.根据权利要求4所述的复合制剂，其特征在于，所述复合制剂中所述重组工程菌的发酵液与胆汁酸、杜仲叶提取物的质量比为1:8~12:2~5。
6.权利要求1所述的木聚糖酶突变体在酶解玉米皮中应用。
7.根据权利要求6所述的木聚糖酶突变体在酶解玉米皮中应用，其特征在于，所述木聚糖酶突变体的用量为100U/g~500U/g。
8.权利要求1所述的木聚糖酶突变体或者权利要求4所述的复合制剂在制备提高饲料利用率的饲料添加剂中的应用。
9.根据权利要求8所述的应用，其特征在于，所述木聚糖酶突变体或者复合制剂的用量为饲料重量的0.01%~0.05%。
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| JP2001231585A (ja) * | 1994-12-21 | 2001-08-28 | Oji Paper Co Ltd | 耐熱性キシラナーゼ |
| CN1177639A (zh) * | 1996-09-09 | 1998-04-01 | 加拿大国立研究院 | 提高木聚糖酶嗜热性、嗜碱性和热稳定性的修饰 |
| CN106676086A (zh) * | 2015-11-05 | 2017-05-17 | 深圳市绿微康生物工程有限公司 | 一种热稳定性改良的木聚糖酶xyn-LVK及其基因 |
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