CN116103267B - 一种中温淀粉酶突变体及其应用 - Google Patents
一种中温淀粉酶突变体及其应用 Download PDFInfo
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Abstract
本发明属于基因工程与蛋白质改造技术领域,具体提供了一种高比活的中温淀粉酶突变体及其应用。本发明以野生型中温淀粉酶ZD2为基础,提供了包含R104Q、E185G、G308V、Y347R中至少一个突变位点的突变体。与野生型相比,本发明提供的淀粉酶单点突变体的比活力普遍提高了41.0%‑230.9%,有利于降低该酶的生产成本,促进其在工业领域中的广泛应用。
Description
技术领域
本发明涉及基因工程和蛋白质改造技术领域,具体涉及一种中温淀粉酶突变体及其应用。
背景技术
中温淀粉酶是一种α-淀粉酶,酶的分类名称是1,4-α-D型葡萄糖水解酶。 中温淀粉酶可以为液体或固体,可以水解直链淀粉和支链淀粉中1,4-α-糖苷键的内切型淀粉酶,迅速降低糊化淀粉的粘度,其分解产品是不同链长的糊精以及低聚糖。α-淀粉酶是动物饲料中常用的一种淀粉酶,它以随机方式水解淀粉分子中的α-1,4糖苷键,淀粉酶水解效率较高,α-淀粉酶虽然对淀粉分子链内的α-1,4糖苷键具有良好的水解作用却不能水解支链淀粉分支点处的a-1,6糖苷键和紧靠α-1,6糖苷键的α-1,4糖苷键,但能跨越α-1,6糖苷键而继续切开支链内部的α-1,4糖苷键,因此,α-淀粉酶作用于直链淀粉时,其终产物是麦芽糖和萄糖,作用于支链淀粉时其终产物除麦芽糖和葡萄糖外还有α-极限糊精。这种传统α-淀粉酶可以70-90℃(158-194℉)的较高温度下使用。
淀粉酶可高效地水解淀粉,在饲料中添加淀粉酶可以补充内源淀粉酶的不足,促进禽畜对淀粉的消化吸收和能量利用,淀粉酶在猪鸡等单胃动物饲料和反刍动物饲料中的应用中都具有非常重要的意义和价值。据报道,在肉鸡日粮中添加α-淀粉酶,日增重和饲料利用率均显著提高,可见淀粉酶在饲料配方中重要性是很高的。目前,饲料中使用的α-淀粉酶有细菌α-淀粉酶和真菌α-淀粉酶,其酶活力和酶的热稳定性等不能满足饲料工业的应用需要,国内外学者主要是通过以下两种途径以求获得更适用于饲料工业的α-淀粉酶:(1)寻找新的酶活高、热稳定性好的α-淀粉酶;(2)对现有的α-淀粉酶进行有关提高酶活和酶分子耐热性的分子改造。
通过自然筛选和重组表达等技术得到的α-淀粉酶,存在获得难度大、酶活力低等问题。国内外学者利用多种分子改良的方法对α-淀粉酶进行理性设计,这些研究结果表明通过对蛋白质进行理性设计可以在不影响其最适反应条件的基础上改善其稳定性,为蛋白质的理性设计的发展提供借鉴,同时也为中温淀粉酶的工业化应用奠定了基础。
发明内容
本发明为解决现有技术问题,提供了一种中温淀粉酶突变体。所述突变体的比活力比野生型得到显著提高,有利于其在工业领域的广泛应用。
为了实现上述发明目的,本发明提供以下技术方案:
本发明涉及一种淀粉酶突变体,其包含与SEQ ID NO:2具有至少90%同一性的氨基酸序列,且与SEQ ID NO:2相比在选自下组中的至少一个位置上包含氨基酸的取代:104,185,308,347。
在本发明的一些实施例中,所述突变体的氨基酸序列与SEQ ID NO:2相比具有至少91%,92%,93%,94%,95%,96%,97%,98%,或至少99%的同一性。
在一些更具体的实施例中,所述突变体的氨基酸序列与SEQ ID NO:2相比具有至少99.1%,99.2%,99.3%,99.4%,99.5%,99.6%,99.7%,99.8%,或至少99.9%的同一性。
在本发明的一些实施例中,所述突变体包含下组中至少一个氨基酸的取代:R104Q、E185G、G308V、Y347R。
本发明还涉及编码上述淀粉酶突变体的DNA分子。
本发明还涉及包含上述DNA分子的重组表达载体。
本发明还涉及一种宿主细胞,包含上述重组表达载体。
将上述的质粒转入宿主细胞中,重组表达的淀粉酶突变体的比活力得到显著提升。
在本发明的一些实施例中,宿主细胞为毕赤酵母(Pichia pastoris)。
本发明还提供了上述淀粉酶突变体在饲料领域中的应用。
本发明以野生型中温淀粉酶ZD2为基础,提供了包含R104Q、E185G、G308V、Y347R中至少一个突变位点的突变体。与野生型相比,本发明提供的淀粉酶突变体的比活力普遍提高了41.0%-230.9%;其中,含E185G单点突变的突变体ZD2-2,其比活力最高,达3397.5 U/mg,取得了意料不到的技术效果。
综上,本发明提供的中温淀粉酶突变体的比活力得到显著提高,从而有利于降低该酶的生产成本,促进其在工业领域中的广泛应用。
具体实施方式
本发明公开了一种比活力提高的淀粉酶突变体、其制备方法及应用、编码该淀粉酶突变体的DNA分子、载体、宿主细胞,本领域技术人员可以借鉴本文内容,适当改进工艺参数实现。本发明的方法及应用已经通过较佳实施例进行了描述,相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合,来实现和应用本发明技术。
本发明用到了遗传工程和分子生物学领域使用的常规技术和方法,例如MOLECΜLAR CLONING:A LABORATORY MANUAL,3nd Ed. (Sambrook, 2001)和CURRENT PROTOCOLSIN MOLECΜLAR BIOLOGY (Ausubel, 2003)中所记载的方法。这些一般性参考文献提供了本领域技术人员已知的定义和方法。但是,本领域的技术人员可以在本发明所记载的技术方案的基础上,采用本领域其它常规的方法、实验方案和试剂,而不限于本发明具体实施例的限定。例如,本发明可选用如下实验材料和试剂:
菌株与载体:大肠杆菌DH5α本公司保藏,毕赤酵母GS115、载体pPIC9k、pPICZA、Amp、G418、Zeocin购自Invitrogen公司。
培养基:
LB培养基(大肠杆菌培养基):0.5%酵母提取物,1%蛋白胨,1%NaCl,pH7.0;
LB+Amp培养基:LB培养基加100μg/ml氨苄青霉素;
YPD培养基(酵母培养基):1%酵母提取物,2%蛋白胨,2%葡萄糖;
YPD+Zeocin培养基:YPD培养基加100μg/ml Zeocin;
MD培养基(酵母筛选培养基):1.34% YNB,4×10-5生物素,1%甘油、2%琼脂糖;
BMGY培养基:2%蛋白胨,1%酵母提取物,100 mM磷酸钾缓冲液(pH6.0),1.34% YNB,4×10-5 %生物素,1%甘油;
BMMY培养基:2%蛋白胨,1%酵母提取物,100 mM磷酸钾缓冲液(pH6.0),1.34% YNB,4×10-5 %生物素,0.5%甲醇;
实验试剂:DNA聚合酶购自Takara公司,T4连接酶、限制性内切酶购自Fermentas公司,质粒提取试剂盒和胶纯化回收试剂盒购自Omega公司,GeneMorph II随机诱变试剂盒购自北京博迈斯生物科技有限公司。
本发明实施例中所述淀粉酶的酶活检测方法参照GB/T24401-2009。
下面结合具体实施方式,对本发明做进一步阐述。
实施例1 中温淀粉酶ZD2的基因合成
以中温淀粉酶基因(GenBank号为AAX84031.1)的氨基酸序列为基础,分析该中温淀粉酶的氨基酸序列,去除其自身的信号肽,再根据毕赤酵母的密码子偏好性,对其进行密码子优化,由华大基因公司进行全基因合成。申请人将合成的中温淀粉酶基因命名为ZD2,其核苷酸序列为SEQ ID NO:1,编码的氨基酸序列为SEQ ID NO:2。
实施例2 高比活中温淀粉酶突变体的筛选
为了提高中温淀粉酶ZD2的比活力,申请人通过定向进化技术进行了大量突变的筛选。
设计PCR引物1(F)和引物1(R):
引物1(F):GCGCGAATTCGAAACTGTTCATAAAGGTAAATCTC(下划线为限制性内切酶EcoRI识别位点);
引物1(R):TAAAGCGGCCGCTTATTTAATCAAAACAGAAGTATTA(下划线为限制性内切酶Not I识别位点)。
以中温淀粉酶ZD2的基因片段为模板,利用上述引物用GeneMorph II随机突变PCR试剂盒(博迈斯)进行PCR扩增;胶回收PCR产物,EcoRI、Not I进行酶切处理后与经同样酶切后的pET21a载体连接,转化至大肠杆菌BL21(DE3)中,涂布于LB+Amp平板,37℃倒置培养;待转化子出现后,用牙签逐个挑至96孔板,每个孔中加入150 ul含有0.1mM IPTG的LB+Amp培养基,37℃、220 rpm培养6 h左右,离心弃上清,菌体用缓冲液重悬,反复冻融破壁,获得含有中温淀粉酶的大肠杆菌细胞裂解液;再将细胞裂解液离心,取上清液,分别进行淀粉酶活力测定和蛋白含量测定,计算不同突变子的比活力水平。
实验结果表明,有些突变对中温淀粉酶ZD2的比活力没有影响,有些突变甚至使其比活力变得更低了。最终,申请人筛选到能显著提高中温淀粉酶ZD2比活力的突变位点:R104Q,E185G,G308V和Y347R。
将含R104Q单点突变的淀粉酶突变体命名为ZD2-1,其氨基酸序列为SEQ ID NO:3;
将含E185G单点突变的淀粉酶突变体命名为ZD2-2,其氨基酸序列为SEQ ID NO:4;
将含G308V单点突变的淀粉酶突变体命名为ZD2-3,其氨基酸序列为SEQ ID NO:5;
将含Y347R单点突变的淀粉酶突变体命名为ZD2-4,其氨基酸序列为SEQ ID NO:6。
用引物1(F)和引物1(R)对上述各突变体进行PCR扩增,PCR条件为:94℃变性5min;然后94℃变性30s,56℃复性30s,72℃延伸1min30s,35个循环后,72℃保温10min。各突变体基因长度与ZD2基因相同,全长1488bp。
实施例3、表达重组中温淀粉酶基因的毕赤酵母工程菌的构建
1、重组质粒的构建
将中温淀粉酶基因ZD2以及四个突变体基因(ZD2-1、ZD2-2、ZD2-3、ZD2-4)分别用限制性内切酶EcoR I和Not I进行双酶切,将表达载体pPIC9K使用同样的限制性内切酶进行双酶切,100 μl酶切体系如下:基因片段/载体40 μl、10×H buffer 10 μl、10×BSA 10μl、EcoR I 5 μl、Not I 5 μl、ddH2O 30 μl。37℃酶切4 h后,琼脂糖凝胶电泳回收。
将双酶切后的中温淀粉酶基因片段分别与表达载体pPIC9K连接,连接体系如下:表达载体pPIC9K双酶切产物5 μl、中温淀粉酶基因双酶切产物3 μl、10×T4 ligasebuffer 1 μl、T4 ligase 1 μl。22 ℃连接过夜,转化到大肠杆菌DH5α,挑取转化子测序验证。将测序验证正确的转化子转接到LB+Amp液体培养基中,37℃过夜培养,提取质粒,即为重组酵母表达质粒。
2、转化与筛选
将上述构建的重组酵母表达质粒分别用Sal I进行线性化,线性化产物用柱纯化试剂盒纯化后,通过电穿孔法转化毕赤酵母GS115,涂布MD平板。在MD平板上生长出的菌落即为重组表达中温淀粉酶ZD2或其突变体的毕赤酵母工程菌株,然后涂布含不同浓度遗传霉素G418的YPD平板上筛选多拷贝的转化子。
3、摇瓶发酵验证
挑取单个多拷贝转化子分别接入BMGY培养基中,30℃、220rpm振荡培养24小时后,再转入BMMY培养基中,30℃、220rpm振荡培养,每24h添加0.5%的甲醇。诱导表达4d后,离心去除菌体,将上清液分别进行中温淀粉酶活力测定和蛋白含量测定,计算比活力,具体结果见表1。
表1 中温淀粉酶突变体的比活力
中温淀粉酶 | 比活力(U/mg) |
野生型ZD2 | 1026.7 |
突变体ZD2-1 | 1447.5 |
突变体ZD2-2 | 3397.5 |
突变体ZD2-3 | 2925.9 |
突变体ZD2-4 | 2426.2 |
从表1的结果可知,在摇瓶水平下,表达野生型中温淀粉酶ZD2的转化子中发酵上清液的比活力最高达到1026.7U/mg;而表达淀粉酶单点突变体的转化子中发酵上清液的比活力最高达到1447.5-3397.5U/mg,比野生型提高了41.0%-230.9%;其中,含E185G单点突变的突变体ZD2-2,其比活力最高,达3397.5 U/mg,取得了意料不到的技术效果。
本发明提供的R104Q,E185G,G308V和Y347R四个突变位点能显著提高中温淀粉酶ZD2的比活力,从而有利于降低该酶的生产成本,促进其在工业领域的广泛应用。
(一)淀粉酶酶活检测方法
参考GB/T24401-2009方法。
(二)考马斯亮蓝法检测蛋白含量
1、试剂
(1)考马斯亮蓝G-250染色液:取考马斯亮蓝G-250 100mg溶于50ml 95%乙醇中,加100ml 85%磷酸,加水稀释至1升,常温可使用1个月;
(2)标准蛋白溶液:用牛血清蛋白,预先经微量凯氏定氮法测定蛋白质含量,根据其纯度,配制成1 mg/ml的蛋白质标准溶液;
(3)标准原液的配制:在分析天平上精确称取0.05g结晶牛血清蛋白,于小烧杯中,加入少量蒸馏水溶解后转入50ml容量瓶中,烧杯内残液用少量蒸馏水冲洗数次,冲洗液一并倒入容量瓶中,最后用蒸馏水定容至刻度。配制成标准原液,其中牛血清蛋白浓度为1000μg/ml。
2、标准曲线的绘制。
(1)分别取6支试管,编号,按下表加入试剂,混匀。
管号 | 1 | 2 | 3 | 4 | 5 | 6 |
样品(ml) | 0 | 0.1 | 0.2 | 0.3 | 0.4 | 0.5 |
水(ml) | 2.0 | 1.9 | 1.8 | 1.7 | 1.6 | 1.5 |
蛋白质含量(mg/ml) | 0 | 0.05 | 0.1 | 0.15 | 0.2 | 0.25 |
准确吸取2.5ml 考马斯亮蓝溶液于6支干净试管中,准确吸取上述各管溶液0.1ml,对应放于各自编号的试管中,涡旋混匀,室温放置5min后,以1号试管调零,测定在595nm处比色,记录吸光值。
(2)绘制标准曲线:记录1-6管所读吸光值,以蛋白质含量(μg)为横坐标,以吸光度为纵坐标,绘出标准曲线。注意,由于考马斯亮蓝染色能力强,比色杯一定要洗干净。不可用石英杯测定。
3、样品的测定
样品的准备:
(1)液体样品:将待测样品稀释至蛋白含量0.1-0.3mg/ml,控制除去空白后的吸光值(减去空白以后)在0.2-0.4之间;
(2)固体样品:准确称取1.0000g样品于100ml三角瓶中,用移液枪加入20ml去离子水,磁力搅拌10min,4000rpm离心10min,取上清进一步稀释测定蛋白含量,稀释方法参见液体样品。
样品检测:
取干净试管,加入到含有2.5ml考马斯亮蓝溶液,然后加入待测样品,涡旋震荡摇匀,室温放置5min,以标准曲线空白作对照,用1cm光径的微量比色杯在595nm测定吸光度,根据标曲求得蛋白含量。
4、蛋白含量计算
蛋白含量=X×稀释倍数×标样折算系数。
X:根据标曲求出的蛋白含量(mg/ml);
标样折算值:标样为47mg/ml,根据实测值折算一个系数。
(三)比活力的计算
“比活力 (Specific Activity)”是指:单位重量的蛋白质中所具有酶的活力单位数,一般用U/mg蛋白质来表示。一般来说,酶的比活力越高,酶越纯。
比活力计算公式:比活力(U/mg)=酶活(U/mL)/ 蛋白含量(mg/mL)。
Claims (6)
1.一种淀粉酶突变体,其特征在于,所述突变体是氨基酸序列为SEQ ID NO:2的淀粉酶的第185位氨基酸由Glu变为Gly。
2.编码权利要求1所述的淀粉酶突变体的DNA分子。
3.包含权利要求2所述的DNA分子的重组表达质粒。
4.一种宿主细胞,其特征在于,所述宿主细胞包含权利要求3所述的重组表达质粒。
5.如权利要求4所述的宿主细胞,其特征在于,所述宿主细胞为毕赤酵母(Pichia pastoris)。
6.权利要求4或5所述的宿主细胞在淀粉酶生产中的应用。
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