CN117925565A - 高比活植酸酶突变体及其应用 - Google Patents
高比活植酸酶突变体及其应用 Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
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- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
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- A—HUMAN NECESSITIES
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
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- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
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- C12Y—ENZYMES
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
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Abstract
本发明属于基因工程和蛋白质改造技术领域,具提供了一种高比活的植酸酶突变体及其应用。所述植酸酶突变体包含L69F、N113E、L258V和Q350A四个突变位点,其摇瓶发酵上清液的比活力比野生型提高了133.9%,效果显著,有利于大幅降低该酶的生产成本,可广泛应用于饲料加工技术领域。
Description
技术领域
本发明涉及基因工程和蛋白质工程改造技术领域,具体涉及一种高比活植酸酶突变体及其应用。
背景技术
植酸的化学名称是肌醇六磷酸酯,它基本不能以游离状态存在。植酸或植酸盐主要存于植物籽实中,是磷在植物中的主要储藏形式。植酸一般与钙、镁、钾、钠、铁等离子结合成复合物,不溶于水,难以被单胃动物消化利用,直接排出体外,对环境形成较大污染,还影响机体对钙、锌、铁等矿物元素的吸收,并且可螯合蛋白分子,严重降低了蛋白质的生物效价和利用率,属于一种广谱性的抗营养因子。大米、豆粕、麸皮、棉籽粕等饲料中所含2/3左右的磷以植酸磷形式储藏。单胃动物消化道内难以分泌植酸酶,难以对植酸进行分解,所含磷也难以利用,与其结合的钙、镁、钾、钠、蛋白质与氨基酸等营养成分难以消化吸收,既浪费资源,又对环境形成污染。家禽养殖中急待解决饲料中植酸抗营养效果及粪污中磷含量对环境污染的问题。
植酸酶是一种磷酸单酯水解酶,属于酸性磷酸酶,可以水解植酸释放出无机磷。国内外许多实验均证明,在饲料中拌入植酸酶,能够有效降解饲料中的植酸,改变无机磷形式,提升饲料中的植酸磷消化率,减少粪污中磷含量的排放,降低对环境的污染,同时也解除饲料中植酸的抗营养效果。伴随着人们对环保意识的提高和养殖业的绿色发展,植酸酶的应用将会愈加广泛。
发明内容
本发明为解决现有技术问题,提供了一种高比活植酸酶突变体。所述突变体的比活力得到显著提升,能大幅降低该酶的生产成本,可广泛应用于饲料加工技术领域。
本发明一方面涉及一种植酸酶突变体,其包含与SEQ ID NO:2具有至少95%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:69,113,258,350。
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96%,97%,98%,或至少99%的同一性。
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:L69F,N113E,L258V,Q350A。
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或取代组合:L69F/N113E,L69F/L258V,L69F/Q350A,N113E/L258V,N113E/Q350A,L258V/Q350A,L69F/N113E/L258V,L69F/N113E/Q350A,N113E/L258V/Q350A,N113E/L258V/Q350A,L69F/N113E/L258V/Q350A。
本发明还涉及编码上述植酸酶突变体的DNA分子。
本发明还涉及包含上述DNA分子的重组表达质粒。
本发明还涉及一种宿主细胞,包含上述重组表达质粒。
将上述的质粒转入宿主细胞中,重组表达的植酸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植酸酶突变体在饲料领域中的应用。
本发明提供的L69F、N113E、L258V和Q350A四个突变位点能显著提高植酸酶A3的比活力。与植酸酶A3相比,突变体A3-2发酵上清液的比活力提高了133.9%,达到435U/mg,取得了意料不到的技术效果。从而有利于降低植酸酶的生产成本,促进其在饲料领域的广泛应用。
具体实施方式
本发明公开了一种高比活植酸酶突变体、其制备方法及应用、编码该植酸酶突变体的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。
酶与试剂盒:DNA聚合酶购买自Takara公司,T4连接酶、限制性内切酶购自Fermentas公司,质粒提取试剂盒及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。
培养基配方:
大肠杆菌培养基(LB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;
酵母培养基(YPD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;
酵母筛选培养基(MD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。
实施例1 植酸酶基因A3的密码子优化和克隆
申请人将来源于Cronobacter sakazakii的植酸酶基因命名为A3,其基因序列为SEQ ID NO:1,编码的氨基酸序列为SEQ ID NO:2。
以A3基因的氨基酸序列为基础,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。优化后的新基因由华大基因公司全基因合成。
采用PCR反应克隆植酸酶基因A3片段,引物和反应条件如下:
引物(F):GCGCGAATTCTTGCACGCCCAGGGTGCTGATAAGA;
引物(R):TAAAGCGGCCGCTTATCTATTGATCTCAGCCATAACA。
PCR条件为:94℃变性5min;然后94℃变性30s,56℃复性30s,72℃延伸1min 30s,35个循环后,72℃保温10min。A3基因全长1254bp。
实施例2 植酸酶A3突变体基因的扩增及合成
为了进一步提高植酸酶A3的比活力,通过定向进化技术对该酶的基因进行了大量突变的筛选;以植酸酶基因A3为模板,利用引物(F)和引物(R)用GeneMorph II随机突变PCR试剂盒(Stratagene)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,获得含有植酸酶的大肠杆菌细胞裂解液。再离心去除菌体,将上清液进行植酸酶活和蛋白含量测定,计算比活力。
实验结果表明,有些突变对植酸酶A3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,申请人得到比活力显著提高的突变位点:L69F,N113E,L258V,Q350A。
将含L69F、N113E、L258V和Q350A四点突变的植酸酶突变体基因命名为A3-2,其基因序列为SEQ ID NO:3,编码的氨基酸序列为SEQ ID NO:4。
用引物(F)和引物(R)对A3-2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。A3-2基因长度与A3基因相同,全长1254bp。
实施例3、表达重组植酸酶基因的毕赤酵母工程菌的构建
1、重组质粒的构建
将密码子优化后克隆得到的植酸酶基因A3和突变体基因A3-2,用限制性内切酶EcoR I和Not I进行双酶切,100 μl酶切体系如下:植酸酶基因的 PCR产物40 μl、10×Hbuffer 10 μl、10×BSA 10 μl、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。
将表达载体pPIC9K先用限制性内切酶EcoR I进行单酶切,100 μl酶切体系如下:表达载体pPIC9K 20 μl、10×H buffer 10 μl、EcoR I 5 μl、ddH2O 65 μl。37℃酶切4 h后,琼脂糖凝胶电泳回收。将回收片段再用限制性内切酶Not I进行单酶切,100 μl酶切体系如下:pPIC9K回收片段 20 μl、10×H buffer 10 μl、10×BSA 10 μl、10×Triton 10 μl、Not I 5 μl、ddH2O 45 μl。37℃酶切4 h后,琼脂糖凝胶电泳回收。
将经EcoR I和Not I双酶切的植酸酶基因片段分别与表达载体pPIC9K连接,构建表达载体。连接体系如下:表达载体pPIC9K双酶切产物5 μl、植酸酶基因双酶切产物3 μl、10×T4 ligase buffer 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。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,提质粒,即为重组酵母表达质粒。
2、转化与筛选
将重组酵母表达质粒用Sal I进行线性化,线性化产物用柱纯化试剂盒纯化后,通过电穿孔法分别转化毕赤酵母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
分别挑取转化子接入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24小时添加0.5%的甲醇。诱导表达4天后,离心去除菌体,将上清液进行植酸酶活和蛋白含量测定,计算比活力。
结果显示,表达植酸酶A3的转化子中发酵上清液的比活力最高达到186U/mg;而表达植酸酶突变体A3-2的转化子中发酵上清液的比活力最高达到435U/mg,比野生型提高了133.9%,取得了意料不到的技术效果。
本发明提供的L69F、N113E、L258V和Q350A四个突变位点能显著提高植酸酶A3的比活力,从而有利于降低植酸酶的生产成本,促进其在饲料领域的广泛应用。
(一)植酸酶酶活测定
(1)酶活单位的定义:
在温度为37℃、pH为5.5的条件下,每分钟从浓度为5.0mmol/L植酸钠中释放1μmol无机磷,即为一个植酸酶活性单位,以U表示。
(2)植酸酶酶活测定方法
取甲、乙两支25mL比色管,各加入1.8mL乙酸缓冲液(pH5.0)、0.2mL样品反应液,混匀,37℃预热5min。在甲管中加入4mL底物溶液,乙管中加入4mL终止液,混匀,37℃反应30min,反应结束后甲管中加入4mL终止液,乙管中加入4mL底物溶液,混匀。静置10min,分别在415nm波长处测定吸光值。每种样品作3个平行,取吸光值的平均值,通过标准曲线用回归直线方程计算植酸酶活性。
酶活X=F×C/(m×30)。
其中:X——酶活力单位,U/g(mL);
F——试样溶液反应前的总稀释倍数;
C——根据实际样液的吸光值由直线回归方程计算出的酶活性,U;
m——试样质量或体积,g/mL;
30——反应时间。
(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。
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 (10)
1.一种植酸酶突变体,其特征在于,所述突变体包含与SEQ ID NO:2具有至少95%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:69,113,258,350。
2.如权利要求1所述的突变体,其特征在于,所述突变体的氨基酸序列与SEQ ID NO:2相比具有至少96%,97%,98%,或至少99%的同一性。
3.如权利要求2所述的突变体,其特征在于,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。
4.如权利要求1所述的突变体,其特征在于,所述突变体包含下组中至少一个氨基酸的取代:L69F,N113E,L258V,Q350A。
5.如权利要求4所述的突变体,其特征在于,所述突变体包含下组中至少一个氨基酸的取代或取代组合:L69F,N113E,L258V,Q350A ,L69F/N113E,L69F/L258V,L69F/Q350A,N113E/L258V,N113E/Q350A,L258V/Q350A,L69F/N113E/L258V,L69F/N113E/Q350A,N113E/L258V/Q350A,N113E/L258V/Q350A,L69F/N113E/L258V/Q350A。
6.编码权利要求5所述的植酸酶突变体的DNA分子。
7.包含权利要求6所述的DNA分子的重组表达质粒。
8.一种宿主细胞,其特征在于,所述宿主细胞包含权利要求7所述的重组表达质粒。
9.如权利要求8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)。
10.权利要求1-5任一所述的植酸酶突变体在饲料中的应用。
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