CN112342209B - 一种氨基甲酸乙酯水解酶突变体及其应用 - Google Patents
一种氨基甲酸乙酯水解酶突变体及其应用 Download PDFInfo
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- CN112342209B CN112342209B CN202011348306.8A CN202011348306A CN112342209B CN 112342209 B CN112342209 B CN 112342209B CN 202011348306 A CN202011348306 A CN 202011348306A CN 112342209 B CN112342209 B CN 112342209B
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- ethyl carbamate
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 65
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Abstract
本发明公开了一种氨基甲酸乙酯水解酶突变体及其应用。所述氨基甲酸乙酯水解酶突变体由根癌农杆菌(Agrobacterium tumefaciens)d3的酰胺酶基因经过突变得到，野生型酰胺酶的氨基酸序列如SEQ ID No.1所示，所述氨基甲酸乙酯水解酶突变体由野生型酰胺酶经过R94P、I97L、S177C或G195A中的至少一个位点突变所得。发明筛选得到来自Agrobacterium tumefaciens d3的酰胺酶，在乙醇浓度0％‑20％之间，剩余酶活高于90％，高于25％之后酶活下降迅速，该酶对低浓度乙醇表现出极好的耐受性。通过半理性改造，构建了一个21个点突变的突变体库，并从中筛选得到4个酶活有提高的突变位点(R94P、I97L、S177C、G195A)，组合突变株I97L/G195A酶活提高了5.2倍，由原来的395U/L提高到2442U/L。
Description
技术领域
本发明涉及生物技术领域，特别是涉及一种氨基甲酸乙酯水解酶突变体及其应用。
背景技术
氨基甲酸乙酯(Ethyl carbamate；C2H5OCONH2)，简称EC。氨基甲酸乙酯是化工生产的重要原料，具有广泛的用途，其具有麻醉神经的作用，在医疗上用作镇静及缓和的催眠药。氨基甲酸乙酯也广泛存在于传统的发酵食品泡菜、酱油、醋和酒精饮料清酒、葡萄酒、黄酒中。1943年EC的潜在致癌作用被Nettleship等人通过研究发现，1974年国际癌症研究机构(IARC)将EC划分为2B级致癌物，2007年IARC正式将EC的致癌级别由2B类提高为2A类。实验证明氨基甲酸乙酯对所有实验动物具有致癌性，对人有潜致癌性。为了解除氨基甲酸乙酯对食品安全的危害，目前主要是通过原料修改，发酵工艺优化，发酵酒中EC前体的运输调节以及添加酸性脲酶来降低EC含量。但是，由于EC有许多前体，例如尿素，瓜氨酸，精氨酸，氢氰酸，碳酸二乙酯2-4，上述方法不能完全避免EC的形成，且对已经形成的EC没有降解作用，因此使用氨基甲酸乙酯水解酶是降解EC的最直接，最有效的方法。
氨基甲酸乙酯水解酶(EC 3.5.1.75)能将EC催化为乙醇，二氧化碳和氨(图1)，是降解EC最直接有效的办法。目前筛选得到的氨基甲酸乙酯水解酶对乙醇的耐受性差，对酸的耐受性差，对EC的亲和力低。其中现筛选得到的耐乙醇能力高的菌株分别是Micrococcussp.在20％的乙醇浓度下，保留82％酶活(Mohapatra,B.R.；Bapuji,M.,Characterizationof urethanase from Micrococcus species associated with the marine sponge(Spirastrella species).Lett.Appl.Microbiol.1997,25(6),393-396.)和Candidaparapsilosis在20％的乙醇浓度下，保留73％酶活(Masaki,K.；Mizukure,T.；Kakizono,D.；Fujihara,K.；Fujii,T.；Mukai,N.,New urethanase from the yeast Candidaparapsilosis.Journal of bioscience and bioengineering 2020.)。由于氨基甲酸乙酯水解酶主要用于发酵酒中，但是传统的发酵酒酒精浓度在20％左右，呈酸性；因此目前筛选得到的氨基甲酸乙酯水解酶适用性不高。
发明内容
针对现发现的氨基甲酸乙酯水解酶存在的问题，本发明的目的在于筛选得到一株耐乙醇且具有高活力的氨基甲酸乙酯水解酶，并且对其催化活性进行改造。
一种氨基甲酸乙酯水解酶突变体，由根癌农杆菌(Agrobacterium tumefaciens)d3的酰胺酶基因经过突变得到，野生型酰胺酶的氨基酸序列如SEQ ID No.1所示，所述氨基甲酸乙酯水解酶突变体由野生型酰胺酶经过R94P、I97L、S177C或G195A中的至少一个位点突变所得。优选的，所述氨基甲酸乙酯水解酶突变体由野生型酰胺酶经过以下任意一种突变所得：G195A/R94P、G195A/S177C、I97L/S177C或I97L/G195A。
本发明又提供了编码所述的氨基甲酸乙酯水解酶突变体的基因。
本发明又提供了所述的氨基甲酸乙酯水解酶突变体或所述的基因在催化水解氨基甲酸乙酯中的应用。
本发明还提供了一种水解氨基甲酸乙酯的方法，使用所述氨基甲酸乙酯水解酶突变体催化水解氨基甲酸乙酯。优选的，催化水解时的pH为6.5～8.5，温度为40℃-65℃。更优选的，pH为7～7.5，温度为50～55℃。优选的，催化水解时体系中乙醇浓度为不高于25％。更优选的，催化水解时体系中乙醇浓度为不高于20％。进一步优选的，催化水解时体系中乙醇浓度为不高于10％。
本发明筛选得到来自Agrobacterium tumefaciens d3的酰胺酶，在pH7.5时具有最高酶活，在pH 7.5，37℃时，其氨基甲酸乙酯水解酶酶活高达473U/L。在pH6.5-8.5的范围内，展现出相对较高的酶活力，其残余酶活大于70％。该酶经过镍柱纯化之后，比酶活为0.62mg/mL，在乙醇浓度0％-20％之间，剩余酶活高于90％，高于25％之后酶活下降迅速，该酶对低浓度乙醇表现出极好的耐受性。通过半理性改造，构建了一个21个点突变的突变体库，并从中筛选得到4个酶活有提高的突变位点(R94P、I97L、S177C、G195A)，组合突变株I97L/G195A酶活提高了5.2倍，由原来的395U/L提高到2442U/L。
附图说明
图1为氨基甲酸乙酯水解酶的反应方程式。
图2为氨基甲酸乙酯水解酶的最适pH检测结果图。
图3为氨基甲酸乙酯水解酶的最适温度检测结果图。
图4为乙醇浓度对氨基甲酸乙酯水解酶活性影响检测结果图。
图5为氨基甲酸乙酯水解酶的活性中心图。
图6为氨基甲酸乙酯水解突变位点对催化活力的影响检测结果图。
图7为突变酶与野生酶的最适pH比较结果图。
图8为突变酶与野生酶的最适温度比较结果图。
图9为乙醇浓度对突变酶与野生酶活性影响比较结果图。
图10为突变酶与野生酶的温度稳定性比较结果图。
具体实施方式
实施例1：Agrobacterium tumefaciens d3酰胺酶异源表达
(1)用引物序列D3-F，D3-R(表1)扩增根癌农杆菌d3的酰胺酶基因(委托擎科生物合成，基因序列为GenBank：AF315580，氨基酸序列如SEQ ID No.1所示)，用引物pET30-F，pET30-R扩增得到线性化载体。然后使用ClonExpress II一步克隆试剂盒(购自诺唯赞公司，货号C112-02)将基因片段连接至载体的NdeI、HindIII两个位点之间。构建的质粒命名为pET-30a(+)/AtUTNase。所构建质粒上的基因具有His-tag，便于纯化。
表1引物
(2)将质粒pET-30a(+)/AtUTNase转化大肠杆菌BL21(DE3)，将携带pET-30a(+)/AtUTNase的大肠杆菌BL21(DE3)接种含有卡那霉素的试管中培养过夜后，转接到50mL含有卡那霉素(50μg/mL)的LB液体培养基中，在37℃的振荡器上孵育。当OD600达到0.6-0.8时，加入IPTG终浓度为0.5mM，18℃诱导表达18h。
(3)培养好的菌体4000RPM，离心10min，用缓冲液清洗两遍，再用缓冲液重悬，使用超声粉碎仪进行破壁(400W，工作3s，间隔7s)。破胞液用于酶活测定。
实施例2：氨基甲酸乙酯水解酶的酶活检测方法
氨基甲酸乙酯水解酶酶活检测方法的原理是：在一定条件下，氨基甲酸乙酯水解酶将氨基甲酸乙酯分解生成乙醇，氨气和二氧化碳。氨与苯酚-次氯酸钠反应形成靛酚蓝显蓝色，用分光光度计在625nm处测其吸光值，分析氨的生成量，从而得到氨基甲酸乙酯水解酶的酶活。酶活力定义：在常压、37℃条件下每分钟分解底物产生1μmol氨为一个酶活力单位。
(1)酶活测定试剂的配制：
显色剂I：称取15g苯酚和0.625g亚硝基铁氰化钠用超纯水定容至250mL。
显色剂II：称取13.125g氢氧化钠和7.5mL次氯酸钠用超纯水定容至250mL。
终止剂：称取10g三氯乙酸用超纯水定容至100mL。
NH4 +标准溶液制备：0.535g氯化铵溶于超纯水并定容至100mL，配成0.1mol·L-1的NH4 +溶液，并以此为母液，用超纯水配制成0.1mmol·L-1、0.2mmol·L-1、0.3mmol·L-1、0.4mmol·L-1、0.5mmol·L-1的NH4+标准溶液。
氨离子标准曲线的绘制：用移液管准确移取1mL NH4 +标准梯度液分别置于顺序编号的10mL比色管中。37℃下恒温保温15min，立即用吸1mL终止剂于比色管中，振荡混匀。再依次加入1mL显色剂I和显色剂II，强烈振荡，使之充分混匀，反应20min。超纯水定容至10mL，在625nm下比色测定OD值，以OD值为纵坐标，NH4 +梯度为横坐标作图，得到标准曲线。
(2)酶活的测定：
取两支10mL比色管，分别加入1mL酶液和超纯水。然后在两管中分别加入1mL模拟酒样溶液(1mL 1％的EC溶液)，在37℃恒温水浴箱中反应15min后，在两管各加入1mL终止剂，混匀后加入1mL的显色剂I和1mL显色剂II，强烈震荡，继续在37℃恒温水浴箱中保温20min后取出，用超纯水稀释到10mL，625nm处比色，并记录OD值，计算酶活。
酶活计算公式：酶活力＝ΔOD625×n×k/15，
式中，ΔOD625：酶反应后样品测定与空白试验光密度值之差；n：酶活测定液稀释倍数；k：标准曲线斜率的倒数；15：酶与底物EC反应的时间(min)。
实施例3：酰胺酶的纯化
(1)配制溶液：
结合缓冲液(Binding Buffer)：称取咪唑1.7g，NaCl 14.6g于烧杯中，加入50mL0.2M Na2HPO4-NaHPO4缓冲液，使用磷酸调节pH，最后用去离子水定容至500mL。
洗脱缓冲液(Elution Buffer)：分别称取咪唑5.1、5.97、6.8、8.5、9.35、10.2、17g，NaCl14.6g于烧杯中，加入50mL 0.2M Na2HPO4-NaHPO4缓冲液，使用磷酸调节pH，最后用去离子水定容至500mL，从而分别配制咪唑浓度150mM、175mM、200mM、250mM、275mM、300mM、500mM的洗脱缓冲液。
(2)操作流程：
预处理：10倍柱体积ddH2O冲洗镍柱，10倍柱体积50mM咪唑缓冲液平衡镍柱；
加样：破胞液反复上样五次；
冲洗：50mM咪唑缓冲液除杂；
洗脱：进行梯度洗脱。
跑蛋白胶确定洗脱浓度。确定洗杂浓度为50mM咪唑缓冲液，洗脱浓度为175mM咪唑缓冲液，洗脱下的液体超滤，最后使用磷酸缓冲洗去咪唑，测得所得纯酶(野生型)的比酶活为0.62U/mg。
实施例4：酰胺酶最适pH，最适温度的测定
(1)最适pH的检测，取一定量纯化的酶加入到一系列的反应缓冲液中制成0.1mg/mL：0.1mol/L柠檬酸-0.2mol/L磷酸氢二钠缓冲液(pH 4-8)；1/15M磷酸氢二钠-磷酸二氢钾(pH8-9)；0.1mol/L甘氨酸-0.1mol/L NaOH缓冲液(pH 9-11)，设置三组平行。底物用不同pH缓冲溶液配制，反应15min后，终止反应，加入显色剂。选取不同的缓冲液体系，不同的pH值来测定酰胺酶的最适pH，结果如图2所示。该酰胺酶的最适反应条件为pH值7.5的0.1柠檬酸-0.2磷酸氢二钠缓冲液，其氨基甲酸乙酯水解酶酶活高达395U/L；在pH6.5-8.5的范围内，酰胺酶，大于70％展现出相对较高的酶活力；当pH值低于5.5时酶活迅速降低。因此此酶适用于酸碱度较中性的酒精饮料中。
(2)最适温度的检测，取一定量纯化的酶加入最适pH缓冲液中，分别在20，25，30，35，40，45，50，55，60，75℃进行酶催化反应。如图3所示，反应最适温度为55℃，在40℃-65℃之间，酶活大于75％，高于65℃之后酶迅速失活。
实施例5：不同乙醇浓度对酰胺酶的氨基甲酸乙酯水解酶酶活的影响
将纯化得到的氨基甲酸乙酯水解酶在不同乙醇浓度(体积浓度，0％、5％、10％、20％、25％、30％、35％、40％、45％)的缓冲液中保温1h(对照组在同样条件下温浴)，测定相应条件下的酶活，以最高酶活为100％，计算各乙醇条件下相对酶活，确定乙醇对酶活稳定性的影响。如图4所示，酶在乙醇浓度0％-20％之间，剩余酶活大于90％，高于25％之后酶活下降迅速，该酶对低浓度乙醇表现出极好的耐受性。
实施例6：氨基甲酸乙酯水解酶的催化活性改造
在SWISS-MODEL中使用具有41％序列相似性的3a1K.1.A作为建模模板进行建模，通过序列分析和与模板的结构比对，确定催化三联体为Lys98，Ser173，Ser197(图5)。为了验证该模型的准确性，将催化三联体突变为Ala，结果表明用Ala代替催化残基会大大降低活性并几乎失活。这些结果证明这三氨基酸是催化活性位点，并且该模型也是可靠的。选取催化三联体附近21个点进行饱和突变，得到21个点突变的突变体库。
表2引物
| 引物名称 | 序列5′-3′ |
| R94-F | TCAAAGGGGATNNKATAGCCATCAAGGACGTCGTCTG |
| R94-R | GCTATMNNATCCCCTTTGAGCGGCCCTTCCTT |
| I97-F | CATAGCCNNKAAGGACGTCGTCTGCGTCGCTG |
| I97-R | CGTCCTTMNNGGCTATGCGATCCCCTTTGAGC |
| S177-F | CNNKGCTGTGCTGATATCGACGGGACAAGTCG |
| S177-R | ATATCAGCACAGCMNNGCCGTTCGAGGATGCGCC |
| G195-F | GACCAGNNKGGCTCCATCCGGCTACCGTCTGC |
| G195-R | ATGGAGCCMNNCTGGTCGCCACCGATAGCGAG |
通过半理性改造，构建了一个21个点突变的突变体库，并从中筛选得到4个酶活(测定条件：37℃，pH7磷酸缓冲液)提高的突变位点：R94P、I97L、S177C、G195A(四个突变对应的引物如表2所示，其中，简并碱基N表示A/T/C/G，M表示A/C，K表示G/T)，在菌体浓度OD600差别不大的情况下，其中R94P提高6％，I97L提高53％，S177C提高12％，G195A提高375％。
对上述筛选到的4点突变位点进行组合突变后检测酶活(测定条件：37℃，pH7磷酸缓冲液)，组合突变G195A/R94P提高177％，G195A/S177C提高293％，I97L/S177C提高51％，I97L/G195A提高518％。虽然R94P和S177C单点突变都提高了酶活，但是与其他点组合后酶活的提高相比较单点突变反而降低了，因此S177C与R94P不再与其他点进行组合。本次半理性改造获得了组合突变株I97L/G195A，酶活提高了5.2倍(即，是野生型酶活的6.2倍)，由原来的395U/L提高到2442U/L(图6)。
将出发菌株，I97L，G195A，I97L/G195A的粗酶液进行纯化，测量突变株I97L，G195A，I97L/G195A纯酶的最适pH，最适温度，耐醇性质，温度稳定性和动力学参数。发现I97L的最适pH与出发菌株相同都是pH7.5，而G195A与I97L/G195A的最适pH为pH7，其最适pH变化幅度不大(图7)。I97L的最适温度与出发菌株相同都是55℃，而G195A与I97L/G195A的最适温度为50℃，且G195A与I97L/G195A在60℃时残余酶活低于70％，相比较出发菌株与I97L在60℃时残余酶活90％，酶活下降迅速，对温度的耐受性更低(图8)。将酶与不同浓度的乙醇37℃保温1h后测量残余酶活，发现I97L的乙醇耐受性略高于出发菌株，而G195A的乙醇耐受性大幅度下降，在乙醇浓度为30％时，出发菌株残余酶活为56％，I97L为80％，而G195A为39％，组合突变体在30％的乙醇浓度下残余酶活为68％，20％的乙醇浓度下残余酶活为79％低于出发菌株在20％乙醇浓度下的90％，因此G195A与I97L/G195A酶活的提高使得出发菌株氨基甲酸乙酯水解酶的耐醇性下降(图9)。在60℃下测酶的热稳定性，发现保留40min后初始菌株氨基甲酸乙酯水解酶残余酶活为83％，I97L为86％，G195A为38％，I97L/G195A为39％，说明点突变G195A导致酶的热稳定性下降(图10)，这也与酶的活力增加稳定性下降的规律相适应。
表3
| 菌株 | K<sub>m</sub>(mm) | V<sub>m</sub>(umol·mg<sup>-1</sup>·min<sup>-1</sup>) |
| WT | 0.964 | 0.887 |
| I97L | 0.885 | 0.889 |
| G195A | 3.29 | 2.21 |
| I97L/G195A | 4.31 | 2.89 |
从表3，可以看出I97L突变导致酶的km降低，提高了对底物的亲和力，而G195A和I97L/G195A突变导致酶的亲和力下降，但是其最大反应速率较野生型提高2.5倍和3.3倍。
序列表
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Claims (9)
1.一种氨基甲酸乙酯水解酶突变体，其特征在于，由根癌农杆菌（Agrobacterium tumefaciens）d3的酰胺酶基因经过突变得到，野生型酰胺酶的氨基酸序列如SEQ ID No.1所示，所述氨基甲酸乙酯水解酶突变体由野生型酰胺酶经过R94P、I97L、S177C或G195A中的一个位点突变所得，或者，所述氨基甲酸乙酯水解酶突变体由野生型酰胺酶经过以下任意一种突变所得：G195A/R94P、G195A/S177C、I97L/S177C或I97L/G195A。
2.编码如权利要求1所述的氨基甲酸乙酯水解酶突变体的基因。
3.如权利要求1所述的氨基甲酸乙酯水解酶突变体或如权利要求2所述的基因在催化水解氨基甲酸乙酯中的应用。
4.一种水解氨基甲酸乙酯的方法，其特征在于，使用如权利要求1所述氨基甲酸乙酯水解酶突变体催化水解氨基甲酸乙酯。
5.如权利要求4所述的方法，其特征在于，催化水解时的pH为6.5~8.5，温度为40℃-65℃。
6.如权利要求5所述的方法，其特征在于，催化水解时的pH为7~7.5，温度为50~55℃。
7.如权利要求4所述的方法，其特征在于，催化水解时体系中乙醇浓度为不高于25%。
8.如权利要求7所述的方法，其特征在于，催化水解时体系中乙醇浓度为不高于20%。
9.如权利要求8所述的方法，其特征在于，催化水解时体系中乙醇浓度为不高于10%。
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| CN105420210A (zh) * | 2015-12-21 | 2016-03-23 | 江南大学 | 热稳定性提高的氨基甲酸乙酯水解酶突变体 |
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| Title |
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| 定点突变改造提高纺锤形赖氨酸芽孢杆菌氨基甲酸乙酯水解酶稳定性;刘晓慧等;《生物工程学报》;20160925(第09期);第1233-1242页 * |
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