CN111254135A

CN111254135A - 一种应用性能提高的脲酶突变体

Info

Publication number: CN111254135A
Application number: CN202010139462.7A
Authority: CN
Inventors: 方芳; 贾云耀; 陈坚; 堵国成
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2020-03-03
Filing date: 2020-03-03
Publication date: 2020-06-09
Anticipated expiration: 2040-03-03
Also published as: CN111254135B

Abstract

本发明公开了一种应用性能提高的脲酶突变体。通过定点突变将野生型脲酶的第325位甲硫氨酸突变为缬氨酸，或将第373位甲硫氨酸突变为丙氨酸或苏氨酸。脲酶突变体M325V，M373A和M373T的K_m相较于野生型脲酶分别降低了41.24％、50.82％和37.47％，对于黄酒中EC的降解率较野生型分别提高了64％、50％和94％，其中降解效果最好的突变体M373T可将黄酒中的EC从513.90μg/L降至393.57μg/L。本发明得到突变体能够适应黄酒的pH及乙醇条件，并对尿素和EC均有良好的降解效果，适用于黄酒的处理及保存中。

Description

一种应用性能提高的脲酶突变体

技术领域

本发明涉及一种应用性能提高的脲酶突变体，属于基因工程和酶工程技术领域。

背景技术

氨基甲酸乙酯(Ethyl carbamate，EC)是一种在传统发酵食品中检测出的氨类危害物。 2007年EC被IARC正式归类为2A类致癌物质，即对人类很可能有致癌性。

在酒精饮料中，EC主要由尿素和乙醇反应生成。目前消除黄酒中的EC主要有三种策略。策略一：工艺优化法，主要集中在三个方面，1、对原材料的精炼，减少原材料中的尿素；2、对酒精饮料发酵过程进行优化，减少因菌株代谢及酶促反应而生成的前体物质；3、对酒精饮料后处理过程进行优化，比如缩短灭菌加热时间、加快灭菌后的降温速度等。策略二：代谢工程改造法，利用代谢工程技术改造酿酒酵母，使其在发酵过程中减少尿素和EC的生成。策略三：微生物酶法，氨基甲酸乙酯水解酶可以降解EC生成氨、二氧化碳和乙醇，但其在酸性条件及高浓度乙醇的条件下不稳定，导致氨基甲酸乙酯水解酶难以应用于酒精饮料中。

脲酶具有尿素酶和氨基甲酸乙酯降解酶两种酶的活性，利用脲酶同时降解EC及其前体尿素被认为是最有希望彻底消除酒精饮料中EC的方法。提高脲酶对氨基甲酸乙酯的亲和力及降解率对于推动脲酶应用于黄酒中降解氨基甲酸乙酯具有重要意义。

发明内容

本发明的目的在于克服现有技术中的不足，提供一种应用性能提高的脲酶突变体及其应用。

本发明提供的应用性能提高的脲酶突变体是以来源于解淀粉芽孢杆菌、氨基酸序列如 SEQ ID NO.1所示的脲酶为亲本，在亲本脲酶氨基酸的基础上，将其第325位或第373位进行突变。

在本发明的一种实施方式中，编码所述亲本脲酶的核苷酸序列如SEQ ID NO.2所示。

在本发明的一种实施方式中，将亲本脲酶氨基酸的第325位甲硫氨酸突变为缬氨酸，将第373位的甲硫氨酸突变为丙氨酸或苏氨酸，得到脲酶突变体M325V、M373A、M373T。

在本发明的一种实施方式中，所述脲酶突变体M325V、M373A、M373T的氨基酸序列如SEQ ID NO.3、SEQ ID NO.5、SEQ ID NO.7所示，编码所述脲酶突变体M325V、M373A、M373T的核苷酸序列如SEQ ID NO.4、SEQ ID NO.6、SEQ ID NO.8所示。

本发明提供一种所述的脲酶突变体的制备方法，所述方法包括以下步骤：

(1)质粒构建：设计突变所用引物，以脲酶亲本基因为模板进行突变并构建突变体的质粒载体；

(2)突变菌株构建：将突变质粒转化至大肠杆菌感受态细胞，得到重组突变菌株；

(3)目标蛋白的表达：挑选含有脲酶突变基因的突变菌株进行发酵培养、诱导、纯化，进而获得脲酶突变体的蛋白，用于后续实验。

在本发明的一种实施方式中，步骤(2)中所述的大肠杆菌为BL-21。

在本发明的一种实施方式中，步骤(3)为所述重组菌株30～40℃培养至OD₆₀₀为0.6～1.2 时加入0.01～0.6mM的IPTG和3～10mM的Ni₂SO₄诱导酶的表达，诱导温度为25～35℃，诱导时间10～20h得到发酵液；再将发酵液进行破碎、过滤、纯化，得到脲酶突变体。

本发明提供了一种表达所述脲酶突变体M325V、M373A、M373T基因的重组表达载体，所述重组表达载体为pET-15或pET-19或pET-20或pET-24或pET-28或pET-32，所述Duet系列载体包括pRSFDuet-1或pACYCDuet-1或pCDFDuet-1，所述pGEX系列载体包括 pGEX-4T-2或pGEX-6P。

本发明提供了一种表达所述脲酶突变体的宿主细胞，所述宿主细胞包括枯草芽孢杆菌、酿酒酵母、大肠杆菌。

本发明提供一种降低黄酒中氨基甲酸乙酯的方法，所述方法是将脲酶突变体M325V、 M373A、M373T添加至含有氨基甲酸乙酯的培养基中进行处理。

在本发明的一种实施方式中，所述脲酶突变体的添加量为3000～10000U/L，在30～40℃下反应40～60h。

在本发明的一种实施方式中，所述脲酶突变体的添加量为5000～70000U/L，在35～40℃下反应45～55h。

本发明提供了一种所述脲酶突变体在降解食品中的尿素及氨基甲酸乙酯中的应用，所述食品包括发酵酒精饮料。

本发明还提供了所述突变体、基因、宿主细胞或表达载体在降解食品中的尿素及氨基甲酸乙酯中的应用，所述食品包括发酵酒精饮料。

本发明的有益效果：本发明通过对来源于解淀粉芽孢杆菌JP-21的脲酶进行改造，通过定点突变将野生型脲酶UreC亚基的第325位甲硫氨酸突变为缬氨酸，或将第373位甲硫氨酸突变为丙氨酸或苏氨酸，突变后的脲酶提高了对EC的亲和力，K_m相较于野生型脲酶分别降低了41.24％，50.82％和37.47％；同时还提高了脲酶对黄酒中氨基甲酸乙酯的降解率，较野生型脲酶分别提高了64％、50％和94％。

附图说明

图1为脲酶及其突变体温度的稳定性。

图2为脲酶及其突变体的最适pH。

图3为脲酶及其突变体的pH稳定性。

图4为脲酶及其突变体的乙醇耐受性。

图5为脲酶及对黄酒中尿素和氨基甲酸乙酯的降解量；*和**代表显著差异(p<0.05)和及其显著差异(p<0.01)；尿素⁺和EC⁺分别为热处理结束之后黄酒中的尿素和EC含量。

具体实施方式

LB培养基：蛋白胨10g/L，酵母粉5g/L，氯化钠10g/L，在121℃灭菌20min。

TB培养基：蛋白胨12g/L，酵母粉24g/L，甘油4mL/L，磷酸盐溶液：KH₂PO₄ 0.017mol/L， H₂KPO₄ 0.017mol/L，磷酸盐溶液与其他组分在121℃下分开灭菌20min。

20mmol/L pH 7.4的磷酸盐缓冲溶液：5.8g/L Na₂HPO₄·12H₂O，0.5928g/LNaH₂PO₄·2 H₂O，8.5g/L NaCl。

尿素测定：先采用二乙酰单氧化反应法处理样品，再利用高效液相色谱法进行测定(参见文献Determination of urea using high-performance liquid chromatographywith fluorescence detection after automated derivatisation with xanthydrol)。

EC测定：采用固相微萃取法处理样品，再利用GC-MS的方法进行测定(参见文献Determination of EC content in rice wine by GC/MS and HPLC-FLD.Food andFermentation Industries)。

实施例1：含脲酶基因重组菌的制备

使用引物P1-F/P1-R，用PCR的方法对质粒pRSF-Duet-1进行线性化，PCR反应体系为： 2×Phanta Max Master Mix 25μL，上游引物(10μM)2μL，下游引物(10μM)2μL，模板DNA 1μL，ddH₂O 20μL(PCR试剂购自于南京诺唯赞生物科技有限公司)，得到线性化的pRSF-Duet-1。

化学合成如SEQ ID NO.2所示的核苷酸序列，将其与线性化质粒pRSF-Duet-1进行连接 (利用ClonExpress II One Step Cloning Kit，片段与线性化质粒各100μg、70μg，在37℃连接30min)得到重组质粒P-1-WT，并将该重组质粒转化至大肠杆菌JM109(具体构建步骤参照北京索莱宝科技有限公司JM109感受态细胞使用说明)。利用阳性转化子提取质粒，将质粒通过热激转化法转化至大肠杆菌BL-21(DE3)，将转化产物涂布在LB固体培养基(含有 0.05mg/mL卡那霉素)上，于37℃恒温培养箱中倒置培养8～12h，得到转化子。

热激转化法：

(1)将10μL连接产物导入100μL BL-21感受态细胞；

(2)冰浴15-30min；

(3)42℃水浴热激90s，取出后迅速放入冰中静置冰浴3-5min；

(4)加入800μL无抗性LB培养基吹吸混匀，于37℃，220rpm培养1h；

(5)5000rpm离心2min收菌；

(6)移去上清，剩余100-200μL吹吸混匀涂布至含0.05mg/mL卡那霉素抗性平板上，37℃恒温培养12h左右，得到单克隆菌落。

表1脲酶表达载体构建引物

引物	引物序列(5’-3’)
			P1-F	TTAACCTAGGCTGCTGCCAC	SEQ ID NO.9
P1-R	GGTATATCTCCTTATTAAAGTTAAACAAAATTA	SEQ ID NO.10

实施例2：突变体M325V、M373A、M373T的制备

通过对脲酶蛋白与底物氨基甲酸乙酯分子对接的结构进行分析，我们选择对其第325、373位的甲硫氨酸进行突变，设计相应的定点突变引物(表2)。以重组质粒P-1-WT为模板，利用表2中的引物对重组质粒P-1-WT进行扩增。

表2脲酶突变引物序列

引物	引物序列(5’-3’)
			M373mut-A	GCAGGCG<u>NNK</u>GGCAG	SEQ ID NO.11
M373mut-S	CTGCC<u>MNN</u>CGCCTGC	SEQ ID NO.12
			M325mut-A	TGATATG<u>NNK</u>ATGGTCTGCCATC	SEQ ID NO.13
M325mut-S	ACCAT<u>MNN</u>CATATCAAGATGCTCG	SEQ ID NO.14

PCR反应体系为：2×Phanta Max Master Mix 25μL，上游引物(10μM)2μL，下游引物 (10μM)2μL，模板DNA 1μL，ddH₂O 20μL。

PCR扩增条件为：95℃预变性3min；随后34个循环(95℃15s，56℃15s，72℃3min)；72℃延伸5min。

PCR产物经限制性内切酶DpnI消化(消化体系为：DpnI 0.5μL、上述反应PCR产物45μL、10×T Buffer 5μL；在37℃孵育3h以消化PCR产物)，转化大肠杆菌BL-21(DE3)感受态细胞，将转化产物涂布在LB固体培养基(含有0.05mg/mL卡那霉素)上，于37℃恒温培养箱中倒置培养8～12h，得到转化子单克隆菌落，挑选单克隆送苏州金唯智测序，测序正确的转化子即为相应的脲酶突变体菌株。

实施例3：脲酶突变体的表达与纯化

将脲酶突变体菌株接种于液体LB培养基(含50μg/mL卡那霉素)，37℃，220rpm培养12h得到种子液，按体积比1％接种量将种子液接种到TB培养基(含50μg/mL卡那霉素)， 37℃，220rpm培养到OD₆₀₀＝0.6-0.8时，加入0.1mM的IPTG和6mM的Ni₂SO₄，30℃， 220rpm诱导培养15h得到发酵液。发酵液于4℃、8000r/min离心15min，收集菌体沉淀物，用20mmol/L pH7.4的磷酸盐缓冲溶液将菌体洗涤2遍，添加50mL的Binding Buffer(20 mmol/L pH 7.4磷酸盐缓冲溶液、0.5mol/L NaCl)重悬菌体，冰水浴超声破碎(破碎条件为 130w、2s/4s、20min)后于4℃、12000r/min离心20min，收集上清液即为脲酶粗酶液；粗酶液用0.45μm微孔滤膜过滤，备用。

采用镍柱亲和层析法对粗酶液进行纯化。准备镍离子亲和层析柱，首先，在4℃环境下利用恒流泵，向柱子里泵入去离子水冲洗柱子(约4-5倍柱体积)，然后用ElutionBuffer(pH 7.4 20mM PBS、0.5M NaCl、0.5M咪唑)平衡柱子环境；待紫外曲线平衡时泵入Binding Buffer (pH 7.4 20mM PBS，0.5M NaCl)，待紫外曲线平衡时，将得到的过膜粗酶液加入到柱子中；先用Binding Buffer冲洗杂蛋白至基线平衡，再用Elution Buffer洗脱；收集吸收峰的洗脱液，并测定其酶活，获得达到电泳纯的目的蛋白。

实施例4：脲酶酶活测定及酶学动力学参数测定

(1)脲酶酶活测定的反应体系及方法

采用Berthelot比色法测定脲酶酶活：

①取经适当稀释n₁倍的酶液200μL，添加到200μL含有30g/L尿素或30g/L EC的50mmol/L pH 6.0柠檬酸-柠檬酸钠缓冲液中，混匀后在37℃条件下反应15min；

②反应结束后立即加入200μL终止剂(体积比为10％的三氯乙酸)，震荡混匀后，再依次加入200μL显色剂Ⅰ(含60g/L苯酚和2.5g/L亚硝基铁氰化钠)和200μL显色剂Ⅱ(含52.5g/L氢氧化钠和30mL/L安替福民)，进行混匀后，于37℃继续反应20min；

③反应液适当稀释n₂倍后，测定其OD₆₂₅的变化，以灭活的酶液作为空白对照。

利用0、0.1mmol/L、0.2mmol/L、0.3mmol/L、0.4mmol/L NH₄Cl溶液作为铵离子标准曲线，根据氯化铵标准曲线即可计算反应体系生成铵的总量，以此来计算酶活。

酶活单位定义：在常压、37℃及pH 6.0的条件下，每分钟分解1μmol EC或尿素所需要的酶量为一个酶活力单位；酶活(U/mL)＝ΔOD₆₂₅×n×k/15

式中15为步骤①中的反应时间(min)，ΔOD₆₂₅为样品与空白对照吸光值之差，k为标准曲线斜率的倒数，n为从酶液到最终测定时酶液总的稀释倍数(n₁+n₂)。

从突变体中筛选出酶活最高的突变体，并根据其突变的位点分别将其命名为M325V、 M373A和M373T

(2)脲酶酶学动力学参数测定

通过在37℃下，分别测定不同底物浓度下的催化反应速率，利用GraphPad Prism7对结果进行非线性回归曲线拟合，以此计算K_m和V_max。以尿素为底物时的浓度范围为2-80mmol/L，以EC为底物时的浓度范围为100-1800mmo/L。

测定结果显示，突变体M373A、M373T、M325V以尿素为底物时各参数没有明显变化，而以EC为底物的Km比野生脲酶分别降低了50.82％、37.47％、41.24％，说明脲酶突变体对于EC的亲和力有明显提高(见表3)。

表3脲酶及其突变体的酶学动力学参数

实施例5：脲酶热稳定性的研究

将脲酶纯酶(1 000-1 200U/L)置于不同温度下(30℃、40℃、50℃、60℃、70℃) 保温30min后测定酶活。以未经保温处理的酶活为100％，计算不同温度下的相对酶活。

如图1，实验结果显示：

野生脲酶和脲酶突变体在低于50℃的条件下均保持了近100％的酶活。

在60℃时，M373A和M325V的热稳定性显著高于野生型，M373A在以尿素为底物和以EC为底物时分别可以保持82.24％和86.57％的酶活，M325V在以尿素为底物和以EC为底物时分别可以保持79.51％和76.64％的酶活，而野生型仅有71.64％和69.39％的酶活。

M325V在70℃下，在以尿素为底物和以EC为底物时酶热稳定性较好，比野生脲酶的相对酶活分别提高了9.34％和7.12％。

实施例6：脲酶突变体最适pH及pH稳定性研究

测定最适pH时，使用的缓冲液为50mmol/L，pH 3.0-7.0的柠檬酸-Na₂HPO₄缓冲液。利用同pH缓冲液配制底物溶液，在不同pH条件下测定酶活，以测得的最高酶活为100％，分别计算其他pH条件下酶活的相对值。

测定pH稳定性时，将脲酶纯酶置于pH 3.0-7.0的缓冲液中，4℃放置6h后测定其残余酶活力。以测得的最高酶活为100％，分别计算其他pH条件下酶活的相对值。

如图2、图3，实验结果显示：野生脲酶和脲酶突变体的最适pH均为6。虽然最适反应pH没有改变，但M373A和野生脲酶及其它突变体相比，具有更宽的pH稳定性范围，尤其是在pH 3.0-5.5时，M373A的尿素酶和EC酶活性分别比野生脲酶高30％和20％。此外，野生脲酶在pH 6时稳定性最好，而突变体在pH 5.5时稳定性最佳，并且在pH 4.5时，M373A 的脲酶相对酶活相比野生脲酶提高了39％。

实施例7：脲酶突变体对乙醇的耐受性研究

将脲酶与乙醇(混合体积比为0-40％)混合，在37℃下放置2h后测定酶活。以不含乙醇的酶液所测酶活力为100％，分别计算不同乙醇浓度下的相对酶活。

如图4，实验结果显示：野生型脲酶及其突变体在20％乙醇条件下保存2h，其尿素酶和 EC酶活力仍有60％和50％以上，各突变体之间无明显差异。这说明，此酶对的乙醇耐受性较好，具有降解酒精饮料中EC及尿素的潜力。

实施例8：脲酶半衰期测定

脲酶半衰期的测定利用Arrhenius方程的原理进行测定和计算。将稀释的脲酶纯酶在70℃下保温，每隔2min取样测定其残余酶活力。取残余酶活力最高的为100％。记脲酶残余酶活力的百分比为A_r，通过绘制作保温时间与lnA_r的线性拟合，所得直线斜率的相反数为K_d，即热失活速率常数。脲酶半衰期(t_1/2)的计算公式为t_1/2＝ln2/K_d。

如表4，实验结果显示，野生脲酶和M373位点突变体的半衰期没有显著差异，而突变体 M325V在以尿素和EC为底物时半衰期与野生脲酶相比略有延长，分别是野生脲酶的1.10倍和1.16倍。

表4脲酶及其突变体在70℃的半衰期

实施例9：脲酶尿素及氨基甲酸乙酯降解率测定

(1)将市售黄酒(经测定，所购市售黄酒含35mg/L尿素，320μg/L EC)中尿素和EC含量分别调整至50mg/L和513.25μg/L。向此黄酒中添加终浓度为6000U/L的野生型及脲酶突变体，37℃下反应50h，取样测定样品中尿素及EC的含量。尿素测定采用二乙酰单氧化反应法用高效液相色谱进行测定，EC的测定采用固相微萃取结合GC-MS的方法。

结果显示：经野生型脲酶及脲酶突变体M325V、M373A、M373T处理后的黄酒中的尿素含量分别为2.5mg/L、1.6mg/L、1.9mg/L、1.7mg/L，去除率分别为95.2％、96.9％、96.3％、96.7％(图5A)，黄酒中尿素基本被降解；经野生型脲酶及脲酶突变体M325V、M373A、M373T处理后的黄酒中的EC含量分别为452.21μg/L、412.11μg/L、420.68μg/L、393.57μg/L，降解率分别为12.0％、19.7％、18.0％、23.3％，脲酶突变体M325V、M373A、M373T对EC的降解率较野生型分别提高了64％、50％和94％。

(2)由于尿素与乙醇可自发反应生成EC，因此黄酒存放一段时间后EC含量会逐渐增加。为考察脲酶对黄酒中EC的全面减控效果，对黄酒采用热处理方法加速EC生成过程，来评估脲酶对EC的减控效果，具体操作步骤为：将步骤(1)中经野生型及脲酶突变体处理过后的黄酒在90℃保温30min，热处理结束之后测定黄酒中的尿素及EC含量。

结果表明：在经过热处理后的黄酒中，未经脲酶处理的黄酒样品中的EC由513.90μg/L 增至595.07μg/L，而经脲酶野生型及其突变体M325V、M373A、M373T处理过的黄酒中的EC分别为452.20μg/L、419.62μg/L、428.68μg/L、397.77μg/L，含量基本没有增加(图5B)。说明通过具有双酶活性(EC酶和尿素酶)的脲酶处理，基本消除了尿素并显著减少了EC。

虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。

SEQUENCE LISTING

<110> 江南大学

<120> 一种应用性能提高的脲酶突变体

<160> 14

<170> PatentIn version 3.3

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<211> 804

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<213> Bacillus amyloliquefaciens

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Thr Thr Cys Thr Ser Gly Val Trp Tyr Met Ser Arg Met Leu Glu Ala

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Ala Glu Glu Phe Pro Ile Asn Val Gly Phe Leu Gly Lys Gly Asn Ala

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Ser Asp Lys Ala Pro Leu Ile Glu Gln Val Glu Ala Gly Ala Ile Gly

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Leu Lys Leu His Glu Asp Trp Gly Ser Thr Pro Ser Ala Ile Lys Ala

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Asp Thr Ile Asn Glu Ala Gly Phe Leu Glu Asn Thr Leu Asp Ala Ile

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Gly Asp Arg Val Ile His Thr Tyr His Ile Glu Gly Ala Gly Gly Gly

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His Ala Pro Asp Ile Met Lys Leu Ala Ser Tyr Ala Asn Ile Leu Pro

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Ser Ser Thr Thr Pro Thr Ile Pro Tyr Thr Val Asn Thr Met Asp Glu

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His Leu Asp Met Met Met Val Cys His His Leu Asp Ser Lys Val Pro

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Asn Gly Asn Asp Asn Val Arg Ala Lys Arg Tyr Ile Ala Lys Tyr Thr

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Ile Asn Pro Ala Val Thr His Gly Leu Ser His Glu Val Gly Ser Val

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Glu Lys Gly Lys Leu Ala Asp Leu Val Leu Trp Asp Pro Val Phe Phe

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Gln Met Gly Asp Pro Asn Ala Ser Ile Pro Thr Pro Glu Pro Val Phe

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Ile Thr Phe Met Ser Gln Ala Gly Ile Ala Asn Gly Val Pro Glu Lys

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Ser Lys Leu Asp Met Lys Leu Asn Asp Ala Met Pro Asn Ile Arg Val

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Asp Pro Lys Thr Tyr Gln Val Phe Ala Asp Gly Glu Glu Leu Ala Cys

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Gln Pro Val Ser Tyr Val Pro Leu Gly Gln Arg Tyr Phe Leu Phe Met

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Lys Leu Thr Pro Val Glu Gln Glu Lys Leu Leu Ile Phe Thr Ala Gly

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Glu Leu Ala Lys Gln Arg Lys Ala Arg Gly Val Leu Leu Asn Tyr Pro

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Glu Ala Ala Ala Tyr Leu Thr Cys Tyr Leu Met Glu Gly Ala Arg Asp

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Gly Lys Ser Val Ala Glu Leu Met Glu Ser Gly Arg Asn Val Leu Thr

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Glu Lys Asp Val Met Glu Gly Val Ala Glu Met Leu Asp Ser Ile Gln

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ttcacgaatt acggggaaga aatgattttc ggcggaggga aaacaatccg ggacggcatg 180

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ttcagtcatt cacgcatcag agcggccacc attgcggcgg aggatattct gcacgatatc 1080

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ccggttgaac aagaaaaatt gcttattttt acggcgggag agctcgctaa gcagcggaag 1800

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Met His His His His His His Lys Met Ser Arg Glu Gln Tyr Ala Glu

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Leu Phe Gly Pro Thr Thr Gly Asp Lys Val Arg Leu Gly Asp Thr Asp

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Leu Trp Ile Glu Val Glu Lys Asp Phe Thr Asn Tyr Gly Glu Glu Met

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Ile Phe Gly Gly Gly Lys Thr Ile Arg Asp Gly Met Gly Gln Asn Gly

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Arg Ile Thr Gly Lys Asp Gly Ala Leu Asp Leu Val Ile Thr Asn Ala

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Val Ile Leu Asp Tyr Thr Gly Ile Val Lys Ala Asp Ile Gly Val Lys

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Asp Gly Arg Ile Val Gly Val Gly Lys Ser Gly Asn Pro Asp Met Met

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Asp Gly Val Asp Pro His Met Ile Ile Gly Ala Gly Thr Glu Val Ile

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Ser Gly Glu Gly Lys Ile Val Thr Ala Gly Gly Val Asp Thr His Ile

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His Phe Ile Cys Pro Gln Gln Met Glu Val Ala Leu Ser Ser Gly Val

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Thr Thr Leu Leu Gly Gly Gly Thr Gly Pro Ala Thr Gly Ser Lys Ala

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Thr Thr Cys Thr Ser Gly Val Trp Tyr Met Ser Arg Met Leu Glu Ala

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Ala Glu Glu Phe Pro Ile Asn Val Gly Phe Leu Gly Lys Gly Asn Ala

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Gly Asp Arg Val Ile His Thr Tyr His Ile Glu Gly Ala Gly Gly Gly

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His Ala Pro Asp Ile Met Lys Leu Ala Ser Tyr Ala Asn Ile Leu Pro

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Ser Ser Thr Thr Pro Thr Ile Pro Tyr Thr Val Asn Thr Met Asp Glu

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His Leu Asp Met Val Met Val Cys His His Leu Asp Ser Lys Val Pro

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Glu Asp Val Ala Phe Ser His Ser Arg Ile Arg Ala Ala Thr Ile Ala

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Ala Glu Asp Ile Leu His Asp Ile Gly Ala Ile Ser Met Thr Ser Ser

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Asp Ser Gln Ala Met Gly Arg Val Gly Glu Val Ile Ile Arg Thr Trp

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Gln Val Ala Asp Lys Met Lys Lys Gln Arg Gly Ala Leu Ser Gly Glu

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Asn Gly Asn Asp Asn Val Arg Ala Lys Arg Tyr Ile Ala Lys Tyr Thr

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Ile Asn Pro Ala Val Thr His Gly Leu Ser His Glu Val Gly Ser Val

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Glu Lys Gly Lys Leu Ala Asp Leu Val Leu Trp Asp Pro Val Phe Phe

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Gly Val Lys Pro Glu Leu Val Leu Lys Gly Gly Met Ile Ala Arg Ala

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Gln Met Gly Asp Pro Asn Ala Ser Ile Pro Thr Pro Glu Pro Val Phe

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Met Arg Gln Met Tyr Ala Ser Tyr Gly Lys Ala Asn Arg Asn Thr Ser

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Ile Thr Phe Met Ser Gln Ala Gly Ile Ala Asn Gly Val Pro Glu Lys

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Leu Gly Leu Glu Lys Met Ile Ser Pro Val Arg Asn Ile Arg Lys Leu

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Ser Lys Leu Asp Met Lys Leu Asn Asp Ala Met Pro Asn Ile Arg Val

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Asp Pro Lys Thr Tyr Gln Val Phe Ala Asp Gly Glu Glu Leu Ala Cys

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Gln Pro Val Ser Tyr Val Pro Leu Gly Gln Arg Tyr Phe Leu Phe Met

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Lys Leu Thr Pro Val Glu Gln Glu Lys Leu Leu Ile Phe Thr Ala Gly

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Glu Leu Ala Lys Gln Arg Lys Ala Arg Gly Val Leu Leu Asn Tyr Pro

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Glu Ala Ala Ala Tyr Leu Thr Cys Tyr Leu Met Glu Gly Ala Arg Asp

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Gly Lys Ser Val Ala Glu Leu Met Glu Ser Gly Arg Asn Val Leu Thr

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Glu Lys Asp Val Met Glu Gly Val Ala Glu Met Leu Asp Ser Ile Gln

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Val Glu Ala Thr Phe Pro Asp Gly Val Lys Leu Val Thr Val His Gln

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Pro Ile Lys Ala Glu Val Lys Ser Met Lys Pro Gly Ala Ile Gln Val

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Ala Lys Gly Thr Ile Thr Ile Asn Glu Gly Arg Lys Thr Leu Glu Val

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Ser Val Thr Asn Asn Gly Thr Arg Ser Val Gln Val Gly Ser His Phe

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His Phe Ala Glu Ala Asn Gly Ala Leu Ser Phe Asn Arg Asp Lys Ala

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Ile Gly Met Arg Leu Asp Ile Pro Ser Gly Thr Ser Val Arg Phe Glu

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Pro Gly Glu Glu Lys Thr Val Thr Leu Val Glu Ile Gly Gly Arg Lys

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Thr Val Arg Gly Leu Asn Gly Met Ala Asp Thr Tyr Met Asp Glu Arg

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Glu Ala Ile Arg

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cacatagagg gagccggcgg aggccatgca ccggatatta tgaaactcgc atcttacgcc 900

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atgcggcaga tgtacgcatc atacggtaaa gcaaaccgca acacctctat tacatttatg 1500

tcccaggccg gtatcgcaaa cggtgtgccg gaaaagctcg gccttgaaaa aatgatttct 1560

cccgtacgga atatccgtaa gctgagtaag ctcgacatga agctgaatga cgcgatgccg 1620

aatatacgtg tcgatccgaa aacctatcag gtgttcgccg acggagaaga gctggcatgc 1680

cagcccgtca gctatgttcc gctaggacag cgttatttct tattttaaat gaaactgaca 1740

ccggttgaac aagaaaaatt gcttattttt acggcgggag agctcgctaa gcagcggaag 1800

gcgcgcggcg ttctgctgaa ttatcccgaa gccgccgcat atttgacctg ttatctgatg 1860

gaaggcgcga gagacggaaa aagcgttgct gagctgatgg aatccggccg caatgtattg 1920

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acgttcccgg acggggttaa gcttgtcacc gttcatcagc cgatcaaagc ggaggtgaag 2040

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cggaagacgc tggaggtgtc agtcaccaat aacggaacgc ggtcagtgca ggtcggatcg 2160

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atggaggagg cgatccgatg a 2421

<210> 5

<211> 804

<212> PRT

<213> 人工序列

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Met His His His His His His Lys Met Ser Arg Glu Gln Tyr Ala Glu

1 5 10 15

Leu Phe Gly Pro Thr Thr Gly Asp Lys Val Arg Leu Gly Asp Thr Asp

20 25 30

Leu Trp Ile Glu Val Glu Lys Asp Phe Thr Asn Tyr Gly Glu Glu Met

35 40 45

Ile Phe Gly Gly Gly Lys Thr Ile Arg Asp Gly Met Gly Gln Asn Gly

50 55 60

Arg Ile Thr Gly Lys Asp Gly Ala Leu Asp Leu Val Ile Thr Asn Ala

65 70 75 80

Val Ile Leu Asp Tyr Thr Gly Ile Val Lys Ala Asp Ile Gly Val Lys

85 90 95

Asp Gly Arg Ile Val Gly Val Gly Lys Ser Gly Asn Pro Asp Met Met

100 105 110

Asp Gly Val Asp Pro His Met Ile Ile Gly Ala Gly Thr Glu Val Ile

115 120 125

Ser Gly Glu Gly Lys Ile Val Thr Ala Gly Gly Val Asp Thr His Ile

130 135 140

His Phe Ile Cys Pro Gln Gln Met Glu Val Ala Leu Ser Ser Gly Val

145 150 155 160

Thr Thr Leu Leu Gly Gly Gly Thr Gly Pro Ala Thr Gly Ser Lys Ala

165 170 175

Thr Thr Cys Thr Ser Gly Val Trp Tyr Met Ser Arg Met Leu Glu Ala

180 185 190

Ala Glu Glu Phe Pro Ile Asn Val Gly Phe Leu Gly Lys Gly Asn Ala

195 200 205

Ser Asp Lys Ala Pro Leu Ile Glu Gln Val Glu Ala Gly Ala Ile Gly

210 215 220

Leu Lys Leu His Glu Asp Trp Gly Ser Thr Pro Ser Ala Ile Lys Ala

225 230 235 240

Cys Met Glu Ala Ala Asp Glu Ala Asp Ile Gln Val Ala Ile His Thr

245 250 255

Asp Thr Ile Asn Glu Ala Gly Phe Leu Glu Asn Thr Leu Asp Ala Ile

260 265 270

Gly Asp Arg Val Ile His Thr Tyr His Ile Glu Gly Ala Gly Gly Gly

275 280 285

His Ala Pro Asp Ile Met Lys Leu Ala Ser Tyr Ala Asn Ile Leu Pro

290 295 300

Ser Ser Thr Thr Pro Thr Ile Pro Tyr Thr Val Asn Thr Met Asp Glu

305 310 315 320

His Leu Asp Met Met Met Val Cys His His Leu Asp Ser Lys Val Pro

325 330 335

Glu Asp Val Ala Phe Ser His Ser Arg Ile Arg Ala Ala Thr Ile Ala

340 345 350

Ala Glu Asp Ile Leu His Asp Ile Gly Ala Ile Ser Met Thr Ser Ser

355 360 365

Asp Ser Gln Ala Ala Gly Arg Val Gly Glu Val Ile Ile Arg Thr Trp

370 375 380

Gln Val Ala Asp Lys Met Lys Lys Gln Arg Gly Ala Leu Ser Gly Glu

385 390 395 400

Asn Gly Asn Asp Asn Val Arg Ala Lys Arg Tyr Ile Ala Lys Tyr Thr

405 410 415

Ile Asn Pro Ala Val Thr His Gly Leu Ser His Glu Val Gly Ser Val

420 425 430

Glu Lys Gly Lys Leu Ala Asp Leu Val Leu Trp Asp Pro Val Phe Phe

435 440 445

Gly Val Lys Pro Glu Leu Val Leu Lys Gly Gly Met Ile Ala Arg Ala

450 455 460

Gln Met Gly Asp Pro Asn Ala Ser Ile Pro Thr Pro Glu Pro Val Phe

465 470 475 480

Met Arg Gln Met Tyr Ala Ser Tyr Gly Lys Ala Asn Arg Asn Thr Ser

485 490 495

Ile Thr Phe Met Ser Gln Ala Gly Ile Ala Asn Gly Val Pro Glu Lys

500 505 510

Leu Gly Leu Glu Lys Met Ile Ser Pro Val Arg Asn Ile Arg Lys Leu

515 520 525

Ser Lys Leu Asp Met Lys Leu Asn Asp Ala Met Pro Asn Ile Arg Val

530 535 540

Asp Pro Lys Thr Tyr Gln Val Phe Ala Asp Gly Glu Glu Leu Ala Cys

545 550 555 560

Gln Pro Val Ser Tyr Val Pro Leu Gly Gln Arg Tyr Phe Leu Phe Met

565 570 575

Lys Leu Thr Pro Val Glu Gln Glu Lys Leu Leu Ile Phe Thr Ala Gly

580 585 590

Glu Leu Ala Lys Gln Arg Lys Ala Arg Gly Val Leu Leu Asn Tyr Pro

595 600 605

Glu Ala Ala Ala Tyr Leu Thr Cys Tyr Leu Met Glu Gly Ala Arg Asp

610 615 620

Gly Lys Ser Val Ala Glu Leu Met Glu Ser Gly Arg Asn Val Leu Thr

625 630 635 640

Glu Lys Asp Val Met Glu Gly Val Ala Glu Met Leu Asp Ser Ile Gln

645 650 655

Val Glu Ala Thr Phe Pro Asp Gly Val Lys Leu Val Thr Val His Gln

660 665 670

Pro Ile Lys Ala Glu Val Lys Ser Met Lys Pro Gly Ala Ile Gln Val

675 680 685

Ala Lys Gly Thr Ile Thr Ile Asn Glu Gly Arg Lys Thr Leu Glu Val

690 695 700

Ser Val Thr Asn Asn Gly Thr Arg Ser Val Gln Val Gly Ser His Phe

705 710 715 720

His Phe Ala Glu Ala Asn Gly Ala Leu Ser Phe Asn Arg Asp Lys Ala

725 730 735

Ile Gly Met Arg Leu Asp Ile Pro Ser Gly Thr Ser Val Arg Phe Glu

740 745 750

Pro Gly Glu Glu Lys Thr Val Thr Leu Val Glu Ile Gly Gly Arg Lys

755 760 765

Thr Val Arg Gly Leu Asn Gly Met Ala Asp Thr Tyr Met Asp Glu Arg

770 775 780

Gly Lys Glu Lys Thr Leu Ser Asn Leu Lys Lys Ala Gly Trp Met Glu

785 790 795 800

Glu Ala Ile Arg

<210> 6

<211> 2421

<212> DNA

<213> 人工序列

<400> 6

atgcaccacc accaccacca caaaatgtcg cgtgagcaat acgcagaact gttcggaccg 60

acaacgggag acaaagtcag actcggagat acggatttat ggattgaagt cgaaaaagat 120

ttcacgaatt acggggaaga aatgattttc ggcggaggga aaacaatccg ggacggcatg 180

gggcagaacg ggcgtatcac cgggaaagac ggagcgcttg atctggtcat tacaaacgcc 240

gtcatcctgg attataccgg aatcgtcaag gcggatatcg gggtgaagga cggccggatt 300

gtcggcgtcg ggaaaagcgg caaccctgat atgatggatg gggtggaccc gcacatgatt 360

atcggtgccg gaacagaagt catttccggc gaagggaaaa tcgtaacggc cggcggggtg 420

gatacgcata tccactttat ctgcccgcag cagatggaag tcgcgctttc ttcaggcgtg 480

actacgcttc tcggcggcgg aacaggtcct gcgacgggaa gtaaagcgac gacttgtaca 540

tccggtgtat ggtacatgtc gagaatgctg gaagcggccg aggagtttcc gatcaatgtc 600

ggtttcttag gaaaaggaaa tgcatccgat aaagcgccgc tgatcgagca ggtggaagca 660

ggcgcaatcg gcctgaagct gcatgaagat tggggatcaa cgccaagcgc tattaaagct 720

tgcatggaag cagcggatga ggcggacatt caggtggcga tccacacaga cacgataaat 780

gaagcgggct ttttagaaaa tacgcttgat gcgatcggcg accgggttat ccatacatat 840

cacatagagg gagccggcgg aggccatgca ccggatatta tgaaactcgc atcttacgcc 900

aatatcctgc cgtcctctac gacgccgacg attccatata ccgtcaacac gatggacgag 960

catcttgata tgatgatggt ctgccatcat ttagattcaa aagtgcctga agacgtggcg 1020

ttcagtcatt cacgcatcag agcggccacc attgcggcgg aggatattct gcacgatatc 1080

ggcgcgatca gcatgacgtc atctgactcg caggcggctg gcagggtggg agaagtgatt 1140

atccggacat ggcaggtggc cgataaaatg aaaaaacagc gcggtgctct atcgggagaa 1200

aacggcaatg acaatgtgcg cgccaaacgc tatatcgcca aatacacgat caacccggct 1260

gtcactcacg gtctgagcca tgaagtcggt tccgttgaaa aaggaaagct cgccgacctc 1320

gtactatggg acccggtttt cttcggcgtc aaacctgaac ttgtgctcaa aggcggcatg 1380

attgcccgcg cccagatggg agatccgaat gcttccattc cgacgcctga gcccgtgttt 1440

atgcggcaga tgtacgcatc atacggtaaa gcaaaccgca acacctctat tacatttatg 1500

tcccaggccg gtatcgcaaa cggtgtgccg gaaaagctcg gccttgaaaa aatgatttct 1560

cccgtacgga atatccgtaa gctgagtaag ctcgacatga agctgaatga cgcgatgccg 1620

aatatacgtg tcgatccgaa aacctatcag gtgttcgccg acggagaaga gctggcatgc 1680

cagcccgtca gctatgttcc gctaggacag cgttatttct tattttaaat gaaactgaca 1740

ccggttgaac aagaaaaatt gcttattttt acggcgggag agctcgctaa gcagcggaag 1800

gcgcgcggcg ttctgctgaa ttatcccgaa gccgccgcat atttgacctg ttatctgatg 1860

gaaggcgcga gagacggaaa aagcgttgct gagctgatgg aatccggccg caatgtattg 1920

acggaaaaag acgttatgga aggcgttgcg gaaatgctgg acagcattca ggtggaagcg 1980

acgttcccgg acggggttaa gcttgtcacc gttcatcagc cgatcaaagc ggaggtgaag 2040

tcatgaatga agccgggagc gattcaagtc gcaaagggga ccatcaccat taacgaaggc 2100

cggaagacgc tggaggtgtc agtcaccaat aacggaacgc ggtcagtgca ggtcggatcg 2160

cattttcatt ttgccgaagc caacggcgcc ctttccttca atcgggacaa agccatcggc 2220

atgcgccttg atatcccgtc aggcacatct gtccgttttg aaccgggaga agagaaaacc 2280

gtcacgctcg tggaaatcgg agggcgaaaa acggtcagag gtctcaacgg catggccgat 2340

acgtacatgg atgagcgggg aaaagagaag acgctgtcaa atcttaaaaa agccggatgg 2400

atggaggagg cgatccgatg a 2421

<210> 7

<211> 804

<212> PRT

<213> 人工序列

<400> 7

Met His His His His His His Lys Met Ser Arg Glu Gln Tyr Ala Glu

1 5 10 15

Leu Phe Gly Pro Thr Thr Gly Asp Lys Val Arg Leu Gly Asp Thr Asp

20 25 30

Leu Trp Ile Glu Val Glu Lys Asp Phe Thr Asn Tyr Gly Glu Glu Met

35 40 45

Ile Phe Gly Gly Gly Lys Thr Ile Arg Asp Gly Met Gly Gln Asn Gly

50 55 60

Arg Ile Thr Gly Lys Asp Gly Ala Leu Asp Leu Val Ile Thr Asn Ala

65 70 75 80

Val Ile Leu Asp Tyr Thr Gly Ile Val Lys Ala Asp Ile Gly Val Lys

85 90 95

Asp Gly Arg Ile Val Gly Val Gly Lys Ser Gly Asn Pro Asp Met Met

100 105 110

Asp Gly Val Asp Pro His Met Ile Ile Gly Ala Gly Thr Glu Val Ile

115 120 125

Ser Gly Glu Gly Lys Ile Val Thr Ala Gly Gly Val Asp Thr His Ile

130 135 140

His Phe Ile Cys Pro Gln Gln Met Glu Val Ala Leu Ser Ser Gly Val

145 150 155 160

Thr Thr Leu Leu Gly Gly Gly Thr Gly Pro Ala Thr Gly Ser Lys Ala

165 170 175

Thr Thr Cys Thr Ser Gly Val Trp Tyr Met Ser Arg Met Leu Glu Ala

180 185 190

Ala Glu Glu Phe Pro Ile Asn Val Gly Phe Leu Gly Lys Gly Asn Ala

195 200 205

Ser Asp Lys Ala Pro Leu Ile Glu Gln Val Glu Ala Gly Ala Ile Gly

210 215 220

Leu Lys Leu His Glu Asp Trp Gly Ser Thr Pro Ser Ala Ile Lys Ala

225 230 235 240

Cys Met Glu Ala Ala Asp Glu Ala Asp Ile Gln Val Ala Ile His Thr

245 250 255

Asp Thr Ile Asn Glu Ala Gly Phe Leu Glu Asn Thr Leu Asp Ala Ile

260 265 270

Gly Asp Arg Val Ile His Thr Tyr His Ile Glu Gly Ala Gly Gly Gly

275 280 285

His Ala Pro Asp Ile Met Lys Leu Ala Ser Tyr Ala Asn Ile Leu Pro

290 295 300

Ser Ser Thr Thr Pro Thr Ile Pro Tyr Thr Val Asn Thr Met Asp Glu

305 310 315 320

His Leu Asp Met Met Met Val Cys His His Leu Asp Ser Lys Val Pro

325 330 335

Glu Asp Val Ala Phe Ser His Ser Arg Ile Arg Ala Ala Thr Ile Ala

340 345 350

Ala Glu Asp Ile Leu His Asp Ile Gly Ala Ile Ser Met Thr Ser Ser

355 360 365

Asp Ser Gln Ala Thr Gly Arg Val Gly Glu Val Ile Ile Arg Thr Trp

370 375 380

Gln Val Ala Asp Lys Met Lys Lys Gln Arg Gly Ala Leu Ser Gly Glu

385 390 395 400

Asn Gly Asn Asp Asn Val Arg Ala Lys Arg Tyr Ile Ala Lys Tyr Thr

405 410 415

Ile Asn Pro Ala Val Thr His Gly Leu Ser His Glu Val Gly Ser Val

420 425 430

Glu Lys Gly Lys Leu Ala Asp Leu Val Leu Trp Asp Pro Val Phe Phe

435 440 445

Gly Val Lys Pro Glu Leu Val Leu Lys Gly Gly Met Ile Ala Arg Ala

450 455 460

Gln Met Gly Asp Pro Asn Ala Ser Ile Pro Thr Pro Glu Pro Val Phe

465 470 475 480

Met Arg Gln Met Tyr Ala Ser Tyr Gly Lys Ala Asn Arg Asn Thr Ser

485 490 495

Ile Thr Phe Met Ser Gln Ala Gly Ile Ala Asn Gly Val Pro Glu Lys

500 505 510

Leu Gly Leu Glu Lys Met Ile Ser Pro Val Arg Asn Ile Arg Lys Leu

515 520 525

Ser Lys Leu Asp Met Lys Leu Asn Asp Ala Met Pro Asn Ile Arg Val

530 535 540

Asp Pro Lys Thr Tyr Gln Val Phe Ala Asp Gly Glu Glu Leu Ala Cys

545 550 555 560

Gln Pro Val Ser Tyr Val Pro Leu Gly Gln Arg Tyr Phe Leu Phe Met

565 570 575

Lys Leu Thr Pro Val Glu Gln Glu Lys Leu Leu Ile Phe Thr Ala Gly

580 585 590

Glu Leu Ala Lys Gln Arg Lys Ala Arg Gly Val Leu Leu Asn Tyr Pro

595 600 605

Glu Ala Ala Ala Tyr Leu Thr Cys Tyr Leu Met Glu Gly Ala Arg Asp

610 615 620

Gly Lys Ser Val Ala Glu Leu Met Glu Ser Gly Arg Asn Val Leu Thr

625 630 635 640

Glu Lys Asp Val Met Glu Gly Val Ala Glu Met Leu Asp Ser Ile Gln

645 650 655

Val Glu Ala Thr Phe Pro Asp Gly Val Lys Leu Val Thr Val His Gln

660 665 670

Pro Ile Lys Ala Glu Val Lys Ser Met Lys Pro Gly Ala Ile Gln Val

675 680 685

Ala Lys Gly Thr Ile Thr Ile Asn Glu Gly Arg Lys Thr Leu Glu Val

690 695 700

Ser Val Thr Asn Asn Gly Thr Arg Ser Val Gln Val Gly Ser His Phe

705 710 715 720

His Phe Ala Glu Ala Asn Gly Ala Leu Ser Phe Asn Arg Asp Lys Ala

725 730 735

Ile Gly Met Arg Leu Asp Ile Pro Ser Gly Thr Ser Val Arg Phe Glu

740 745 750

Pro Gly Glu Glu Lys Thr Val Thr Leu Val Glu Ile Gly Gly Arg Lys

755 760 765

Thr Val Arg Gly Leu Asn Gly Met Ala Asp Thr Tyr Met Asp Glu Arg

770 775 780

Gly Lys Glu Lys Thr Leu Ser Asn Leu Lys Lys Ala Gly Trp Met Glu

785 790 795 800

Glu Ala Ile Arg

<210> 8

<211> 2421

<212> DNA

<213> 人工序列

<400> 8

atgcaccacc accaccacca caaaatgtcg cgtgagcaat acgcagaact gttcggaccg 60

acaacgggag acaaagtcag actcggagat acggatttat ggattgaagt cgaaaaagat 120

ttcacgaatt acggggaaga aatgattttc ggcggaggga aaacaatccg ggacggcatg 180

gggcagaacg ggcgtatcac cgggaaagac ggagcgcttg atctggtcat tacaaacgcc 240

gtcatcctgg attataccgg aatcgtcaag gcggatatcg gggtgaagga cggccggatt 300

gtcggcgtcg ggaaaagcgg caaccctgat atgatggatg gggtggaccc gcacatgatt 360

atcggtgccg gaacagaagt catttccggc gaagggaaaa tcgtaacggc cggcggggtg 420

gatacgcata tccactttat ctgcccgcag cagatggaag tcgcgctttc ttcaggcgtg 480

actacgcttc tcggcggcgg aacaggtcct gcgacgggaa gtaaagcgac gacttgtaca 540

tccggtgtat ggtacatgtc gagaatgctg gaagcggccg aggagtttcc gatcaatgtc 600

ggtttcttag gaaaaggaaa tgcatccgat aaagcgccgc tgatcgagca ggtggaagca 660

ggcgcaatcg gcctgaagct gcatgaagat tggggatcaa cgccaagcgc tattaaagct 720

tgcatggaag cagcggatga ggcggacatt caggtggcga tccacacaga cacgataaat 780

gaagcgggct ttttagaaaa tacgcttgat gcgatcggcg accgggttat ccatacatat 840

cacatagagg gagccggcgg aggccatgca ccggatatta tgaaactcgc atcttacgcc 900

aatatcctgc cgtcctctac gacgccgacg attccatata ccgtcaacac gatggacgag 960

catcttgata tgatgatggt ctgccatcat ttagattcaa aagtgcctga agacgtggcg 1020

ttcagtcatt cacgcatcag agcggccacc attgcggcgg aggatattct gcacgatatc 1080

ggcgcgatca gcatgacgtc atctgactcg caggcgactg gcagggtggg agaagtgatt 1140

atccggacat ggcaggtggc cgataaaatg aaaaaacagc gcggtgctct atcgggagaa 1200

aacggcaatg acaatgtgcg cgccaaacgc tatatcgcca aatacacgat caacccggct 1260

gtcactcacg gtctgagcca tgaagtcggt tccgttgaaa aaggaaagct cgccgacctc 1320

gtactatggg acccggtttt cttcggcgtc aaacctgaac ttgtgctcaa aggcggcatg 1380

attgcccgcg cccagatggg agatccgaat gcttccattc cgacgcctga gcccgtgttt 1440

atgcggcaga tgtacgcatc atacggtaaa gcaaaccgca acacctctat tacatttatg 1500

tcccaggccg gtatcgcaaa cggtgtgccg gaaaagctcg gccttgaaaa aatgatttct 1560

cccgtacgga atatccgtaa gctgagtaag ctcgacatga agctgaatga cgcgatgccg 1620

aatatacgtg tcgatccgaa aacctatcag gtgttcgccg acggagaaga gctggcatgc 1680

cagcccgtca gctatgttcc gctaggacag cgttatttct tattttaaat gaaactgaca 1740

ccggttgaac aagaaaaatt gcttattttt acggcgggag agctcgctaa gcagcggaag 1800

gcgcgcggcg ttctgctgaa ttatcccgaa gccgccgcat atttgacctg ttatctgatg 1860

gaaggcgcga gagacggaaa aagcgttgct gagctgatgg aatccggccg caatgtattg 1920

acggaaaaag acgttatgga aggcgttgcg gaaatgctgg acagcattca ggtggaagcg 1980

acgttcccgg acggggttaa gcttgtcacc gttcatcagc cgatcaaagc ggaggtgaag 2040

tcatgaatga agccgggagc gattcaagtc gcaaagggga ccatcaccat taacgaaggc 2100

cggaagacgc tggaggtgtc agtcaccaat aacggaacgc ggtcagtgca ggtcggatcg 2160

cattttcatt ttgccgaagc caacggcgcc ctttccttca atcgggacaa agccatcggc 2220

atgcgccttg atatcccgtc aggcacatct gtccgttttg aaccgggaga agagaaaacc 2280

gtcacgctcg tggaaatcgg agggcgaaaa acggtcagag gtctcaacgg catggccgat 2340

acgtacatgg atgagcgggg aaaagagaag acgctgtcaa atcttaaaaa agccggatgg 2400

atggaggagg cgatccgatg a 2421

<210> 9

<211> 20

<212> DNA

<213> 人工序列

<400> 9

ttaacctagg ctgctgccac 20

<210> 10

<211> 33

<212> DNA

<213> 人工序列

<400> 10

ggtatatctc cttattaaag ttaaacaaaa tta 33

<210> 11

<211> 15

<212> DNA

<213> 人工序列

<220>

<221> misc_feature

<222> (8)..(9)

<223> n is a, c, g, or t

<400> 11

gcaggcgnnk ggcag 15

<210> 12

<211> 15

<212> DNA

<213> 人工序列

<220>

<221> misc_feature

<222> (7)..(8)

<223> n is a, c, g, or t

<400> 12

ctgccmnncg cctgc 15

<210> 13

<211> 23

<212> DNA

<213> 人工序列

<220>

<221> misc_feature

<222> (8)..(9)

<223> n is a, c, g, or t

<400> 13

tgatatgnnk atggtctgcc atc 23

<210> 14

<211> 24

<212> DNA

<213> 人工序列

<220>

<221> misc_feature

<222> (7)..(8)

<223> n is a, c, g, or t

<400> 14

accatmnnca tatcaagatg ctcg 24

Claims

1.一种脲酶突变体，其特征在于，是以氨基酸序列如SEQ ID NO.1所示的脲酶为亲本脲酶，将亲本脲酶氨基酸的第325位或第373位进行突变。

2.根据权利要求1所述的脲酶突变体，其特征在于，将亲本脲酶氨基酸的第325位甲硫氨酸突变为缬氨酸，或将373位的甲硫氨酸突变为丙氨酸或苏氨酸。

3.编码权利要求1或2所述脲酶突变体的基因。

4.表达权利要求1或2所述脲酶突变体的宿主细胞。

5.根据权利要求4所述的宿主细胞，其特在在于，所述宿主细胞为真菌或细菌。

6.携带权利要求3所述基因的表达载体。

7.根据权利要求6所述的表达载体，其特在在于，所述表达载体为pET系列、Duet系列、pGEX系列、pPIC3K或pPIC9K系列中的任意一种。

8.一种降低氨基甲酸乙酯的方法，其特征在于，所述方法是将权利要求1或2所述脲酶突变体添加至含有氨基甲酸乙酯的体系中进行处理。

9.根据权利要求8所述的方法，其特征在于，所述脲酶突变体的添加量为3000～10000U/L，在30～40℃下反应40～60h。

10.权利要求1或2所述脲酶突变体或权利要求3所述基因或权利要求5所述的宿主细胞或权利要求7所述表达载体在降解食品中的尿素及氨基甲酸乙酯中的应用。