CN114196659B - 酰胺酶突变体、编码基因、工程菌及其应用 - Google Patents
酰胺酶突变体、编码基因、工程菌及其应用 Download PDFInfo
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- CN114196659B CN114196659B CN202111496213.4A CN202111496213A CN114196659B CN 114196659 B CN114196659 B CN 114196659B CN 202111496213 A CN202111496213 A CN 202111496213A CN 114196659 B CN114196659 B CN 114196659B
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Abstract
本发明公开了一种酰胺酶突变体、编码基因、工程菌及其在拆分2,2‑二甲基环丙烷甲酰胺制备(S)‑2,2‑二甲基环丙烷甲酰胺中的应用，所述酰胺酶突变体是将SEQ ID NO.2所示氨基酸序列第154位甘氨酸替换为天冬酰胺、苯丙氨酸或谷氨酸获得的。本发明提供的酰胺酶突变体较亲本活力提高1.8倍，对映体选择率(E值)由42提高至162，进一步提高了酰胺酶法生产(S)‑2,2‑二甲基环丙烷甲酰胺的效率。
Description
(一)技术领域
本发明涉及酶工程技术领域，具体涉及一种酰胺酶突变体、编码基因及其在催化合成(S)-2,2-二甲基环丙烷甲酰胺应用。
(二)背景技术
酰胺酶是一类重要工业酶，能催化各类天然、非天然酰胺类化合物水解生成相应羧酸。因其高立体选择性、广底物谱等优势，以酰胺酶为催化剂的(手性)羧酸和(手性)酰胺及其衍生物的生物合成日益受到重视。如瑞士Lonza公司利用Klebsiella terrigenaDSM9174酰胺酶拆分外消旋哌嗪-2-甲酰胺制备S-哌嗪-2-甲酸，光学纯度达99.4％(US5945534)；来源于Rhodococcus erythropolis AJ270酰胺酶立体选择性水解氧杂环内消旋二酰胺合成相应羧酸，产物光学纯度达99.5％以上(ACS Catal.2021,11:6900-6907)。
根据氨基酸序列特征，酰胺酶可分为酰胺酶标签家族(Amidase SignatureFamily)和腈水解酶家族(Nitrilase Family)两大类。标签家族酰胺酶的氨基酸序列含有130个左右高度保守氨基酸区域，并含有催化三联体Lys-Ser-Ser，作用底物范围较广，可水解脂肪族、芳香族及杂环类酰胺。目前发现的具有立体选择性的酰胺酶绝大多数都来源于标签家族。
亚胺培南/西司他丁钠是美国默克公司研制开发的一种广谱β-内酰胺抗生素，具有抗菌谱广、抗菌活性强等优点。来源于Delftia tsuruhatensis标签家族酰胺酶可催化外消旋2,2-二甲基环丙烷甲酰胺合成亚胺培南/西司他丁钠关键手性中间体(S)-2,2-二甲基环丙烷甲酰胺(ZL200510061680.9；ZL200910155659.3)。本发明在此基础上，利用半理性设计方法进一步获得立体选择性和酶活力提高的突变体，提高生物法合成(S)-2,2-二甲基环丙烷甲酰胺的效率。
(三)发明内容
本发明目的是提供一种酰胺酶突变体、编码基因及其在催化合成(S)-2,2-二甲基环丙烷甲酰胺中的应用，通过定点饱和突变技术对来源于D.tsuruhatensis的酰胺酶Dt-Ami6(GenBank NO.KP943494)进行分子改造，筛选获得一种突变体蛋白，提高其对外消旋2,2-二甲基环丙甲酰胺的水解活力和立体选择性，从而有利于该酰胺酶在(S)-2,2-二甲基环丙烷甲酰胺制备中的应用。
本发明采用的技术方案是：
本发明提供一种酰胺酶突变体，所述酰胺酶突变体是将SEQ ID NO.2所示氨基酸序列第154位丙氨酸替换为天冬酰胺、苯丙氨酸或谷氨酸获得的，优选天冬酰胺。
本发明涉及所述酰胺酶突变体的编码基因，编码基因构建的重组载体以及重组载体转化制备的重组基因工程菌。所述重组载体以载体pET28为基础载体，基因在载体中的嵌入位点为Nco I和EcoRI，所述重组基因工程菌以E.coli BL21(DE3)为宿主菌。
本发明还涉及一种所述酰胺酶突变体在拆分2,2-二甲基环丙烷甲酰胺制备(S)-2,2-二甲基环丙烷甲酰胺中的应用，所述的应用为：以酰胺酶突变体工程菌经发酵培养获得的湿菌体或者湿菌体破碎后提取的酶作为催化剂，以2,2-二甲基环丙烷甲酰胺为底物，以pH为7.5～8.5(优选pH值为8.0)的缓冲液(优选Tris-HCl缓冲液)为反应介质构成转化体系，在30～50℃、150～500r/min条件下(优选30℃、200r/min)进行转化反应，反应结束后，取反应液分离纯化，获得(R)-2,2-二甲基环丙烷甲酸和(S)-2,2-二甲基环丙烷甲酰胺。所述反应体系中，底物的初始浓度为10～50mM(优选50mM)，催化剂的用量以菌体质量(细胞干重)计为0.25～0.75g/L(优选0.5g/L)。
本发明所述湿菌体按如下方法制备：将酰胺酶突变体工程菌接种到含有终浓度50mg/L卡那霉素的LB培养基中，37℃、150r/min培养12h，随后以体积浓度1％接种量转接到新鲜的含终浓度50mg/L卡那霉素的LB培养基中，37℃、150r/min培养至菌体浓度OD600为0.4～0.8，再向培养基中加入终浓度为0.1～1.0mM的IPTG(优选0.1mM)，28℃、150r/min诱导培养12h，取培养物离心，收集沉淀即获得湿菌体。LB液体培养基组成为：蛋白胨10g/L，酵母提取物5g/L，NaCl 10g/L，pH值为7.0；LB平板培养基组成为：蛋白胨10g/L，酵母提取物5g/L，NaCl 10g/L，琼脂15g/L，溶剂为去离子水，pH值为7.0。
本发明所述的酰胺酶突变体可以以全细胞形式使用，也可以是未经纯化的粗酶形式使用，还可以是经部分纯化或完全纯化的酶蛋白形式使用。如果需要，还可以利用本领域已知的固定化技术将本发明的酰胺酶突变体制成固定化酶或者固定化细胞形式进行使用。
本发明的酰胺酶突变体较亲本活力和立体选择性大幅提高，在使用该酶的粗提取物或工程菌全细胞进行催化时，反应酶活仍然保持较高状态。
与现有技术相比，本发明的有益效果主要体现在：本发明提供的酰胺酶突变体较亲本活力提高1.8倍，对映体选择率(E值)由42提高至162，进一步提高了酰胺酶法生产(S)-2,2-二甲基环丙烷甲酰胺的效率。
(四)附图说明
图1为实施例2突变体A154N细胞催化外消旋2,2-二甲基环丙烷甲酰胺水解气相色谱分析图谱。
图2为实施例3中0.25g/L突变体A154N细胞催化外消旋2,2-二甲基环丙烷甲酰胺水解反应进程。
图3为实施例4中0.5g/L突变体A154N细胞催化外消旋2,2-二甲基环丙烷甲酰胺水解反应进程。
图4为实施例5中0.75g/L突变体A154N细胞催化外消旋2,2-二甲基环丙烷甲酰胺水解反应进程。
(五)具体实施方式
下面结合具体实施例对本发明进行进一步描述，但本发明的保护范围并不仅限于此：
LB液体培养基组成为：蛋白胨10g/L，酵母提取物5g/L，NaCl 10g/L，pH值为7.0。
LB平板培养基组成为：蛋白胨10g/L，酵母提取物5g/L，NaCl 10g/L，琼脂15g/L，溶剂为去离子水，pH值为7.0。
实施例1.酰胺酶突变文库构建
定点饱和突变技术参考(Applied Microbiology and Biotechnology,2014,98(6):2473-2483)的描述，阳性突变子的高通量筛选模型参考(ZL200510062182.6；AppliedMicrobiology and Biotechnology,2007,74:256-262)的描述，具体过程如下：
步骤一：酰胺酶定点突变
从基因文库中选取戴尔福特菌(D.tsuruhatensis)来源的酰胺酶Dt-Ami6标签序列(GenBank NO.KP943494)，以克隆有Dt-Ami6编码基因(核苷酸序列如SEQ ID NO.1所示，氨基酸序列如SEQ ID NO.2所示)的质粒pET28-Dt-Ami6为模板(基因在载体中的嵌入位点为Nco I和EcoRI)，设计全饱和突变引物(表1)对154位点进行全饱和突变，进行全质粒扩增。用0.9％琼脂糖凝胶电泳分析PCR产物，取PCR产物20μL，加入1μL的DpnI，37℃酶切3h去除模板质粒DNA，于65℃下灭活10min。
PCR体系为：2×phanta Max buffer 25μL，dNTP mixture(10mM)1μL，如表1所示的突变引物10μM各1μL，质粒pET28-Dt-Ami6 0.5μL，Phanta Max DNA聚合酶0.5μL，ddH2O补至50μL。
PCR条件为：95℃预变性2min；95℃变性30s，60℃退火20s，72℃延伸4.5min，进行30个循环；最后72℃延伸10min。
步骤二：酰胺酶突变体的转化
取感受态细胞E.coli BL21(DE3)，加入10μL步骤一中的PCR产物，冰上静置30min，在42℃热激90s，加入600μL不含卡那霉素的LB培养基，37℃下以180r/min转速培养1h，涂布于含卡那霉素(50mg/L)的LB平板，37℃培养过夜。
步骤三：菌体培养及高通量筛选
突变菌：挑取步骤二单菌落分别接种到96孔培养板(装有1mL含50mg/L卡那霉素的LB培养基)中，37℃、150r/min培养至OD600＝0.5，然后加入终浓度为0.1mM的IPTG，28℃下培养12h，分别获得突变菌菌液。
含野生型酰胺酶的工程菌：同样条件下，制备含Dt-Ami6编码基因(核苷酸序列如SEQ ID NO.1所示)的E.coli BL21(DE3)的菌液为对照。
取新的96孔培养板，分别逐孔对应加入100μL培养的菌液(突变菌菌液和对照菌液)，终浓度为50mM的(R)-2,2-二甲基环丙烷甲酰胺、终浓度为50mM的(S)-2,2-二甲基环丙烷甲酰胺和终浓度为50mM的盐酸羟胺，30℃、200r/min反应300min，向培养孔中加入100μL浓度为96g/L的FeCl3盐酸水溶液(将48g FeCl3溶于25mL质量浓度36％的浓盐酸，再加入475mL水配制)为显色剂，以含野生型酰胺酶的工程菌细胞为参照，根据显色颜色变化(黄绿色→深红色)程度判定酰胺酶突变体对(R)-2,2-二甲基环丙烷甲酰胺和(S)-2,2-二甲基环丙烷甲酰胺的活力高低，筛选出对(R)-2,2-二甲基环丙烷甲酰胺底物变色程度高于对照组或对(S)-2,2-二甲基环丙烷甲酰胺底物变色程度低于对照组的菌株作为初筛阳性菌。
表1定点饱和突变引物设计表
注：N＝A/G/C/T，K＝G/T，M＝A/C，
实施例2.酰胺酶阳性突变体的复筛
将实施例1所得阳性菌接种到100mL含50μg/mL卡那霉素的LB摇瓶培养基中，37℃，150r/min培养至菌体浓度OD600至0.6，加入终浓度为0.1mM的IPTG，28℃诱导培养12h后，4℃、12000rpm离心10min收集菌体，用0.85％的生理盐水清洗后重复上述离心步骤，置于-20℃保藏备用，即为静息细胞，用于水解反应。
称取静息细胞加入20mM Tris-HCl(pH 8.0)缓冲液打匀，制成细胞浓度为100g/L的菌悬液。突变体活力测定的反应体系为：总体系10mL，细胞终浓度为2g/L，20mM Tris-HCl(pH 8.0)以及20mM 2,2-二甲基环丙烷甲酰胺。在30℃，200r/min振荡反应10min后，取1mL加入100μL 2.0M盐酸终止反应，加入800μL乙酸乙酯萃取，离心，取上清，加入少量无水硫酸钠后使用气相色谱检测(图1)。取全细胞酶活和立体选择性高于野生型的菌株，提取质粒进行测序，结果见表2。活力和立体选择性提高的阳性克隆DNA测序显示其第154位的丙氨酸替换为天冬酰胺(核苷酸序列如SEQ ID No.3所示，氨基酸序列如SEQ ID No.4所示)、苯丙氨酸(核苷酸序列如SEQ ID No.5所示，氨基酸序列如SEQ ID No.6所示)和谷氨酸(核苷酸序列如SEQ ID No.7所示，氨基酸序列如SEQ ID No.8所示)。
气相色谱检测方法：BGB-174毛细管气相色谱柱(柱长30m，内径0.25mm，液膜厚度0.25μm)；载气为高纯氦气，流量为1.0mL/min，进样量1μL，分流比为30:1；检测器及进样口温度均为220℃，柱温100℃保持5min，然后以5℃·min-1升温至160℃并保持2min。
酶活单位(U)定义：在30℃、pH 8.0条件下，每分钟生成1μmol(R)-2,2-二甲基环丙烷甲酸所需的酶量作为一个活力单位(U)。
表2、酰胺酶突变体对2,2-二甲基环丙烷甲酰胺的立体选择性和比活力
实施例3.酰胺酶突变体A154N全细胞催化2,2-二甲基环丙烷甲酰胺水解(一)
按实施例2方法制备重组菌E.coli BL21(DE3)/pET28-A154N静息细胞作为催化剂，以2,2-二甲基环丙烷甲酰胺为底物进行拆分反应。反应体系(10mL)：20mM Tris-HCl缓冲液(pH 8.0)，50mM 2,2-二甲基环丙烷甲酰胺，0.25g/L细胞(细胞干重)，于30℃、200r/min下进行反应。定时取样，用2.0M盐酸终止反应，同实施例2方法进行气相色谱检测。结果表明，反应20min后，转化率可达50.6％，(R)-2,2-二甲基环丙烷甲酸ee为97.58％，(S)-2,2-二甲基环丙烷甲酰胺ee>99.9％(图2)。
实施例4.酰胺酶突变体A154N全细胞催化2,2-二甲基环丙烷甲酰胺水解(二)
将实施例3中催化剂用量改为0.5g/L细胞(细胞干重)，其他操作相同。结果表明，反应20min后，反应转化率可达54.9％，(R)-2,2-二甲基环丙烷甲酸ee为82.0％，(S)-2,2-二甲基环丙甲烷酰胺ee>99.9％(图3)。
实施例5.酰胺酶突变体A154N全细胞催化2,2-二甲基环丙烷甲酰胺水解(三)
将实施例3中催化剂用量改为0.75g/L细胞(细胞干重)，其他操作相同。结果表明，反应20min后，转化率可达56.3％，(R)-2，2-二甲基环烷丙甲酸ee为77.5％，(S)-2,2-二甲基环丙烷甲酰胺ee>99.9％(如图4)。
序列表
<110> 浙江工业大学
<120> 酰胺酶突变体、编码基因、工程菌及其应用
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1398
<212> DNA
<213> 未知(Unknown)
<400> 1
atgaacgatt ctgaactgca tcacctggaa ctgctggaag tgggtcgcga aattcagtct 60
cgtcgtatct cttctgagga agttactcgc cacatgctgg cgcgtattga ggcagtcgac 120
gcgcgcctgc attcctacgt tactgtgatg gcgcagcagg cactggaaga cgctcgccgt 180
gcggacgctg agattgcaca gggtcgtcgt cgtggcgcac tgcacggtgt accgctggcg 240
ctgaaagatc tgctgtggac tcgcggcgtg ccgactaccc acggtatgac cctgcaccgt 300
gaacatcgtc cgaccgaaga cgcgactgta gtccgtcgtc tgcgtgaggc tggtgccgtt 360
atcctgggta aactgcagca gactgaaggc gcttttgctg atcatcatcc ggaaatcacc 420
gcaccggtca atccgtgggg tgcccagctg tggccgggtg cgtctagcag cggttctggc 480
gttgcgaccg cggctggcct gtgtttcggt tccctgggta ccgataccgg cggcagcatc 540
cgctttccat ctgccgcgaa cggtatcacg ggcctgaaac cgacctgggg ccgcgtgtcc 600
cgtcacggcg ctttcgaact ggcagcaagc ctggaccata tcggcccgat ggcgcgctct 660
gccgcggacg cagcggcgat gctggcggct attgccggtg cagatccgct ggacccgacc 720
gcatctcagt gttccgtgcc ggattacctg gctatgatga cccgtggctt ctccggtctg 780
cgcctgggta tggaccgtca gtgggcgctg gatggcgtgg acgctccgtc ccgtcaagcg 840
gttgaacagg cgctggcggt tgctcagcgc ctgggtgcga gcgttcagga agtacgtttc 900
ccggacgcga cccaggcggt acaggattgg ccagcactgt gcgcagtgga aaccgctgtg 960
gcacatggcg caacctttcc agctcgtcgc gaagcctacg gtccgggtct ggctggcctg 1020
atcgatctgg gtctgggtct gtctgcaacc gactatcaac gtctgctgct gcgccgtgct 1080
gacttcactg gccgtgttcg tgcactgttt gcccaagtgg atctgctgct ggtgccggct 1140
actgctttcg ctgcgccaac cctgcaacgc atggcgcatt tcggtagcga cgccgaactg 1200
ttctctggca tgctgcgtta cacctgtcct ttcgacctga cgggttctcc gactatcact 1260
ctgccaggcg gtcgtactcc ggagggcgca ccggttgctt tccagttcgt agccccggat 1320
ttccgtgaag atctgctggt gcgtgcgggc tgggccttcc agcaggcgac cgactggcac 1380
cgtcagcacc cggctgct 1398
<210> 2
<211> 466
<212> PRT
<213> 未知(Unknown)
<400> 2
Met Asn Asp Ser Glu Leu His His Leu Glu Leu Leu Glu Val Gly Arg
1 5 10 15
Glu Ile Gln Ser Arg Arg Ile Ser Ser Glu Glu Val Thr Arg His Met
20 25 30
Leu Ala Arg Ile Glu Ala Val Asp Ala Arg Leu His Ser Tyr Val Thr
35 40 45
Val Met Ala Gln Gln Ala Leu Glu Asp Ala Arg Arg Ala Asp Ala Glu
50 55 60
Ile Ala Gln Gly Arg Arg Arg Gly Ala Leu His Gly Val Pro Leu Ala
65 70 75 80
Leu Lys Asp Leu Leu Trp Thr Arg Gly Val Pro Thr Thr His Gly Met
85 90 95
Thr Leu His Arg Glu His Arg Pro Thr Glu Asp Ala Thr Val Val Arg
100 105 110
Arg Leu Arg Glu Ala Gly Ala Val Ile Leu Gly Lys Leu Gln Gln Thr
115 120 125
Glu Gly Ala Phe Ala Asp His His Pro Glu Ile Thr Ala Pro Val Asn
130 135 140
Pro Trp Gly Ala Gln Leu Trp Pro Gly Ala Ser Ser Ser Gly Ser Gly
145 150 155 160
Val Ala Thr Ala Ala Gly Leu Cys Phe Gly Ser Leu Gly Thr Asp Thr
165 170 175
Gly Gly Ser Ile Arg Phe Pro Ser Ala Ala Asn Gly Ile Thr Gly Leu
180 185 190
Lys Pro Thr Trp Gly Arg Val Ser Arg His Gly Ala Phe Glu Leu Ala
195 200 205
Ala Ser Leu Asp His Ile Gly Pro Met Ala Arg Ser Ala Ala Asp Ala
210 215 220
Ala Ala Met Leu Ala Ala Ile Ala Gly Ala Asp Pro Leu Asp Pro Thr
225 230 235 240
Ala Ser Gln Cys Ser Val Pro Asp Tyr Leu Ala Met Met Thr Arg Gly
245 250 255
Phe Ser Gly Leu Arg Leu Gly Met Asp Arg Gln Trp Ala Leu Asp Gly
260 265 270
Val Asp Ala Pro Ser Arg Gln Ala Val Glu Gln Ala Leu Ala Val Ala
275 280 285
Gln Arg Leu Gly Ala Ser Val Gln Glu Val Arg Phe Pro Asp Ala Thr
290 295 300
Gln Ala Val Gln Asp Trp Pro Ala Leu Cys Ala Val Glu Thr Ala Val
305 310 315 320
Ala His Gly Ala Thr Phe Pro Ala Arg Arg Glu Ala Tyr Gly Pro Gly
325 330 335
Leu Ala Gly Leu Ile Asp Leu Gly Leu Gly Leu Ser Ala Thr Asp Tyr
340 345 350
Gln Arg Leu Leu Leu Arg Arg Ala Asp Phe Thr Gly Arg Val Arg Ala
355 360 365
Leu Phe Ala Gln Val Asp Leu Leu Leu Val Pro Ala Thr Ala Phe Ala
370 375 380
Ala Pro Thr Leu Gln Arg Met Ala His Phe Gly Ser Asp Ala Glu Leu
385 390 395 400
Phe Ser Gly Met Leu Arg Tyr Thr Cys Pro Phe Asp Leu Thr Gly Ser
405 410 415
Pro Thr Ile Thr Leu Pro Gly Gly Arg Thr Pro Glu Gly Ala Pro Val
420 425 430
Ala Phe Gln Phe Val Ala Pro Asp Phe Arg Glu Asp Leu Leu Val Arg
435 440 445
Ala Gly Trp Ala Phe Gln Gln Ala Thr Asp Trp His Arg Gln His Pro
450 455 460
Ala Ala
465
<210> 3
<211> 1398
<212> DNA
<213> 未知(Unknown)
<400> 3
atgaacgatt ctgaactgca tcacctggaa ctgctggaag tgggtcgcga aattcagtct 60
cgtcgtatct cttctgagga agttactcgc cacatgctgg cgcgtattga ggcagtcgac 120
gcgcgcctgc attcctacgt tactgtgatg gcgcagcagg cactggaaga cgctcgccgt 180
gcggacgctg agattgcaca gggtcgtcgt cgtggcgcac tgcacggtgt accgctggcg 240
ctgaaagatc tgctgtggac tcgcggcgtg ccgactaccc acggtatgac cctgcaccgt 300
gaacatcgtc cgaccgaaga cgcgactgta gtccgtcgtc tgcgtgaggc tggtgccgtt 360
atcctgggta aactgcagca gactgaaggc gcttttgctg atcatcatcc ggaaatcacc 420
gcaccggtca atccgtgggg tgcccagctg tggccgggta actctagcag cggttctggc 480
gttgcgaccg cggctggcct gtgtttcggt tccctgggta ccgataccgg cggcagcatc 540
cgctttccat ctgccgcgaa cggtatcacg ggcctgaaac cgacctgggg ccgcgtgtcc 600
cgtcacggcg ctttcgaact ggcagcaagc ctggaccata tcggcccgat ggcgcgctct 660
gccgcggacg cagcggcgat gctggcggct attgccggtg cagatccgct ggacccgacc 720
gcatctcagt gttccgtgcc ggattacctg gctatgatga cccgtggctt ctccggtctg 780
cgcctgggta tggaccgtca gtgggcgctg gatggcgtgg acgctccgtc ccgtcaagcg 840
gttgaacagg cgctggcggt tgctcagcgc ctgggtgcga gcgttcagga agtacgtttc 900
ccggacgcga cccaggcggt acaggattgg ccagcactgt gcgcagtgga aaccgctgtg 960
gcacatggcg caacctttcc agctcgtcgc gaagcctacg gtccgggtct ggctggcctg 1020
atcgatctgg gtctgggtct gtctgcaacc gactatcaac gtctgctgct gcgccgtgct 1080
gacttcactg gccgtgttcg tgcactgttt gcccaagtgg atctgctgct ggtgccggct 1140
actgctttcg ctgcgccaac cctgcaacgc atggcgcatt tcggtagcga cgccgaactg 1200
ttctctggca tgctgcgtta cacctgtcct ttcgacctga cgggttctcc gactatcact 1260
ctgccaggcg gtcgtactcc ggagggcgca ccggttgctt tccagttcgt agccccggat 1320
ttccgtgaag atctgctggt gcgtgcgggc tgggccttcc agcaggcgac cgactggcac 1380
cgtcagcacc cggctgct 1398
<210> 4
<211> 466
<212> PRT
<213> 未知(Unknown)
<400> 4
Met Asn Asp Ser Glu Leu His His Leu Glu Leu Leu Glu Val Gly Arg
1 5 10 15
Glu Ile Gln Ser Arg Arg Ile Ser Ser Glu Glu Val Thr Arg His Met
20 25 30
Leu Ala Arg Ile Glu Ala Val Asp Ala Arg Leu His Ser Tyr Val Thr
35 40 45
Val Met Ala Gln Gln Ala Leu Glu Asp Ala Arg Arg Ala Asp Ala Glu
50 55 60
Ile Ala Gln Gly Arg Arg Arg Gly Ala Leu His Gly Val Pro Leu Ala
65 70 75 80
Leu Lys Asp Leu Leu Trp Thr Arg Gly Val Pro Thr Thr His Gly Met
85 90 95
Thr Leu His Arg Glu His Arg Pro Thr Glu Asp Ala Thr Val Val Arg
100 105 110
Arg Leu Arg Glu Ala Gly Ala Val Ile Leu Gly Lys Leu Gln Gln Thr
115 120 125
Glu Gly Ala Phe Ala Asp His His Pro Glu Ile Thr Ala Pro Val Asn
130 135 140
Pro Trp Gly Ala Gln Leu Trp Pro Gly Asn Ser Ser Ser Gly Ser Gly
145 150 155 160
Val Ala Thr Ala Ala Gly Leu Cys Phe Gly Ser Leu Gly Thr Asp Thr
165 170 175
Gly Gly Ser Ile Arg Phe Pro Ser Ala Ala Asn Gly Ile Thr Gly Leu
180 185 190
Lys Pro Thr Trp Gly Arg Val Ser Arg His Gly Ala Phe Glu Leu Ala
195 200 205
Ala Ser Leu Asp His Ile Gly Pro Met Ala Arg Ser Ala Ala Asp Ala
210 215 220
Ala Ala Met Leu Ala Ala Ile Ala Gly Ala Asp Pro Leu Asp Pro Thr
225 230 235 240
Ala Ser Gln Cys Ser Val Pro Asp Tyr Leu Ala Met Met Thr Arg Gly
245 250 255
Phe Ser Gly Leu Arg Leu Gly Met Asp Arg Gln Trp Ala Leu Asp Gly
260 265 270
Val Asp Ala Pro Ser Arg Gln Ala Val Glu Gln Ala Leu Ala Val Ala
275 280 285
Gln Arg Leu Gly Ala Ser Val Gln Glu Val Arg Phe Pro Asp Ala Thr
290 295 300
Gln Ala Val Gln Asp Trp Pro Ala Leu Cys Ala Val Glu Thr Ala Val
305 310 315 320
Ala His Gly Ala Thr Phe Pro Ala Arg Arg Glu Ala Tyr Gly Pro Gly
325 330 335
Leu Ala Gly Leu Ile Asp Leu Gly Leu Gly Leu Ser Ala Thr Asp Tyr
340 345 350
Gln Arg Leu Leu Leu Arg Arg Ala Asp Phe Thr Gly Arg Val Arg Ala
355 360 365
Leu Phe Ala Gln Val Asp Leu Leu Leu Val Pro Ala Thr Ala Phe Ala
370 375 380
Ala Pro Thr Leu Gln Arg Met Ala His Phe Gly Ser Asp Ala Glu Leu
385 390 395 400
Phe Ser Gly Met Leu Arg Tyr Thr Cys Pro Phe Asp Leu Thr Gly Ser
405 410 415
Pro Thr Ile Thr Leu Pro Gly Gly Arg Thr Pro Glu Gly Ala Pro Val
420 425 430
Ala Phe Gln Phe Val Ala Pro Asp Phe Arg Glu Asp Leu Leu Val Arg
435 440 445
Ala Gly Trp Ala Phe Gln Gln Ala Thr Asp Trp His Arg Gln His Pro
450 455 460
Ala Ala
465
<210> 5
<211> 1398
<212> DNA
<213> 未知(Unknown)
<400> 5
atgaacgatt ctgaactgca tcacctggaa ctgctggaag tgggtcgcga aattcagtct 60
cgtcgtatct cttctgagga agttactcgc cacatgctgg cgcgtattga ggcagtcgac 120
gcgcgcctgc attcctacgt tactgtgatg gcgcagcagg cactggaaga cgctcgccgt 180
gcggacgctg agattgcaca gggtcgtcgt cgtggcgcac tgcacggtgt accgctggcg 240
ctgaaagatc tgctgtggac tcgcggcgtg ccgactaccc acggtatgac cctgcaccgt 300
gaacatcgtc cgaccgaaga cgcgactgta gtccgtcgtc tgcgtgaggc tggtgccgtt 360
atcctgggta aactgcagca gactgaaggc gcttttgctg atcatcatcc ggaaatcacc 420
gcaccggtca atccgtgggg tgcccagctg tggccgggtt tttctagcag cggttctggc 480
gttgcgaccg cggctggcct gtgtttcggt tccctgggta ccgataccgg cggcagcatc 540
cgctttccat ctgccgcgaa cggtatcacg ggcctgaaac cgacctgggg ccgcgtgtcc 600
cgtcacggcg ctttcgaact ggcagcaagc ctggaccata tcggcccgat ggcgcgctct 660
gccgcggacg cagcggcgat gctggcggct attgccggtg cagatccgct ggacccgacc 720
gcatctcagt gttccgtgcc ggattacctg gctatgatga cccgtggctt ctccggtctg 780
cgcctgggta tggaccgtca gtgggcgctg gatggcgtgg acgctccgtc ccgtcaagcg 840
gttgaacagg cgctggcggt tgctcagcgc ctgggtgcga gcgttcagga agtacgtttc 900
ccggacgcga cccaggcggt acaggattgg ccagcactgt gcgcagtgga aaccgctgtg 960
gcacatggcg caacctttcc agctcgtcgc gaagcctacg gtccgggtct ggctggcctg 1020
atcgatctgg gtctgggtct gtctgcaacc gactatcaac gtctgctgct gcgccgtgct 1080
gacttcactg gccgtgttcg tgcactgttt gcccaagtgg atctgctgct ggtgccggct 1140
actgctttcg ctgcgccaac cctgcaacgc atggcgcatt tcggtagcga cgccgaactg 1200
ttctctggca tgctgcgtta cacctgtcct ttcgacctga cgggttctcc gactatcact 1260
ctgccaggcg gtcgtactcc ggagggcgca ccggttgctt tccagttcgt agccccggat 1320
ttccgtgaag atctgctggt gcgtgcgggc tgggccttcc agcaggcgac cgactggcac 1380
cgtcagcacc cggctgct 1398
<210> 6
<211> 466
<212> PRT
<213> 未知(Unknown)
<400> 6
Met Asn Asp Ser Glu Leu His His Leu Glu Leu Leu Glu Val Gly Arg
1 5 10 15
Glu Ile Gln Ser Arg Arg Ile Ser Ser Glu Glu Val Thr Arg His Met
20 25 30
Leu Ala Arg Ile Glu Ala Val Asp Ala Arg Leu His Ser Tyr Val Thr
35 40 45
Val Met Ala Gln Gln Ala Leu Glu Asp Ala Arg Arg Ala Asp Ala Glu
50 55 60
Ile Ala Gln Gly Arg Arg Arg Gly Ala Leu His Gly Val Pro Leu Ala
65 70 75 80
Leu Lys Asp Leu Leu Trp Thr Arg Gly Val Pro Thr Thr His Gly Met
85 90 95
Thr Leu His Arg Glu His Arg Pro Thr Glu Asp Ala Thr Val Val Arg
100 105 110
Arg Leu Arg Glu Ala Gly Ala Val Ile Leu Gly Lys Leu Gln Gln Thr
115 120 125
Glu Gly Ala Phe Ala Asp His His Pro Glu Ile Thr Ala Pro Val Asn
130 135 140
Pro Trp Gly Ala Gln Leu Trp Pro Gly Phe Ser Ser Ser Gly Ser Gly
145 150 155 160
Val Ala Thr Ala Ala Gly Leu Cys Phe Gly Ser Leu Gly Thr Asp Thr
165 170 175
Gly Gly Ser Ile Arg Phe Pro Ser Ala Ala Asn Gly Ile Thr Gly Leu
180 185 190
Lys Pro Thr Trp Gly Arg Val Ser Arg His Gly Ala Phe Glu Leu Ala
195 200 205
Ala Ser Leu Asp His Ile Gly Pro Met Ala Arg Ser Ala Ala Asp Ala
210 215 220
Ala Ala Met Leu Ala Ala Ile Ala Gly Ala Asp Pro Leu Asp Pro Thr
225 230 235 240
Ala Ser Gln Cys Ser Val Pro Asp Tyr Leu Ala Met Met Thr Arg Gly
245 250 255
Phe Ser Gly Leu Arg Leu Gly Met Asp Arg Gln Trp Ala Leu Asp Gly
260 265 270
Val Asp Ala Pro Ser Arg Gln Ala Val Glu Gln Ala Leu Ala Val Ala
275 280 285
Gln Arg Leu Gly Ala Ser Val Gln Glu Val Arg Phe Pro Asp Ala Thr
290 295 300
Gln Ala Val Gln Asp Trp Pro Ala Leu Cys Ala Val Glu Thr Ala Val
305 310 315 320
Ala His Gly Ala Thr Phe Pro Ala Arg Arg Glu Ala Tyr Gly Pro Gly
325 330 335
Leu Ala Gly Leu Ile Asp Leu Gly Leu Gly Leu Ser Ala Thr Asp Tyr
340 345 350
Gln Arg Leu Leu Leu Arg Arg Ala Asp Phe Thr Gly Arg Val Arg Ala
355 360 365
Leu Phe Ala Gln Val Asp Leu Leu Leu Val Pro Ala Thr Ala Phe Ala
370 375 380
Ala Pro Thr Leu Gln Arg Met Ala His Phe Gly Ser Asp Ala Glu Leu
385 390 395 400
Phe Ser Gly Met Leu Arg Tyr Thr Cys Pro Phe Asp Leu Thr Gly Ser
405 410 415
Pro Thr Ile Thr Leu Pro Gly Gly Arg Thr Pro Glu Gly Ala Pro Val
420 425 430
Ala Phe Gln Phe Val Ala Pro Asp Phe Arg Glu Asp Leu Leu Val Arg
435 440 445
Ala Gly Trp Ala Phe Gln Gln Ala Thr Asp Trp His Arg Gln His Pro
450 455 460
Ala Ala
465
<210> 7
<211> 1398
<212> DNA
<213> 未知(Unknown)
<400> 7
atgaacgatt ctgaactgca tcacctggaa ctgctggaag tgggtcgcga aattcagtct 60
cgtcgtatct cttctgagga agttactcgc cacatgctgg cgcgtattga ggcagtcgac 120
gcgcgcctgc attcctacgt tactgtgatg gcgcagcagg cactggaaga cgctcgccgt 180
gcggacgctg agattgcaca gggtcgtcgt cgtggcgcac tgcacggtgt accgctggcg 240
ctgaaagatc tgctgtggac tcgcggcgtg ccgactaccc acggtatgac cctgcaccgt 300
gaacatcgtc cgaccgaaga cgcgactgta gtccgtcgtc tgcgtgaggc tggtgccgtt 360
atcctgggta aactgcagca gactgaaggc gcttttgctg atcatcatcc ggaaatcacc 420
gcaccggtca atccgtgggg tgcccagctg tggccgggtg aatctagcag cggttctggc 480
gttgcgaccg cggctggcct gtgtttcggt tccctgggta ccgataccgg cggcagcatc 540
cgctttccat ctgccgcgaa cggtatcacg ggcctgaaac cgacctgggg ccgcgtgtcc 600
cgtcacggcg ctttcgaact ggcagcaagc ctggaccata tcggcccgat ggcgcgctct 660
gccgcggacg cagcggcgat gctggcggct attgccggtg cagatccgct ggacccgacc 720
gcatctcagt gttccgtgcc ggattacctg gctatgatga cccgtggctt ctccggtctg 780
cgcctgggta tggaccgtca gtgggcgctg gatggcgtgg acgctccgtc ccgtcaagcg 840
gttgaacagg cgctggcggt tgctcagcgc ctgggtgcga gcgttcagga agtacgtttc 900
ccggacgcga cccaggcggt acaggattgg ccagcactgt gcgcagtgga aaccgctgtg 960
gcacatggcg caacctttcc agctcgtcgc gaagcctacg gtccgggtct ggctggcctg 1020
atcgatctgg gtctgggtct gtctgcaacc gactatcaac gtctgctgct gcgccgtgct 1080
gacttcactg gccgtgttcg tgcactgttt gcccaagtgg atctgctgct ggtgccggct 1140
actgctttcg ctgcgccaac cctgcaacgc atggcgcatt tcggtagcga cgccgaactg 1200
ttctctggca tgctgcgtta cacctgtcct ttcgacctga cgggttctcc gactatcact 1260
ctgccaggcg gtcgtactcc ggagggcgca ccggttgctt tccagttcgt agccccggat 1320
ttccgtgaag atctgctggt gcgtgcgggc tgggccttcc agcaggcgac cgactggcac 1380
cgtcagcacc cggctgct 1398
<210> 8
<211> 466
<212> PRT
<213> 未知(Unknown)
<400> 8
Met Asn Asp Ser Glu Leu His His Leu Glu Leu Leu Glu Val Gly Arg
1 5 10 15
Glu Ile Gln Ser Arg Arg Ile Ser Ser Glu Glu Val Thr Arg His Met
20 25 30
Leu Ala Arg Ile Glu Ala Val Asp Ala Arg Leu His Ser Tyr Val Thr
35 40 45
Val Met Ala Gln Gln Ala Leu Glu Asp Ala Arg Arg Ala Asp Ala Glu
50 55 60
Ile Ala Gln Gly Arg Arg Arg Gly Ala Leu His Gly Val Pro Leu Ala
65 70 75 80
Leu Lys Asp Leu Leu Trp Thr Arg Gly Val Pro Thr Thr His Gly Met
85 90 95
Thr Leu His Arg Glu His Arg Pro Thr Glu Asp Ala Thr Val Val Arg
100 105 110
Arg Leu Arg Glu Ala Gly Ala Val Ile Leu Gly Lys Leu Gln Gln Thr
115 120 125
Glu Gly Ala Phe Ala Asp His His Pro Glu Ile Thr Ala Pro Val Asn
130 135 140
Pro Trp Gly Ala Gln Leu Trp Pro Gly Glu Ser Ser Ser Gly Ser Gly
145 150 155 160
Val Ala Thr Ala Ala Gly Leu Cys Phe Gly Ser Leu Gly Thr Asp Thr
165 170 175
Gly Gly Ser Ile Arg Phe Pro Ser Ala Ala Asn Gly Ile Thr Gly Leu
180 185 190
Lys Pro Thr Trp Gly Arg Val Ser Arg His Gly Ala Phe Glu Leu Ala
195 200 205
Ala Ser Leu Asp His Ile Gly Pro Met Ala Arg Ser Ala Ala Asp Ala
210 215 220
Ala Ala Met Leu Ala Ala Ile Ala Gly Ala Asp Pro Leu Asp Pro Thr
225 230 235 240
Ala Ser Gln Cys Ser Val Pro Asp Tyr Leu Ala Met Met Thr Arg Gly
245 250 255
Phe Ser Gly Leu Arg Leu Gly Met Asp Arg Gln Trp Ala Leu Asp Gly
260 265 270
Val Asp Ala Pro Ser Arg Gln Ala Val Glu Gln Ala Leu Ala Val Ala
275 280 285
Gln Arg Leu Gly Ala Ser Val Gln Glu Val Arg Phe Pro Asp Ala Thr
290 295 300
Gln Ala Val Gln Asp Trp Pro Ala Leu Cys Ala Val Glu Thr Ala Val
305 310 315 320
Ala His Gly Ala Thr Phe Pro Ala Arg Arg Glu Ala Tyr Gly Pro Gly
325 330 335
Leu Ala Gly Leu Ile Asp Leu Gly Leu Gly Leu Ser Ala Thr Asp Tyr
340 345 350
Gln Arg Leu Leu Leu Arg Arg Ala Asp Phe Thr Gly Arg Val Arg Ala
355 360 365
Leu Phe Ala Gln Val Asp Leu Leu Leu Val Pro Ala Thr Ala Phe Ala
370 375 380
Ala Pro Thr Leu Gln Arg Met Ala His Phe Gly Ser Asp Ala Glu Leu
385 390 395 400
Phe Ser Gly Met Leu Arg Tyr Thr Cys Pro Phe Asp Leu Thr Gly Ser
405 410 415
Pro Thr Ile Thr Leu Pro Gly Gly Arg Thr Pro Glu Gly Ala Pro Val
420 425 430
Ala Phe Gln Phe Val Ala Pro Asp Phe Arg Glu Asp Leu Leu Val Arg
435 440 445
Ala Gly Trp Ala Phe Gln Gln Ala Thr Asp Trp His Arg Gln His Pro
450 455 460
Ala Ala
465
Claims (8)
1.一种酰胺酶突变体，所述酰胺酶突变体是将SEQ ID NO.2所示氨基酸序列第154位丙氨酸替换为天冬酰胺、苯丙氨酸或谷氨酸获得的。
2.一种权利要求1所述酰胺酶突变体的编码基因。
3.一种权利要求2所述编码基因构建的重组载体。
4.一种权利要求3所述重组载体转化制备的重组基因工程菌。
5.一种权利要求1所述酰胺酶突变体在拆分2,2-二甲基环丙烷甲酰胺制备(S)-2,2-二甲基环丙烷甲酰胺中的应用。
6.如权利要求5所述的应用，其特征在于所述的应用为：以酰胺酶突变体工程菌经发酵培养获得的湿菌体或者湿菌体破碎后提取的酶作为催化剂，以2,2-二甲基环丙烷甲酰胺为底物，以pH为7.5~8.5的缓冲液为反应介质构成转化体系，在30~50 ℃、150~500 r/min条件下进行转化反应，反应结束后，取反应液分离纯化，获得(S)-2,2-二甲基环丙烷甲酰胺。
7.如权利要求6所述的应用，其特征在于所述转化体系中，底物的初始浓度为10~50mM，催化剂的用量以菌体细胞干重计为0.25~0.75 g/L。
8.如权利要求6所述的应用，其特征在于所述湿菌体按如下方法制备：将酰胺酶突变体工程菌接种到含有终浓度50 mg/L卡那霉素的LB培养基中，37 ℃、150 r/min培养12 h，随后以体积浓度1%接种量转接到新鲜的含终浓度50 mg/L卡那霉素的LB培养基中，37 ℃、150r/min培养至菌体浓度OD600为0.4~0.8，再向培养基中加入终浓度为0.1~1.0 mM的IPTG，28℃、150 r/min诱导培养12 h，取培养物离心，收集沉淀即获得湿菌体。
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