CN114807103B - 一种氨基甲酰基水解酶突变体及基因和应用 - Google Patents
一种氨基甲酰基水解酶突变体及基因和应用 Download PDFInfo
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- CN114807103B CN114807103B CN202210657831.0A CN202210657831A CN114807103B CN 114807103 B CN114807103 B CN 114807103B CN 202210657831 A CN202210657831 A CN 202210657831A CN 114807103 B CN114807103 B CN 114807103B
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Abstract
本发明公开了一种氨基甲酰基水解酶突变体及基因和应用，属于基因工程技术领域。所述突变体是将氨基酸序列如SEQIDNO.2所示的氨基甲酰基水解酶第242位氨基酸由天冬酰胺突变为甘氨酸，第262位氨基酸由苏氨酸突变为丙氨酸。本发明提供的氨基甲酰基水解酶突变体比野生型氨基甲酰基水解酶的抗氧化性更强，在强氧化剂过氧化氢环境中的残余酶活比例为40％，而野生型氨基甲酰基水解酶的残余酶活比例为18％；将本发明提供的氨基甲酰基水解酶突变体应用于D‑对羟基苯甘氨酸的生产，能够显著提高D‑对羟基苯甘氨酸的转化效率，降低生产成本。
Description
技术领域
本发明属于基因工程技术领域，具体涉及一种氨基甲酰基水解酶突变体及基因和应用。
背景技术
D-对羟基苯甘氨酸是合成beta-半合成内酰胺类抗生素如阿莫西林、头孢羟氨苄等药物的关键手性中间体。目前，其生产方法主要有化学法拆分和生物酶法两种。化学法原理为：利用光学活性剂L型(+)与DL-型对羟基苯甘氨酸反应形成其D-型对羟基苯甘氨酸-L拆分剂复盐，然后在酸性溶液中溶解，加碱调pH＝4.0，即析出D型结晶(其过程如下所示)，拆分剂一般有酒石酸、溴化樟脑磺酸、苯磺酸等。其主要缺点为：拆分前需甲酯化，拆分后又要水解去甲氧基，步骤多，收率低，拆分收率一般仅为50％。
上世纪80年代后，由于酶法合成工艺具有环境友好及成本低廉的优势，受到越来越多生产厂家的重视。在酶法转化反应过程中，原料对羟基苯海因首先经过海因酶的开环反应和构型转换得到中间体，再在氨基甲酰基水解酶的作用下进行水解脱氨，生成对羟基苯甘氨酸，其反应原理如下所示。
人们对上述两个酶进行了大量的研究，比如Kim等人利用海因酶作用底物后产生的N-乙酰基化合物的酸碱性，设计了一种基于pH颜色变化的筛选培养基，可以较为高通量地筛选得到产海因酶活力的菌株。Hirokazu Nanba et al，(1998)以这种方法成功地从土壤中筛选到一株产氨基甲酰基水解酶的土壤杆菌Agrobacterium sp.KNK712。
然而，在以上述酶品种进行生物酶法生产过程中，我们发现氨基甲酰基水解酶的抗氧化性较差，导致酶稳定性差，不利于酶的生产、储存及实际转化。因此，本发明提供一种上述酶突变品种，提高了其抗氧化能力，大大稳定酶活力及提高实际生产中的转化效率。
发明内容
本发明的目的在于提供一种氨基甲酰基水解酶突变体及基因和应用，通过定向进化技术对目的基因进行随机突变，并结合高通量筛选方法，获得抗氧化性提高的氨基甲酰基水解酶突变体，以解决背景技术中的问题。
本发明的目的可以通过以下技术方案实现：
本发明要解决的第一个技术问题是提供一种氨基甲酰基水解酶突变体，所述突变体是将氨基酸序列如SEQ ID NO.2所示的氨基甲酰基水解酶第242位氨基酸由天冬酰胺突变为甘氨酸，第262位氨基酸由苏氨酸突变为丙氨酸，其突变体氨基酸序列如SEQ ID NO.4所示。具体序列如下。
SEQ ID NO.2：
MTRQMILAVGQQGPIARAETREQVVVRLLYMLTKAASRGANFIVFPELALTTFFPRWHFTDEAELDSFYETEMPGPVVRPLFEKAAELGIGFNLGYAELVVEGGVKRRFNTSILVDKSGKIVGKYRKIHLPGHKEYEAYRPFQHLEKRYFEPGDLGFPVY
DVDAAKMGMFICNDRRWPEAWRVMGLRGAEIICGGYNTPTHNPPVPQHDHLTSFHHLLSMQAGSYQNGAWSAAAGKVGMEENCMLLGHSCIVAPTGEIVALTTTLEDEVITAAVDLDRCRELREHIFNFKQHRQPQHYGLIAEL
SEQ ID NO.4：
MTRQMILAVGQQGPIARAETREQVVVRLLYMLTKAASRGANFIVFPELALTTFFPRWHFTDEAELDSFYETEMPGPVVRPLFEKAAELGIGFNLGYAELVVEGGVKRRFNTSILVDKSGKIVGKYRKIHLPGHKEYEAYRPFQHLEKRYFEPGDLGFPVY
DVDAAKMGMFICNDRRWPEAWRVMGLRGAEIICGGYNTPTHNPPVPQHDHLTSFHHLLSMQAGSYQNGAWSAAAGKVGMEEGCMLLGHSCIVAPTGEIVALATTLEDEVITAAVDLDRCRELREHIFNFKQHRQPQHYGLIAEL
本发明要解决的第二个技术问题是提供编码所述氨基甲酰基水解酶突变体的基因，所述基因核苷酸序列如SEQ ID NO.3所示，具体如下。
SEQ ID NO.3：
atgacacgtcagatgatacttgcagtgggacaacaaggtccgatcgcgcgcgcggagacacgcgaacaggtcgtcgttcgtcttctctacatgctgacgaaagccgcgagccggggcgcgaatttcattgtcttccccgaactcgcgcttacgaccttcttcccgcgctggcatttcaccgacgaggccgagctcgatagcttctatgagaccgaaatgcccggcccggtggtccgtccactctttgagaaggccgcggaactcgggatcggcttcaatctgggctacgctgaactcgtcgtcgaaggcggcgtcaagcgtcgcttcaacacgtccattttggtggataagtcaggcaagatcgtcggcaagtatcgtaagatccatttgccgggtcacaaggagtacgaggcctaccggccgttccagcatcttgaaaagcgttatttcgagccgggcgatctcggcttcccggtctatgacgtcgacgccgcgaaaatggggatgttcatctgcaacgatcgccgctggcctgaagcctggcgggtgatgggcctcaggggcgccgagatcatctgcggcggctacaacacgccgacccacaatccccctgttccccagcacgaccacctgacgtccttccaccatctcctatcgatgcaggccgggtcttatcagaacggggcctggtccgcggccgcgggcaaggtgggcatggaggagggctgcatgctgctcggccactcctgcatcgtggcgccgaccggggaaatcgtcgctctcgctacgacgctggaagacgaggtgatcaccgccgccgtcgatctcgatcgctgccgggaactgcgtgaacacatcttcaacttcaagcagcatcgtcagccccagcactatggtctgatcgcggaactctga
本发明要解决的第三个技术问题是所述氨基甲酰基水解酶突变体在D-对羟基苯甘氨酸生产中的应用，提高了D-对羟基苯甘氨酸的转化效率，降低D-对羟基苯甘氨酸的生产成本。
本发明的有益效果：
本发明提供的氨基甲酰基水解酶突变体比野生型氨基甲酰基水解酶的抗氧化性更强，在强氧化剂过氧化氢环境中的残余酶活比例为40％，而野生型氨基甲酰基水解酶的残余酶活比例为18％；将本发明提供的氨基甲酰基水解酶突变体应用于D-对羟基苯甘氨酸的生产，能够显著提高D-对羟基苯甘氨酸的转化效率，降低生产成本。
具体实施方式
下面将结合本发明实施例，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其它实施例，都属于本发明保护的范围。
本发明中涉及的DNA序列均采用常规的方法合成，本发明实施例中未提到的试验条件和操作均按本领域常规方法或制造商建议的条件进行。
抗氧化性分析采用的方式是：使用不同浓度(0-1.0mM)的过氧化氢在25℃条件下对粗酶液处理15min后，计算剩余活力与初始酶活的比例。
实施例1
来源于假单胞菌的野生型氨基甲酰基水解酶基因克隆：
设计一对引物：DC-F：5’-catatgacacgtcagatgatac-3’和DC-R：5’-ctcgagtcagagttccgcgatca-3’；
以假单胞菌菌种为出发菌株模板进行聚合酶链式反应(PCR反应)，反应体系：18μL重蒸水，2.5μL PCR缓冲液(PCR buffer)，1μL氯化镁(25mM浓度)，1.5μL dNTP(2.5mM浓度)，0.5μL上述两条引物，20ng上述模板，2个单位的Taq酶(上海sangon公司)。
PCR条件：94℃变性10min后，94℃变性30s，55℃退火30s，72℃延伸1min，共进行30个循环；最后72℃充分延伸10min。
野生型氨基甲酰基水解酶基因的核苷酸序列如SEQ ID NO.1所示，其氨基酸序列如SEQ ID NO.2所示。
SEQ ID NO.1：
atgacacgtcagatgatacttgcagtgggacaacaaggtccgatcgcgcgcgcggagacacgcgaacaggtcgtcgttcgtcttctctacatgctgacgaaagccgcgagccggggcgcgaatttcattgtcttccccgaactcgcgcttacgaccttcttcccgcgctggcatttcaccgacgaggccgagctcgatagcttctatgagaccgaaatgcccggcccggtggtccgtccactctttgagaaggccgcggaactcgggatcggcttcaatctgggctacgctgaactcgtcgtcgaaggcggcgtcaagcgtcgcttcaacacgtccattttggtggataagtcaggcaagatcgtcggcaagtatcgtaagatccatttgccgggtcacaaggagtacgaggcctaccggccgttccagcatcttgaaaagcgttatttcgagccgggcgatctcggcttcccggtctatgacgtcgacgccgcgaaaatggggatgttcatctgcaacgatcgccgctggcctgaagcctggcgggtgatgggcctcaggggcgccgagatcatctgcggcggctacaacacgccgacccacaatccccctgttccccagcacgaccacctgacgtccttccaccatctcctatcgatgcaggccgggtcttatcagaacggggcctggtccgcggccgcgggcaaggtgggcatggaggagaactgcatgctgctcggccactcctgcatcgtggcgccgaccggggaaatcgtcgctctcactacgacgctggaagacgaggtgatcaccgccgccgtcgatctcgatcgctgccgggaactgcgtgaacacatcttcaacttcaagcagcatcgtcagccccagcactatggtctgatcgcggaactctgaSEQ ID NO.2：
MTRQMILAVGQQGPIARAETREQVVVRLLYMLTKAASRGANFIVFPELALTTFFPRWHFTDEAELDSFYETEMPGPVVRPLFEKAAELGIGFNLGYAELVVEGGVKRRFNTSILVDKSGKIVGKYRKIHLPGHKEYEAYRPFQHLEKRYFEPGDLGFPVY
DVDAAKMGMFICNDRRWPEAWRVMGLRGAEIICGGYNTPTHNPPVPQHDHLTSFHHLLSMQAGSYQNGAWSAAAGKVGMEENCMLLGHSCIVAPTGEIVALTTTLEDEVITAAVDLDRCRELREHIFNFKQHRQPQHYGLIAEL
实施例2
氨基甲酰基水解酶表达载体的构建：
PCR扩增反应结束后，使用1％琼脂糖凝胶进行电压为100伏的核苷酸电泳，待条带清晰出现后，对大小为900bp左右的目标条带进行胶条割胶，使用胶回收试剂盒(宝生物公司)回收目标DNA片段，并接着按照宝生物公司(网址：www.takara.com.cn)2009版产品目录说明书描述的方法，将所获得的DN片段和载体pET28b+(Novagen公司)用限制性内切酶NdeI和XhoI进行双酶切。
酶切产物再次进行电压为100伏的DNA电泳，回收相应大小为900bp和5300bp左右的片段和载体，最后将片段和载体按照3:1的比例混合后，加入1.5单位的T4连接酶，16℃连接12h。
连接反应结束后，将连接产物取10μL转化大肠杆菌DH5-alpha感受态细胞，在含有卡那霉素的LB平板上筛选阳性克隆，经过NdeI和XhoI双酶切和DNA测序验证，获得包含有大小为905bp的目标DNA序列的阳性克隆及其重组质粒。
实施例3
表达野生型氨基甲酰基水解酶的基因工程菌构建：
用氯化钙转化法(郝福英等编著，《分子生物学实验技术》，北京大学出版社，1998年，第12-15页)将实施例2中最终得到的重组质粒转化至E.coli BL21(DE3)中(Novagen公司)，即获得野生型氨基甲酰基水解酶大肠杆菌表达菌株。
实施例4
氨基甲酰基水解酶基因的随机突变：
设计一对引物：DC-F:5’-catatgacacgtcagatgatac-3’和DC-R:5’-ctcgagtcagagttccgcgatca-3’；
易错PCR体系为：20ng模版DNA(实施例2中获得的重组质粒)，各30pmoL的上述一对引物，7mM氯化镁，50mM氯化钾，10mM Tris-HCl(pH8.2)，0.2mM dGTP，0.2mM dATP，1mMdCTP，1mM dTTP，0.05mM氯化锰和5个单位的Taq酶(NEB公司)。
PCR反应条件：94℃变性10min后，94℃变性30s，56℃退火30s，72℃延伸60s，共进行30个循环；最后72℃充分延伸10min。
PCR扩增结束后，先使用0.9％琼脂糖凝胶进行电压为100伏的核苷酸电泳。用胶回收试剂盒(上海生工)回收大小为0.9kb左右的DNA目的片段。
以此回收的DNA片段作为引物，重组质粒作为模板，进行PCR扩增，具体反应体系为：20ng DNA模板，10μL引物，10μL 2.5mM dNTP和2个单位的KOD聚合酶(toyobo公司)。PCR反应条件为：95℃变性8min后，95℃变性45s，57℃退火45s，68℃延伸5.5min，共进行25个循环；最后68℃充分延伸10min。最终的PCR产物用DpnI酶于37℃消化1h去除DNA模板，经过65℃处理10min使DpnI失活，以供转化使用。
实施例5
突变体库的构建及筛选：
5.1突变体库的构建
通过电击的方法将实施例4中构建得到的突变载体转化到感受态细胞E.coliDH10B(美国Invitrogen公司)中。转化细胞涂布在含卡那霉素的LB平板(蛋白胨1％，酵母提取物0.5％，氯化钠1％，琼脂2％)上，37℃过夜培养。用该方法能得到超过3×104个克隆的突变体库。
5.2突变体筛选与鉴定
菌体生长12h后，挑取单菌落接种于含有300μL LB培养基的96孔细菌培养板中，37℃、220rpm摇床培养，2h后对菌体进行0.9mM IPTG诱导。诱导后培养温度降为30℃，继续培养16h。
取100μL/孔上述培养后菌液至96孔酶标板中，将其整体置于-70℃环境中2h后，转至37℃环境中复苏，复苏时间30min。
将复苏后的菌液中加入0.6mM过氧化氢，在25℃下对粗酶液处理15min后，与反应液混合于96孔酶标板中，反应液配比为：5％NC-HPG原料，1mM EDTA，0.01％酚红，pH 6.3。放置于37℃下转速220rpm震荡反应2h后，利用高效液相色谱检测产物的生成。
色谱分析条件为：使用色谱柱Shodex Sugar SH1011(8.0mmLDX 300mm)，流动相为5mM稀硫酸溶液，流速0.6mL/min，检测器为示差检测器，柱温50℃，进样量为10μL。
5.3筛选结果
通过上述步骤，从突变体库中筛选得到两株能在强氧化剂条件下明显保持活力的突变体。命名为M42和S63。
5.4突变体测序
对M42和S63两个突变体进行测序，并将得到的氨基酸序列与出发序列相比。结果发现，由于对应位置的基因序列的突变，引起了分别在第242位和262位的氨基酸发生了改变，如表1所示：
表1
| 突变体 | 核苷酸位置 | 核苷酸变化 | 氨基酸位置 | 氨基酸变化 |
| M42 | 724-726 | AAC-GGC | 242 | 天冬酰胺-甘氨酸 |
| S63 | 784-786 | ACT-GCT | 262 | 苏氨酸-丙氨酸 |
实施例6
野生型及突变型氨基甲酰基水解酶表达菌株发酵：
将实施例3中获得的野生型核糖激酶大肠杆菌表达菌株及实施例5获得的两株突变型氨基甲酰基水解酶的单个微生物菌落接种于含有100μg/m卡那霉素的50mL LB培养基(蛋白胨1％，酵母提取物0.5％，氯化钠1％)中。细胞在37℃培养箱中于250rpm振荡生长过夜(至少12h)。在1L培养瓶中装入250mL TB培养基(12g/L胰蛋白胨、24g/L酵母提取物、4mL/L甘油、65mM pH 7.0磷酸钾、1mM硫酸镁、100μg/mL卡那霉素)，按照8％的接种量将上述过夜培养物接种于其中，然后在37℃下振荡培养。当培养物的OD600为0.7至0.8时，降温至28℃，用1mM IPTG(异丙基-β-D-硫代半乳糖苷)诱导核糖激酶基因的表达，继续培养至少12h。
实施例7
野生型及突变型氨基甲酰基水解酶粗酶液的制备：
培养结束后，使用离心机在4℃、5000rpm条件下离心15min，收集细胞并弃去上清液。细胞沉淀物在相等体积的4℃，100mM pH7.0三乙醇胺(氯化物)缓冲液中重悬，5000rpm条件下离心20min后，收集细胞。洗涤的细胞在2倍体积的4℃100mM pH 7.0三乙醇胺(氯化物)缓冲液中重悬，以12000psi在高压匀浆机(ATS公司)中机械破胞两次，全程保持温度为4℃。细胞碎片通过冷冻高速离心机在4℃，10000rpm条件下离心40min去除，上清液为所获得的可溶性氨基甲酰基水解酶粗酶液。
实施例8
野生型和突变体氨基甲酰基水解酶在氧化剂环境中的活力比较：
将实施例7获得的粗酶液加入0.6mM过氧化氢，在25℃下对粗酶液处理15min后，进行残余酶活测定。
方法为：经上述过氧化氢氧化剂处理过的粗酶液400μL加入等体积的N-氨甲酰-D-对羟基苯甘氨酸底物进行水解反应，在40℃水浴摇床中以150r/min的速度震荡。反应30min后，加入800μL 10％盐酸终止反应。将反应物用蒸馏水稀释5倍之后，12000转/min离心5min，取上清进行高效液相色谱测定。
高效液相色谱检测条件如下：
8.1仪器组成及电源
8.1.1本仪器由一台P200II型高压恒流泵，一台UV200II紫外可变波长检测器，一个7725六通进样阀，一根C18色谱柱，色谱工作站组成。
8.1.2电源：各部件均为220伏稳压电源。
8.1.3 1000mL流动性配置方法：1.7g磷酸氢二钾，75mL乙腈，用蒸馏水定容至1000mL，调pH 3.5。
液相条件：C18液相柱，波长230，柱温30，流速1mL/min。
8.2操作
8.2.1检查仪器各部件连接是否正确。
8.2.2接通电源，依次打开P200Ⅱ型高压恒流泵，UV200Ⅱ紫外可变波长检测器，工作站，电脑的开关钮。
8.2.3设定相关参数，按功能键，使限压设定灯亮，按▲(加)▼(减)调限压为30Mpa，再按功能键，使流量设定灯亮，按▲▼调流速为1ml/min。拧检测器波长旋钮调波长为230nm。
8.2.4启动P200Ⅱ型高压恒流泵，使流动相流入液相色谱仪。
8.2.5待基线平稳后，即可将经0.45μm微孔滤后的样品注入进样阀，通过工作站记录图谱和峰面积及计算结果。
8.2.6要关闭色谱仪时，先用甲醇(色谱纯)：水＝20:80混合溶液冲洗色谱柱30min，再用色谱纯甲醇冲洗30min。流量设为0.5mL/min。上述溶液必须用0.45μm微孔滤膜过滤和超声方可使用。
8.2.7待色谱柱洗涤干净后，依次关闭工作站、检测器，泵的开关钮。
8.2.8为保护检测器，一般洗柱时，柱出口与检测器断开。
测试结果按照下列公司计算酶活，1U酶活定义为每min产生1微摩尔D-对羟基苯甘氨酸所需的酶量。
上述式中，A样为样品的面积，A标为标样的面积，M标为标样的质量(g)，10为样品反应体积(mL)，30为反应时间(min)，167.2为苯海因的摩尔质量(μg/μmol)，1000为标样的体积(mL)；
残余酶活的计算公式＝酶经过氧化剂处理后的残余活力/初始活力*100％；结果如表2所示。
表2
| 样品 | 残余酶活比例 |
| 野生型 | 18％ |
| M42突变株 | 26％ |
| S63突变株 | 33％ |
从表2可以看出，相比野生型氨基甲酰基水解酶，M42和S63突变株均显示了对强氧化剂过氧化氢更好的耐受度，分别提高了44％和83％。
实施例9
对M42和S63的突变位点进行组合突变：
本实施例的目的是将实施例5中获得的两个有益突变进行叠加，以考察双突变体在抗氧化性方面的表现。
使用
Site-Directed Mutagenesis Kit试剂盒(Stratagen，Agilient公司)将242位和262位同时分别突变为甘氨酸和丙氨酸。操作方法按试剂盒说明书进行(www.agilent.com)，组合突变后所得的突变体氨基酸序列如SEQ ID NO.4所示，其基因序列如SEQ ID NO.3所示。在获得242和262位叠加突变后(该叠加突变体命名为N242G/T262A)，然后按照实施例3中基因工程菌构建方法进行N242G/T262A酶工程菌的构建，再按照实施例6中的方法进行N242G/T262A酶表达菌株发酵，按照实施例7中的方法进行N242G/T262A酶粗酶液的制备，然后按照实施例8中相同方法分别进行N242G/T262A酶的发酵及残余酶活测定，结果如表3所示。
表3
| 样品 | 残余酶活比例 |
| 野生型 | 18％ |
| N242G/T262A | 40％ |
从表3中的数据可知，相比野生型氨基甲酰基水解酶，N242G/T262A酶显示了对强氧化剂过氧化氢更高的耐受度，提高了122.2％。
SEQ ID NO.3：
atgacacgtcagatgatacttgcagtgggacaacaaggtccgatcgcgcgcgcggagacacgcgaacaggtcgtcgttcgtcttctctacatgctgacgaaagccgcgagccggggcgcgaatttcattgtcttccccgaactcgcgcttacgaccttcttcccgcgctggcatttcaccgacgaggccgagctcgatagcttctatgagaccgaaatgcccggcccggtggtccgtccactctttgagaaggccgcggaactcgggatcggcttcaatctgggctacgctgaactcgtcgtcgaaggcggcgtcaagcgtcgcttcaacacgtccattttggtggataagtcaggcaagatcgtcggcaagtatcgtaagatccatttgccgggtcacaaggagtacgaggcctaccggccgttccagcatcttgaaaagcgttatttcgagccgggcgatctcggcttcccggtctatgacgtcgacgccgcgaaaatggggatgttcatctgcaacgatcgccgctggcctgaagcctggcgggtgatgggcctcaggggcgccgagatcatctgcggcggctacaacacgccgacccacaatccccctgttccccagcacgaccacctgacgtccttccaccatctcctatcgatgcaggccgggtcttatcagaacggggcctggtccgcggccgcgggcaaggtgggcatggaggagggctgcatgctgctcggccactcctgcatcgtggcgccgaccggggaaatcgtcgctctcgctacgacgctggaagacgaggtgatcaccgccgccgtcgatctcgatcgctgccgggaactgcgtgaacacatcttcaacttcaagcagcatcgtcagccccagcactatggtctgatcgcggaactctga
SEQ ID NO.4：
MTRQMILAVGQQGPIARAETREQVVVRLLYMLTKAASRGANFIVFPELALTTFFPRWHFTDEAELDSFYETEMPGPVVRPLFEKAAELGIGFNLGYAELVVEGGVKRRFNTSILVDKSGKIVGKYRKIHLPGHKEYEAYRPFQHLEKRYFEPGDLGFPVY
DVDAAKMGMFICNDRRWPEAWRVMGLRGAEIICGGYNTPTHNPPVPQHDHLTSFHHLLSMQAGSYQNGAWSAAAGKVGMEEGCMLLGHSCIVAPTGEIVALATTLEDEVITAAVDLDRCRELREHIFNFKQHRQPQHYGLIAEL
实施例10
N242G/T262A在D-对羟基苯甘氨酸生产中的应用：
步骤一、将实施例9获得N242G/T262A酶工程菌通过10m3发酵罐发酵，高压破壁机破壁，蝶片高速离心机离心提取N242G/T262A粗酶液，进入冷库保藏；
步骤二、在30m3转化罐中，加入水和底物3000kg，配置成12％的底物溶液。分别加入了海因酶100kg海因酶，待底物全部转化为N-氨甲酰基苯甘氨酸后，加入100kgN242G/T262A粗酶液，转化42小时全部转化完成。
对比例1
野生型氨基甲酰基水解酶在D-对羟基苯甘氨酸生产中的应用：
步骤一、与实施例10步骤一中操作相同，获得野生型氨基甲酰基水解酶的粗酶液；
步骤二、与实施例10步骤一中操作相同，监控同样的转化时间完成底物的全部转化，所需要的野生型氨基甲酰基水解酶的粗酶液的质量，结果显示需要野生型氨基甲酰基水解酶的粗酶液130kg。
由实施例10和对比例1的结果显示可知，通过抗氧化性突变体改造，在同样条件下，酶液加入量减少了30kg，节约了酶的加入了，降低了生产成本。
实施例11
N242G/T262A在D-对羟基苯甘氨酸生产中的应用：
步骤一、与实施例10中的步骤一相同；
步骤二、与实施例10中的步骤二相同，完成底物的全部转化所需要的时间为41.5小时。
对比例2
野生型氨基甲酰基水解酶在D-对羟基苯甘氨酸生产中的应用：
步骤一、与实施例10步骤一中操作相同，获得野生型氨基甲酰基水解酶的粗酶液；
步骤二、与实施例10步骤一中操作相同，且加入的野生型氨基甲酰基水解酶的粗酶液的质量为100kg，监控完成底物的全部转化所需要的时间，结果显示所需时间为63小时。
从实施例11和对比例2的数据可知，通过抗氧化性突变体改造，在同样条件下，同样酶液加入量，转化时间减少了22.5小时，提高了转化效率，降低了生产成本。
需要说明的是，在本文中，诸如第一和第二等之类的关系术语仅仅用来将一个实体或者操作与另一个实体或操作区分开来，而不一定要求或者暗示这些实体或操作之间存在任何这种实际的关系或者顺序。而且，术语“包括”、“包含”或者其任何其他变体意在涵盖非排他性的包含，从而使得包括一系列要素的过程、方法、物品或者设备不仅包括那些要素，而且还包括没有明确列出的其他要素，或者是还包括为这种过程、方法、物品或者设备所固有的要素。
尽管已经示出和描述了本发明的实施例，对于本领域的普通技术人员而言，可以理解在不脱离本发明的原理和精神的情况下可以对这些实施例进行多种变化、修改、替换和变型，本发明的范围由所附权利要求及其等同物限定。
序列表
<110> 合肥利夫生物科技有限公司
<120> 一种氨基甲酰基水解酶突变体及基因和应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 915
<212> DNA
<213> 编码氨基甲酰基水解酶突变体的基因(2 Ambystoma laterale xAmbystoma jeffersonianum)
<400> 1
atgacacgtc agatgatact tgcagtggga caacaaggtc cgatcgcgcg cgcggagaca 60
cgcgaacagg tcgtcgttcg tcttctctac atgctgacga aagccgcgag ccggggcgcg 120
aatttcattg tcttccccga actcgcgctt acgaccttct tcccgcgctg gcatttcacc 180
gacgaggccg agctcgatag cttctatgag accgaaatgc ccggcccggt ggtccgtcca 240
ctctttgaga aggccgcgga actcgggatc ggcttcaatc tgggctacgc tgaactcgtc 300
gtcgaaggcg gcgtcaagcg tcgcttcaac acgtccattt tggtggataa gtcaggcaag 360
atcgtcggca agtatcgtaa gatccatttg ccgggtcaca aggagtacga ggcctaccgg 420
ccgttccagc atcttgaaaa gcgttatttc gagccgggcg atctcggctt cccggtctat 480
gacgtcgacg ccgcgaaaat ggggatgttc atctgcaacg atcgccgctg gcctgaagcc 540
tggcgggtga tgggcctcag gggcgccgag atcatctgcg gcggctacaa cacgccgacc 600
cacaatcccc ctgttcccca gcacgaccac ctgacgtcct tccaccatct cctatcgatg 660
caggccgggt cttatcagaa cggggcctgg tccgcggccg cgggcaaggt gggcatggag 720
gagggctgca tgctgctcgg ccactcctgc atcgtggcgc cgaccgggga aatcgtcgct 780
ctcgctacga cgctggaaga cgaggtgatc accgccgccg tcgatctcga tcgctgccgg 840
gaactgcgtg aacacatctt caacttcaag cagcatcgtc agccccagca ctatggtctg 900
atcgcggaac tctga 915
<210> 2
<211> 304
<212> PRT
<213> 氨基甲酰基水解酶突变体的氨基酸序列(2 Ambystoma laterale xAmbystoma jeffersonianum)
<400> 2
Met Thr Arg Gln Met Ile Leu Ala Val Gly Gln Gln Gly Pro Ile Ala
1 5 10 15
Arg Ala Glu Thr Arg Glu Gln Val Val Val Arg Leu Leu Tyr Met Leu
20 25 30
Thr Lys Ala Ala Ser Arg Gly Ala Asn Phe Ile Val Phe Pro Glu Leu
35 40 45
Ala Leu Thr Thr Phe Phe Pro Arg Trp His Phe Thr Asp Glu Ala Glu
50 55 60
Leu Asp Ser Phe Tyr Glu Thr Glu Met Pro Gly Pro Val Val Arg Pro
65 70 75 80
Leu Phe Glu Lys Ala Ala Glu Leu Gly Ile Gly Phe Asn Leu Gly Tyr
85 90 95
Ala Glu Leu Val Val Glu Gly Gly Val Lys Arg Arg Phe Asn Thr Ser
100 105 110
Ile Leu Val Asp Lys Ser Gly Lys Ile Val Gly Lys Tyr Arg Lys Ile
115 120 125
His Leu Pro Gly His Lys Glu Tyr Glu Ala Tyr Arg Pro Phe Gln His
130 135 140
Leu Glu Lys Arg Tyr Phe Glu Pro Gly Asp Leu Gly Phe Pro Val Tyr
145 150 155 160
Asp Val Asp Ala Ala Lys Met Gly Met Phe Ile Cys Asn Asp Arg Arg
165 170 175
Trp Pro Glu Ala Trp Arg Val Met Gly Leu Arg Gly Ala Glu Ile Ile
180 185 190
Cys Gly Gly Tyr Asn Thr Pro Thr His Asn Pro Pro Val Pro Gln His
195 200 205
Asp His Leu Thr Ser Phe His His Leu Leu Ser Met Gln Ala Gly Ser
210 215 220
Tyr Gln Asn Gly Ala Trp Ser Ala Ala Ala Gly Lys Val Gly Met Glu
225 230 235 240
Glu Gly Cys Met Leu Leu Gly His Ser Cys Ile Val Ala Pro Thr Gly
245 250 255
Glu Ile Val Ala Leu Ala Thr Thr Leu Glu Asp Glu Val Ile Thr Ala
260 265 270
Ala Val Asp Leu Asp Arg Cys Arg Glu Leu Arg Glu His Ile Phe Asn
275 280 285
Phe Lys Gln His Arg Gln Pro Gln His Tyr Gly Leu Ile Ala Glu Leu
290 295 300
Claims (4)
1.一种氨基甲酰基水解酶突变体，其特征在于，所述突变体的氨基酸序列如SEQ IDNO.4所示。
2.一种氨基甲酰基水解酶突变体基因，其特征在于：编码权利要求1所述的氨基甲酰基水解酶突变体。
3.根据权利要求2所述的一种氨基甲酰基水解酶突变体基因，其特征在于：所述基因的核苷酸序列如SEQ ID NO.3所示。
4.一种氨基甲酰基水解酶突变体的应用，其特征在于：权利要求1所述的氨基甲酰基水解酶突变体在D-对羟基苯甘氨酸生产中的应用。
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1168415A (zh) * | 1992-08-10 | 1997-12-24 | 钟渊化学工业株式会社 | 编码可提高耐热性的脱氨基甲酰酶的dna及其用途 |
| WO2001016337A1 (fr) * | 1999-08-31 | 2001-03-08 | Kaneka Corporation | Stereostructure de decarabamylase et procede d'utilisation |
| CN1995336A (zh) * | 2006-01-06 | 2007-07-11 | 中国科学院上海生命科学研究院 | D－氨甲酰水解酶的突变体及其应用 |
| CN102747060A (zh) * | 2011-04-22 | 2012-10-24 | 中国科学院上海生命科学研究院 | D-氨甲酰水解酶的突变体及其制备方法和应用 |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1168415A (zh) * | 1992-08-10 | 1997-12-24 | 钟渊化学工业株式会社 | 编码可提高耐热性的脱氨基甲酰酶的dna及其用途 |
| WO2001016337A1 (fr) * | 1999-08-31 | 2001-03-08 | Kaneka Corporation | Stereostructure de decarabamylase et procede d'utilisation |
| CN1995336A (zh) * | 2006-01-06 | 2007-07-11 | 中国科学院上海生命科学研究院 | D－氨甲酰水解酶的突变体及其应用 |
| CN102747060A (zh) * | 2011-04-22 | 2012-10-24 | 中国科学院上海生命科学研究院 | D-氨甲酰水解酶的突变体及其制备方法和应用 |
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