CN105018506B - 苯并噁唑酮类化合物水解酶、编码该酶的基因、含有该基因的工程菌及其应用 - Google Patents
苯并噁唑酮类化合物水解酶、编码该酶的基因、含有该基因的工程菌及其应用 Download PDFInfo
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Abstract
本发明公开了苯并噁唑酮类化合物水解酶、编码该酶的基因、含有该基因的工程菌及其应用。一种苯并噁唑酮类化合物水解酰胺酶基因cbaA,其核苷酸序列为SEQ ID No.1。所编码的BOA类化合物水解酰胺酶蛋白质,其氨基酸序列为SEQ ID No.2。将该BOA类化合物水解酰胺酶基因构建重组表达载体再导入E.coli BL21(DE3)即获得含有该基因的基因工程菌株。本发明所述的BOA类化合物水解酰胺酶基因及利用该基因生产的酶制剂可用于环境的生物修复、工业废弃物处理、食品加工、医药、洗涤等行业,不仅可以解决BOA类化合物的环境污染问题,还可以取得可观的经济效益。
Description
技术领域
本发明属于基因工程领域,涉及苯并噁唑酮类化合物水解酶、编码该酶的基因、含有该基因的工程菌及其应用。
背景技术
苯并噁唑酮(BOA)类化合物是存在于禾本科、爵床科、毛莨科、玄参科等植物中特有的化学成分,这类成分对细菌、真菌、昆虫等具有防御作用,同时具有除草、植物生长调节和昆虫拒食等生物活性。BOA类化合物作为一种重要的药物中间体,常被用于抗菌药、解热镇痛药、催眠药等的合成与生产,例如:6-氯-苯并噁唑酮具有抗菌活性,5-氯-苯噁唑酮(氯唑沙宗)具有抗炎活性,临床上用于治疗各种急慢性软组织扭伤、挫伤及慢性筋膜炎等。此外,BOA类化合物作为新农药开发的先导化合物被广泛研究,噁唑禾草灵,噁唑酰草胺,伏杀硫磷等都是已经商品化并广泛使用的农药品种。但是,由于以BOA类化合物作为活性结构的药物和农药的大量使用,将会严重污染生态环境,破坏生态平衡。
环境污染物的降解主要依赖于土壤中微生物的作用,而微生物对物质的降解主要由细胞内的酶来完成。微生物是各种酶的重要来源。通过从环境中分离产酶微生物,利用分子克隆技术从产酶微生物中克隆产酶基因,再将其与合适的载体连接并转入相应宿主细胞,可进行酶的大量表达。通过基因工程技术手段进行生产酶,已经成为工业用酶的主导。因此,筛选能够高效降解BOA类化合物的菌株并研究其代谢机制,对降解基因资源的开发利用和环境污染修复非常具有现实意义。
目前仅有少数致病真菌对BOA类化合物具有抗性,例如,小麦冠腐镰刀菌(Fusarium pseudograminearum)、禾谷镰刀菌(Fusarium graminearum)、轮状镰刀菌(Fusarium verticillioides)等,因此,阐述清楚致病真菌对BOA类化合物的脱毒机制尤为重要。研究认为致病真菌对BOA的脱毒过程为:首先BOA在一种β-内酰胺FDB1的作用下转化为邻氨基苯酚,然后再由N-乙酰转移酶FDB2的作用下生成无毒性的N-(2-羟基苯基)丙酰胺酸,邻氨基苯酚也可以被转化为2-乙酰基氨基苯酚。目前对编码FDB2的基因已有明确报道,但是对FDB1对BOA的转化作用仅为一种推测。
发明内容
本发明的目的是针对BOA类化合物严重污染环境的问题,提供一株该类化合物降解菌株。
本发明的另一目的是提供该菌株中克隆得到的降解BOA类化合物的基因、该基因编码的酶、含有该基因的重组表达载体以及含有该基因的工程菌。
本发明的又一目的是提供上述菌株、基因、酶、工程菌的应用。
本发明的目的可通过如下技术方案实现:
一种苯并噁唑酮类化合物水解酰胺酶基因cbaA,其特征在于,核苷酸序列为:SEQID No.1。该基因全长(从起始密码子到终止密码子)为1020bp,G+C含量为51.57%,编码339个氨基酸,其氨基酸序列为:SEQ ID No.2。
所述的苯并噁唑酮类化合物水解酰胺酶酶基因核苷酸序列所编码的BOA类化合物水解酰胺酶蛋白质,其氨基酸序列为:SEQ ID No.2。一种来自于菌株Pigmentiphagasp.VL-1中的BOA类化合物水解酰胺酶CbaA通过硫酸铵沉淀、DEAE-Sepharose离子交换层析和Butyl-650M疏水层析等蛋白质纯化手段,被纯化19倍,回收率达到4%。CbaA纯酶在碱性条件下稳定性较好,温度稳定性强,在pH 7.0-11.0,40℃以下储存24h,酶活力仅损失不到30%;EDTA对酶活无显著影响,表明该酶是不依赖金属离子的非金属酶。CbaA的最适底物为6-氯苯并噁唑酮,比酶活力为5650U·mg protein-1,Km值为0.36μmol,Vmax为10.94。CbaA同时对苯并噁唑酮、6-氯苯并噁唑酮、5-氯苯并噁唑酮、4-羟基-苯并噁唑酮、6-甲氧基-苯并噁唑酮、6-硝基-苯并噁唑酮、6-氨基-苯并噁唑酮、6-溴-苯并噁唑酮、5-溴-苯并噁唑酮等具有酶活性,表明CbaA可能为一种酰胺酶并且对苯并噁唑类化合物有较好的酶活性。
一种含有本发明所述的苯并噁唑酮类化合物水解酰胺酶基因cbaA的重组表达载体。
所述的重组表达载体,优选出发载体为pET-29a(+)。
含所述的BOA类化合物水解酰胺酶基因的基因工程菌Escherich coli BL21(DE3)。
所述的基因工程菌E.coli BL21(DE3)的构建方法:所述的含BOA类化合物水解酰胺酶基因的pET-29a-cbaA重组质粒转化到表达宿主菌E.coli BL21(DE3)获得重组微生物E.coli BL21(DE3-pET-29a-cbaA),再将所获得的重组菌株E.coli BL21(DE3-pET-29a-cbaA)转接到含有50mg/l卡那霉素和24mg/l IPTG的平板,37℃培养16h后,挑取转化子验证降解BOA的能力,经测序验证基因序列无误后,保存。
本发明所述的基因cbaA在水解苯并噁唑酮类化合物中的应用;所述的苯并噁唑酮类化合物优选自苯并噁唑酮、6-氯苯并噁唑酮、5-氯苯并噁唑酮、4-羟基-苯并噁唑酮、6-甲氧基-苯并噁唑酮、6-硝基-苯并噁唑酮、6-氨基-苯并噁唑酮、6-溴-苯并噁唑酮、5-溴-苯并噁唑酮中的任意一种。
本发明所述的苯并噁唑酮类化合物水解酶CbaA在水解苯并噁唑酮类化合物中的应用,所述的苯并噁唑酮类化合物优选自苯并噁唑酮、6-氯苯并噁唑酮、5-氯苯并噁唑酮、4-羟基-苯并噁唑酮、6-甲氧基-苯并噁唑酮、6-硝基-苯并噁唑酮、6-氨基-苯并噁唑酮、6-溴-苯并噁唑酮、5-溴-苯并噁唑酮中的任意一种。
本发明所述的基因工程菌E.coli BL21(DE3-pET-29a-cbaA)在水解苯并噁唑酮类化合物中的应用,所述的苯并噁唑酮类化合物优选自苯并噁唑酮、6-氯苯并噁唑酮、5-氯苯并噁唑酮、4-羟基-苯并噁唑酮、6-甲氧基-苯并噁唑酮、6-硝基-苯并噁唑酮、6-氨基-苯并噁唑酮、6-溴-苯并噁唑酮、5-溴-苯并噁唑酮中的任意一种。
一株降解苯并噁唑酮类化合物的细菌Pigmentiphaga sp.VL-1,保藏于中国典型培养物保藏中心,保藏日期为2014年3月3日,保藏编号为CCTCC NO:M2014057。
本发明所述的细菌Pigmentiphaga sp.VL-1在降解苯并噁唑酮类化合物中的应用;所述的苯并噁唑酮类化合物优选自苯并噁唑酮、6-氯苯并噁唑酮、5-氯苯并噁唑酮、4-羟基-苯并噁唑酮、6-甲氧基-苯并噁唑酮、6-硝基-苯并噁唑酮、6-氨基-苯并噁唑酮、6-溴-苯并噁唑酮、5-溴-苯并噁唑酮中的任意一种。
本发明所述的一种BOA类化合物水解酶在环境的生物修复、工业废弃物的处理、食品加工、医药、洗涤等行业的应用。
本发明的有益效果如下:
1.本发明以6-氯苯并噁唑酮为唯一碳源从土壤中分离筛选到一株高效降解BOA类化合物的菌株Pigmentiphaga sp.VL-1,并以该菌株为材料成功纯化出苯并噁唑酮水解酰胺酶CbaA(BOA类化合物降解的起始酶),该酶能高效水解BOA类化合物6-氯苯并噁唑酮、5-氯苯并噁唑酮、苯并噁唑酮等。
2.本发明成功从菌株Pigmentiphaga sp.VL-1基因组中克隆到苯并噁唑酮水解酰胺酶基因cbaA,并构建该基因的重组表达菌株E.coli BL21(DE3-pET-29a-cbaA),该重组菌株仍然可以水解BOA类化合物,生产的酶制剂可用于环境的生物修复、化工废弃物处理、食品加工、医药和洗涤等行业。
附图说明
图1基于16S rRNA基因构建的菌株VL-1系统发育树
图2菌株VL-1降解苯并噁唑酮过程中的降解曲线和生长曲线
图3苯并噁唑酮水解酶的纯化和鉴定流程图
图4CbaA与PDB数据库中相似氨基酸序列比对分析
图5pH对CbaA酶活性和稳定性的影响
图6温度对CbaA酶活性和稳定性的影响
图7金属离子对CbaA酶活性的影响
图8重组表达菌株静息细胞转化苯并噁唑酮
生物材料保藏信息
Pigmentiphaga sp.VL-1,保藏于中国典型培养物保藏中心,保藏日期为2014年5月17日,保藏地址为中国武汉,武汉大学,保藏编号为CCTCC No.M 2014057。
具体实施方式
下面结合实施对本发明进一步详细的描述,但发明的实施方式不限于此。
实施例1:BOA类化合物降解菌株的分离与鉴定
取5g被BOA化合物污染的土样置于100mL含20mg·L-16-氯苯并噁唑酮(CDHB)的富集培养基中,于30℃、180r·min-1培养5d。用紫外扫描仪测定富集液对CDHB的降解情况,确定CDHB被降解后,以10%的接种量接入到30mg·L-1CDHB的富集培养基中,继续富集并测定降解情况,按此方法直至CDHB浓度提高至50mg·L-1,并传代3次。
将CDHB富集液经梯度稀释后分别涂布于以25mg·L-1CDHB为唯一碳源的MSM平板上,于30℃培养箱中分别培养7d。将平板上长出的菌落形态不同的单菌落分别划线于LB平板纯化,并接种于以25mg·L-1CDHB为唯一碳源的液体无机盐培养基中,于30℃、180r·min-1摇床培养2d后,取2mL样品离心收集上清,在波长200-350nm范围内进行紫外扫描,CDHB最大吸收峰为280nm,根据特征吸收峰的变化情况来确定富集液和菌株对苯酚的降解能力。其中菌株VL-1可以将CDHB降解到检测不到任何物质。
菌株VL-1在LB平板上生长较慢,30℃,96h可形成直径为2mm的微黄色菌落,菌落边缘整齐,有光泽,突出,干燥,褶皱,不透明。其电镜图片中菌体呈短杆状,大小为0.7×1.5μm。基于16S rRNA基因以及生理生化鉴定,将菌株VL-1鉴定为Pigmentiphaga属,其系统发育树见图1所示。菌株VL-1的最适生长温度为37℃,当温度低于30℃或高于37℃,菌体生长量明显下降;菌株VL-1的最适生长pH值为7.0,在pH 6.0-8.0范围内生长情况较好,当超出该范围时,菌体生长量迅速下降,菌株VL-1耐盐性较好,当NaCl浓度在1.5-4%之间时,菌株均可以较好生长,当盐浓度逐渐升高时,菌株生长量逐渐下降;通气量对菌株VL-1的生长没有明显的影响,但随着通气量的减少,菌株的生长量有逐渐减小趋势,这说明菌株VL-1为好氧生长。
实施例2:菌株Pigmentiphaga sp.VL-1对BOA类化合物的降解特性
从LB平板上挑取菌株VL-1单菌落,分别接种于3mL LB液体培养基中于30℃,摇床180r·min-1培养48h。然后将该菌株的培养液转接到100mL新鲜的液体LB培养基中,继续培养48h。6000r·min-1离心10min,收集菌体,用灭菌的MSM洗涤一次后,制成OD600=1.0的菌悬液,作为降解实验种子液。在CDHB终浓度为20mg·L-1的无机盐培养基中,按5%接种量接入OD600=1.0菌株VL-1种子液,于30℃、180r·min-1摇床培养,每隔12h取样一次,测定OD600和CDHB的浓度。由图2可以看出,菌株VL-1接种到培养基中12h,就开始降解CDHB,而没有明显的延滞现象;之后降解速率逐渐增加,到60h就已将20mg·L-1的CDHB基本降解完全。利用平板计数法对培养液中的菌株VL-1进行计数,结果显示接种初期和培养60h的细胞数分别为1.5×106和4.2×106cfu·mL-1,这说明菌株VL-1可以利用CDHB作为唯一碳源进行生长。
菌株VL-1降解CDHB的最适温度为37℃,在30和42℃降解效率略微下降,在20-42℃范围内均可以降解CDHB;菌株VL-1在pH 4.0-10.0范围内均可以降解CDHB,最适pH范围为7.0-10.0,即在碱性条件下降解效果较好,pH=4时降解率为最适pH值的40%;CDHB的降解率随着菌株VL-1接种量的增大而加快,但是低接种量时(1%以下),由于CDHB对菌体存在一定毒性,所以降解速度缓慢,甚至不降解;菌株VL-1可以在72h内将20mg·L-1的CDHB完全降解,但是如果浓度继续升高,CDHB的降解速度非常缓慢,当浓度高于50mg·L-1时基本不降解,这说明尽管菌株VL-1可以降解CDHB,但是高浓度的CDHB依然对其产生抑制效应。
以相同的降解条件测定菌株VL-1对其他BOA类化合物的降解能力。结果表明菌株VL-1可以降解苯并噁唑酮、5-氯苯并噁唑酮、4-羟基-苯并噁唑酮、6-甲氧基-苯并噁唑酮、6-硝基-苯并噁唑酮、6-氨基-苯并噁唑酮、6-溴-苯并噁唑酮、5-溴-苯并噁唑酮中的任意一种。
实施例3:BOA类化合物水解酶的分离纯化及序列分析
挑取经划线活化的菌株VL-1单菌落接种于3mL液体LB试管培养基中,37℃,180rpm培养菌液至对数生长期,按1%接种量接种至1L新鲜LB液体培养基中,37℃恒温180rpm培养至稳定期,4℃,5,000rpm离心10min,收集菌体,弃上清;用适量预冷的20mM Gly-NaOH(pH8.0)重悬菌体,4℃,12,000rpm离心10min,弃上清;以5mL Gly-NaOH(pH 8.0)重悬菌体。菌悬液以180Hz超声波破碎10min,整个过程在冰浴下进行,破碎液在4℃,13,000rpm离心20min,上清液即为粗酶液。粗酶液通过硫酸铵分级沉淀、Butyl-650M疏水层析和DEAE-Sepharose离子交换层析3个步骤处理(图3),成功从菌株VL-1中将BOA类化合物水解酶CbaA纯化19倍,回收率达到4%,得到了电泳纯的条带(表1)。
表1苯并噁唑酮水解酶纯化过程中的蛋白得率和纯化倍数
将各步骤纯化的CbaA进行SDS-PAGE电泳,将SDS-PAGE电泳后的凝胶条带分成两部份,一部份进行考马斯亮蓝染色,另一部份复性后铺于含CDHB的20mM Gly-NaOH(pH 9.0)的琼脂平板上,55℃反应30min后,加适量0.1M 4-氨基替比林及0.2M铁氰化钾进行显色。酶谱结果显示CbaA活性位置,在考马斯亮蓝染色胶相应位置含有一条明显的,且条带单一的蛋白条带A,有弥散现象,达到电泳纯标准,满足肽指纹图谱分析要求,接合菌株VL-1的基因组信息,鉴定CbaA的核苷酸和氨基酸序列。
BOA类化合物水解酰胺酶基因cbaA,其核苷酸序列为SEQ ID No.1。该基因全长(从起始密码子到终止密码子)为1020bp,G+C含量为51.57%,编码339个氨基酸,其氨基酸序列为:SEQ ID No.2。CbaA与假设水解酶蛋白2B0A关系最近,其次就是靛红水解酶4J0N,这是一个新的酰胺水解酶,含有保守的活性中心位点His83、His212和Ser/Glu225,CDHB水解酶CbaA也含有这三个活性中心His137、His288和Glu301(图4)。将三个活性中心分别突变为H137A、H288A、E301A后,BOA类化合物水解酰胺酶CbaA将丧失BOA类化合物水解能力。
实施例3:水解酰胺酶CbaA对BOA类化合物的水解
BOA类化合物被水解后会产生苯胺衍生物。苯胺衍生物可以与4-氨基安替比林和铁氰化钾形成红色反应,并且反应产物在OD545处有最大吸光度,吸光光度值与颜色的深浅成正相关,所以本实施例通过测定水解产物颜色反应后的光度值,计算水解酶酶活力。取适量水解酰胺酶CbaA加至3mL含2mM BOA类化合物的20mM Gly-NaOH(pH 9.0)中,于55℃反应10min后,加入0.1M 4-氨基安替比林和0.2M铁氰化钾各30μL终止酶反应,立刻测定OD545。酶活定义为55℃下每分钟催化1umoL底物所需的蛋白量(mg)。
结果表明,CbaA的最适底物为6-氯苯并噁唑酮,比酶活力为5650U·mg protein-1,Km值为0.36μmol,Vmax为10.94。CbaA对BOA和苯甲酰胺具有酶活性,比酶活分别为3842和1693U·mg protein-1(表2)。同时检测到CbaA可以水解5-氯苯并噁唑酮、4-羟基-苯并噁唑酮、6-甲氧基-苯并噁唑酮、6-硝基-苯并噁唑酮、6-氨基-苯并噁唑酮、6-溴-苯并噁唑酮、5-溴-苯并噁唑酮等BOA类化合物。
CbaA在碱性条件下稳定性较好,在pH 7.0-11.0范围内储存24h,仍然可以残留80%左右的酶活力,但是在酸性条件下稳定性较差,pH为6.0时残留酶活力50%,pH 5.0时残留酶活力仅剩不到20%;该酶对温度的稳定性强,在40℃以下储存12h,酶活力仅损失30%,在50℃时酶活力残留55%,但是当温度达到60℃以上时,储存12h后酶活丧失(图5和6)。Cu2+和Ni2+对CbaA酶活有一定的激活作用,酶活提高达40%,Ba2+的激活作用较弱,酶活仅提高10%;Fe2+、K+、Zn2+、Mg2+、Ca2+、Na+对CDHB水解酶没有没有明显的影响;Al3+、Co2+、Mn2+等对酶活力有一定的抑制作用,含有Al3+和Mn2+的相对酶活有40%,而Co2+的抑制作用最强烈,残留酶活仅有20%(图7)。常用的有机溶剂对CbaA酶活力影响不大,残留酶活力仍然在80%以上;EDTA和EGTA对酶活没有影响,表明该酶应该是一个非金属依赖的水解酶;DMSO、PMSF和1%的SDS可以使酶活力降低20%;TritonX-100和CTAB对酶活力有显著的影响,使酶活力残留50%左右;β-巯基乙醇和DTT几乎完全抑制酶活性(表3)。
表2 CbaA的底物特异性与表观动力学常数
表3不同化学试剂对CbaA活性的影响
实施例4:BOA类化合物水解酶基因在E.coli BL21(DE3)中的高效表达
(1)菌体总DNA的提取
采用高盐法提取Pigmentiphaga sp.VL-1的染色体总DNA:挑取菌株VL-1单菌落接种于3ml LB液体培养基中,37℃、180r·min-1培养至OD600≈1.0,12000r·min-1离心收集菌体;用1.0mL TE缓冲液(10mmol/l Tris·Cl(pH8.0),1mmol/l EDTA,pH 8.0)重悬洗涤菌体,10000r·min-1离心5min收集菌体,加入1.0ml TEN缓冲液(10mmol/l Tris·Cl(pH8.0),1mmol/lEDTA,0.1mol/l NaCl pH 8.0)悬浮菌体,加入5μl的溶菌酶(100mg/mL),37℃水浴1h,加入25-50μl 20%SDS和5μl蛋白酶K(20mg/ml),65℃水浴2h,待液体澄清后,加入340μl饱和NaCl溶液剧烈震荡,12000r·min-1离心10min,将上清液转移至无菌洁净eppendorf管中,用等体积的酚:氯仿:异戊醇(25:24:1)抽提至界面澄清无白色固体物为止,转移上清液于另一无菌洁净eppendort管中,加入0.6倍体积的异丙醇,-20℃放置0.5-1h沉淀DNA,12000r·min-1离心10min,去除上清后用70%乙醇洗涤2次,待乙醇挥发后加入30μl无菌水或TER,放置4℃冰箱过夜溶解,短期4℃保存,长期采用-20℃冻存。
(2)BOA类化合物水解酰胺酶基因的克隆
根据核苷酸序列分析结果设计引物cbaA-F:CATATGACTAAAAAATACCTAGTTG(正向引物,加Nde I酶切位点)和cbaA-R:AAGCTTTCAGTGGTGGTGGTGGTGGTGGTAGATCACGAAAGGTCGAGCAG(反向引物,加Hind III酶切位点和His-tag)以VL-1总DNA为模板,PrimeStar HS DNApolymerase扩增出全长1038bp的cbaA基因序列;将目的片段回收后与pMD19-T载体相连后,转化至E.coli DH5α高效感受态细胞,涂布与含有100mg·L-1Amp的LB固体平板上,37℃培养16h,提取质粒验证酶连效果,0.75%琼脂糖凝胶电泳检测,Nde I/Hind III双酶切验证无误后送至南京金斯瑞生物技术有限公司测序,测序正确的克隆载体命名为pMD19T-cbaA。
(3)克隆载体pMD19T-cbaA和表达载体pET-29a(+)的Nde I和Hind III双酶切
酶切体系:
在37℃水浴中,反应3h以上。酶切产物进行0.75%的琼脂糖凝胶电泳切胶回收cbaA和线性化的pET-29a(+)。
(4)表达菌株的构建
将上述酶切好的目标片段进行酶连得含cbaA基因的pET-29a(+)重组质粒pET-29a-cbaA。将重组质粒pET-29a-cbaA转化到表达宿主菌E.coli BL21(DE3)(获得重组微生物E.coli BL21(DE3-pET-29a-cbaA),涂布含有20mg/l CDHB、50mg/l卡那霉素和24mg/lIPTG的平板,经37℃培养16-20h后,挑取阳性克隆子,经测序验证基因序列无误后,测试重组菌株对BOA类化合物的降解能力。
从LB平板挑取表达菌株E.coli BL21(DE3-pET-29a-cbaA)单菌落,接种至3mL LB液体培养基,37℃,180r·min-1振荡培养12h;然后按1%接种量转移至100mL LB液体培养基进行扩大培养,37℃,180r·min-1振荡培养12h,,5,000r·min-1离心3min,菌体以1mL灭菌MSM液体培养基洗涤2次,菌悬液以10%接种量分别转移至20mL PBS(含有20mg·L-1CDHB或BOA,0.2%酵母膏,10mM葡萄糖)中,37℃,180r·min-1振荡培养24h,检测底物转化情况并对产物进行HPLC-MS鉴定,功能验证过程以E.coli BL21(DE3-pET29a(+))作为对照。
表达菌株E.coli BL21(DE3-pET-29a-cbaA)静息细胞在24h内可以将10%以上的CDHB转化为2A5CP。同时表达菌株也可以在24h转化苯并噁唑类化合物的母体结构BOA,结果见图8。E.coli BL21(DE3-pET29a(+))在24h内不能转化BOA,但是表达菌株E.coli BL21(DE3-pET-29a-cbaA)可以将BOA转化,在HPLC色谱图上有一新峰出现,将该峰进行MS/MS分析,结果显示该物质分子量为109,是BOA在水解酶CbaA作用下转化的产物邻氨基苯酚(2AP)。
Claims (8)
1.一种苯并噁唑酮类化合物水解酰胺酶基因cbaA,其特征在于,核苷酸序列为:SEQ IDNo.1。
2.权利要求1所述的基因cbaA编码的苯并噁唑酮类化合物水解酶CbaA,其特征在于,氨基酸序列为:SEQ ID No.2。
3.一种含有权利要求1所述的苯并噁唑酮类化合物水解酰胺酶基因cbaA的重组表达载体。
4.根据权利要求3所述的重组表达载体,其特征在于,该表达载体的出发载体为pET-29a(+)。
5.含权利要求1所述的苯并噁唑酮类化合物水解酰胺酶基因cbaA的基因工程菌E.coliBL21(DE3-pET-29a-cbaA)。
6.权利要求1所述的基因cbaA在水解苯并噁唑酮类化合物中的应用, 所述的苯并噁唑酮类化合物选自苯并噁唑酮、6-氯苯并噁唑酮、5-氯苯并噁唑酮、4-羟基-苯并噁唑酮、6-甲氧基-苯并噁唑酮、6-硝基-苯并噁唑酮、6-氨基-苯并噁唑酮、6-溴-苯并噁唑酮、5-溴-苯并噁唑酮中的任意一种。
7.权利要求2所述的苯并噁唑酮类化合物水解酶CbaA在水解苯并噁唑酮类化合物中的应用,所述的苯并噁唑酮类化合物选自苯并噁唑酮、6-氯苯并噁唑酮、5-氯苯并噁唑酮、4-羟基-苯并噁唑酮、6-甲氧基-苯并噁唑酮、6-硝基-苯并噁唑酮、6-氨基-苯并噁唑酮、6-溴-苯并噁唑酮、5-溴-苯并噁唑酮中的任意一种。
8.权利要求5所述的基因工程菌E.coli BL21(DE3-pET-29a-cbaA)在水解苯并噁唑酮类化合物中的应用,所述的苯并噁唑酮类化合物选自苯并噁唑酮、6-氯苯并噁唑酮、5-氯苯并噁唑酮、4-羟基-苯并噁唑酮、6-甲氧基-苯并噁唑酮、6-硝基-苯并噁唑酮、6-氨基-苯并噁唑酮、6-溴-苯并噁唑酮、5-溴-苯并噁唑酮中的任意一种。
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