CN114438148A - 一种利用单加氧酶ycnE降解吲哚和/或吲哚酮产生靛红的方法 - Google Patents
一种利用单加氧酶ycnE降解吲哚和/或吲哚酮产生靛红的方法 Download PDFInfo
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- CN114438148A CN114438148A CN202210153248.6A CN202210153248A CN114438148A CN 114438148 A CN114438148 A CN 114438148A CN 202210153248 A CN202210153248 A CN 202210153248A CN 114438148 A CN114438148 A CN 114438148A
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- ycne
- monooxygenase
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Abstract
本发明公开了一种利用单加氧酶ycnE降解吲哚和/或吲哚酮产生靛红的方法。本发明采用基因工程的技术方法获得抗生素生物合成单加氧酶基因ycnE,并构建重组质粒,然后在大肠杆菌中诱导表达,通过亲和层析对细胞破碎液中的抗生素生物合成单加氧酶ycnE组分进行纯化,首次成功表达并纯化了ycnE,并通过该酶首次实现使用酶催化法转化吲哚和/或吲哚酮产生靛红,降解率达25%,表现出良好的应用潜力。
Description
技术领域
本发明属于生物工程技术领域,特别涉及一种利用单加氧酶ycnE降解吲哚和/或吲哚酮产生靛红的方法。
背景技术
吲哚又名苯并吡咯,是一种由苯环和吡咯环并联形成的氮杂环芳香族化合物。吲哚在低浓度时具有花香味,而在浓度高时则具有强烈臭味,是臭味产生的主要原因之一。在吲哚-3-甘油磷酸酯和色氨酸存在的情况下,植物和微生物可以通过色氨酸酶或其类似物产生吲哚,因此吲哚存在于植物和富含细菌的生态位中,如土壤、根际、污泥和肠道等。动物本身不能合成吲哚,但很多动物共生菌尤其是肠道微生物可以转化色氨酸合成吲哚,吲哚通过汗液、尿液和粪便等排泄物排出体外。此外,在煤焦油、动物粪便和污水中都发现了相对较高浓度的吲哚,这是造成气味污染的主要原因。随着煤矿、石油加工和动物集约化养殖的发展,大量含吲哚的废水和烟雾产生并排放到环境中,导致了严重的环境污染和臭味滋扰。
吲哚由于其毒性和潜在的致突变性,长期以来被认为是废水中典型的杂环芳烃污染物。据报道高浓度的吲哚会引发动物脏腑和组织的出血和充血、贫血、白血球异常、溶血病、血红蛋白肾病、暂时性皮肤刺激、肿瘤形成和植物色素沉着减少等问题。对于微生物,高浓度的吲哚会造成细胞膜氧化毒性,通过影响膜电位而阻止细胞分裂,抑制三磷酸腺苷的产生和蛋白质折叠,并导致DNA损伤。
吲哚是一类难以根除的有毒环境污染物,研究开发合适的、没有环境污染的生物酶法降解的处理方法,是一个具有重大生态价值与理论价值的研究课题。目前对于吲哚类化合物的去除主要有物理化学法和生物法。物理化学法,包括超声波法、臭氧氧化等,虽然物理化学法具有较好的吲哚去除效率,但是运行费用高、能耗大,且会产生毒性更大的持久性有机污染物。而微生物转化在吲哚污染系统修复中具有高效、价廉且环境友好的特点,被广泛用于环境污染物的处理中。目前关于微生物降解转化吲哚的研究多集中于群落分析、产物鉴定和途径解析中,而对于吲哚降解的功能基因研究较少。筛选出高效降解吲哚的功能基因、产酶量高且适合工业化生产的菌株及表达系统、寻找合适的发酵工艺、探索使酶活稳定的方法等是推动吲哚降解研究及相关产业发展的重要研究方向。
目前尚无商品化的吲哚降解酶,对于吲哚降解相关的功能基因研究也较少。Guang-Huey Lin等用E.coli CY15000重组表达Acinetobacter baumannii的IacA可在5μM酶浓度下可转化吲哚为靛蓝;Mikas Sadauskas等用E.coli BL21(DE3)重组表达Acinetobacter sp.Strain O153的IifC可在IifD存在下转化吲哚为靛蓝;Yuanyuan Qu等用E.coli BL21(DE3)重组表达Cupriavidus sp.SHE的IndA可转化吲哚为靛蓝;Qiao Ma等用E.coli BL21(DE3)重组表达Burkholderia sp.IDO3的IifCD,重组大肠杆菌可以转化吲哚为靛蓝。与本发明重组表达的抗生素生物合成单加氧酶ycnE相似的序列无功能验证、重组表达、相关降解活性等分析。
发明内容
本发明的首要目的在于克服现有技术的缺点与不足,提供一种利用单加氧酶ycnE降解吲哚和/或吲哚酮产生靛红的方法。本发明采用基因工程的技术方法得抗生素生物合成单加氧酶基因ycnE,并构建重组质粒,然后在大肠杆菌中诱导表达,通过亲和层析对细胞破碎液中的抗生素生物合成单加氧酶ycnE组分进行纯化,首次成功表达并纯化了ycnE,并首次证明该酶能转化吲哚为靛红。本发明首次异源表达的抗生素生物合成单加氧酶ycnE具有吲哚降解活性,而且可以降解吲哚生成靛红,与同类报道的转化产物靛蓝有明显差异。
本发明的目的通过下述技术方案实现:
单加氧酶ycnE在降解吲哚和/或吲哚酮中的应用,所述的单加氧酶ycnE的氨基酸序列如SEQ ID NO.2所示。
一种利用单加氧酶ycnE降解吲哚和/或吲哚酮产生靛红的方法,是将单加氧酶ycnE添加到含有吲哚和/或吲哚酮的样品中,反应,以实现所述的样品中吲哚和/或吲哚酮的降解。
优选的,所述的样品中包含:吲哚0~0.2g/L,和/或吲哚酮0~0.2g/L;进一步选为吲哚0~0.1g/L,和/或吲哚酮0~0.1g/L;最优选为吲哚0~100mg/L,和/或吲哚酮0~100mg/L。
优选的,所述的单加氧酶ycnE的添加量,按其在体系中的终浓度为1~125mg/L计;优选为25~125mg/L;进一步优选为90~110mg/L;最优选为100mg/L。
优选的,所述的反应的条件为温度20~30℃,时间1~30h;优选为温度23~27℃,时间20~30h;进一步优选为温度25℃,时间24h。
优选的,所述的反应的体系中还含有细菌或细菌细胞破碎液,其添加量按5%(v/v)计。
优选的,所述的细菌为大肠杆菌。
所述的单加氧酶ycnE的制备方法,是先将合成的单加氧酶ycnE的编码序列构建到表达载体pet28a,然后将所得重组质粒转化大肠杆菌(E.coli)BL21(DE3),最后诱导表达及纯化。
优选的,所述的单加氧酶ycnE的编码序列如SEQ ID NO.1所示。
优选的,所述的重组质粒的构建过程中的双酶切位点为BamHI和EcoRI;连接体系每10μL含:pet28a为1μL、ycnE为3μL、T4连接酶为0.5μL。
优选的,所述的转化的条件为42℃热击70秒。
优选的,所述的诱导表达的条件为0.5mmol/L IPTG于37℃诱导2.5小时;所述的纯化通过镍柱亲和层析实现。
本发明相对于现有技术具有如下的优点及效果:
1.本发明首次成功表达纯化了单加氧酶ycnE。
2.本发明的方法大幅度提高了ycnE酶的产量。
3.本发明得到的ycnE首次报道可以降解吲哚和/或吲哚酮产生靛红,降解率达25%,表现出良好的应用潜力。
附图说明
图1是重组表达载体pet28a-ycnE的质粒图谱。
图2是重组蛋白的SDS-PAGE凝胶电泳图,A为ycnE诱导表达结果,其中M表示标准分子量蛋白,泳道1是未诱导的大肠杆菌BL21(DE3)全细胞蛋白,泳道2是经过IPTG诱导的大肠杆菌BL21(DE3)全细胞蛋白;B为ycnE纯化结果,泳道1是细胞破碎上清的穿过液,泳道2和泳道3是流出液,泳道3-8是用洗脱液洗脱不同次数收集得到的样品,M表示标准分子量蛋白。结果表明经过诱导纯化可以得到电泳纯级别的ycnE。
图3是经过不同浓度的重组蛋白ycnE处理后吲哚的残留量分析图,结果表明ycnE具有吲哚降解活性(从上到下依次为0mg/L、1mg/L、5mg/L、25mg/L、125mg/L)。
图4是经过ycnE处理后吲哚的降解产物碱化后的HPLC结果分析图,产物中检测到出峰时间与吲哚、吲哚酮或靛红酸相同的成分,表明ycnE可以氧化吲哚生成吲哚酮,还可以继续氧化吲哚酮生成靛红(靛红酸)。
图5是经过ycnE处理后吲哚的降解产物的HPLC-HRMS结果分析图,产物中检测到分子式与吲哚、吲哚酮或靛红一致的成分,表明ycnE可以氧化吲哚生成吲哚酮,还可以继续氧化吲哚酮生成靛红。
具体实施方式
下面结合实施例及附图对本发明作进一步详细的描述,但本发明的实施方式不限于此。
实施例1
(一)重组质粒构建
(1)委托公司合成ycnE基因片段,序列如SEQ ID NO.1所示。使用EcoRI和BamHI(宝日医生物技术有限公司)对ycnE基因片段和pet28a载体按照说明书进行双酶切(双酶切体系如表1所示),用普通DNA回收试剂盒(天根生化科技有限公司)对酶切后的DNA分别进行纯化回收,然后用核酸浓度检测仪测定DNA浓度;
表1双酶切体系
酶切条件37℃,30分钟;
(2)将纯化回收的抗生素生物合成单加氧酶基因ycnE和pet28a载体片段按摩尔比为1:5的比例利用T4连接酶进行体外连接,连接条件22℃,16小时,连接体系如表2所示。
表2 T4连接酶连接体系
(二)基因ycnE转化E.coli BL21(DE3):
将连接后的重组质粒42℃热击70秒转化至大肠杆菌BL21(DE3)菌株中。在含有50μg/mL卡那霉素的LB平板上挑选阳性单菌落,然后使用2×Taq PCR Mix进行菌落PCR筛选阳性菌落,反应体系及程序如表3所示:
表3菌落PCR体系
菌落PCR反应条件为:94℃预变性3分钟;94℃变性30秒;51℃退火30秒;72℃延伸1分钟;32个循环;最后72℃延伸10分钟;然后将产物通过琼脂糖凝胶电泳鉴定,琼脂糖浓度为1%,电泳条件为120V,25分钟,得到目的基因条带大小约为300bp;
(2)挑取阳性克隆加入到含卡那霉素的LB液体培养基于37℃,180rpm摇床上扩大培养12小时。取扩大培养的菌液于离心管中,送至生工生物工程(上海)股份有限公司进行测序鉴定,对构建成功的表达质粒命名为pet28a-ycnE。
(三)ycnE的诱导表达及纯化:
(1)挑取单菌落接种到5ml的含卡那霉素(25μg/mL)的LB液体培养基中,37℃、180rpm培养12-16小时;
(2)吸取0.1mL过夜培养菌液转接至10mL含卡那霉素的LB液体培养基中,37℃、180rpm培养4-6小时至OD600为0.6左右;
(3)取一组作为未诱导菌液对照,向实验组菌液中加入IPTG至终浓度为0.5mmol/L,继续37℃培养2.5小时;
(4)菌液12000rpm离心2分钟收集菌体沉淀,加入1mL裂解液(50mmol/L NaH2PO4,300mmol/L NaCl,10mmol/L Imidazole),于冰上超声破碎,破碎条件为:超声功率300W,开9s关9s,持续30分钟,12000rpm离心2分钟分别收集破碎上清及破碎后沉淀;
(5)将离心获得的沉淀,依次使用0mol/L、2mol/L、4mol/L、6mol/L尿素(均含100mmol/L NaH2PO4,10mmol/L Tris-HCl)进行洗涤,最后溶解在8mol/L尿素中;
(6)使用镍柱对含有目的蛋白的组分进行纯化,步骤如下:
a)将镍填料装入亲和层析管,加入10倍填料体积的平衡溶液过柱;
b)关闭底部开关,加入含有目的蛋白的蛋白样品,4℃结合过夜;
c)打开底部开关,收集流出液;
d)加入10倍填料体积的Wash buffer过柱,收集流出液;
e)重复d步骤一次;
f)加入5倍填料体积Elution buffer过柱,收集洗脱液;
g)收集各组分。
经检测,经过IPTG诱导可以成功重组表达ycnE(图2A);诱导表达的蛋白位于细胞破碎上清中,表明诱导表达的ycnE并非包涵体而是可溶的;细胞破碎上清经过镍柱纯化可以得到电泳纯级别的ycnE(图2B)。
(7)对蛋白进行透析复性,将蛋白溶液装入透析袋,放入盛有PBS(10mmol/LNa2HPO4,137mmol/L NaCl,2.7mmol/L KCl,2mmol/L KH2PO4)溶液中透析48小时,整个复性过程均在4℃进行,期间使用磁力搅拌机进行搅拌。
(四)重组蛋白的SDS-PAGE检测:
利用SDS-PAGE凝胶电泳来确认重组单加氧酶的表达情况、纯度和分子质量的大小。采用的浓缩胶浓度为12%以及分离胶浓度为5%,上样量为10μL,以标准分子量的标准蛋白作为Marker。SDS-PAGE凝胶电泳的操作过程参见《蛋白质电泳实验技术》。
纯化后酶液的SDS-PAGE电泳图如图2所示,结果表明通过本发明的异源表达和纯化方法可以成功获得电泳纯级别的抗生素生物合成单加氧酶ycnE(14.5kDa)。纯化酶液使用BCA试剂盒测定其蛋白浓度,测得纯化酶液的浓度为2g/L,换算至发酵液产量为1.2g/L,远高于ycnE在海氏肠球菌中的表达量(低于10mg/L,海氏肠球菌的蛋白表达量由TMT标记定量蛋白组学分析测得)。
(五)重组蛋白降解吲哚及产物检测:
(1)把表4中的组分加入到洁净的EP管中:
表4吲哚降解反应体系
反应条件25℃,24小时;
(2)吲哚含量测定方法如下:往反应液中加入等体积的甲醇沉淀蛋白,12000rpm离心10分钟,收集上清并使用0.22μm有机相滤膜过滤样品,滤液通过Shimadzu-LC-20A高效液相色谱系统检测产物,色谱柱为ZORBAX Eclipse XDB-C18(4.6×250mm,5μm),流动相为60%(v/v)乙腈,流速1.0mL/min,温度35℃,使用荧光检测器测定吲哚含量,激发波长270nm,发射波长350nm;
经检测发现,反应液中加入有活性的ycnE可以降低吲哚浓度,而且酶添加量越大吲哚浓度越低,结果如表5和图3所示,表明本发明制备的ycnE可以有效降解吲哚,降解率达25%。只含有ycnE的反应体系无法构成循环电子传递系统,这可能是限制吲哚降解率的重要因素,接下来将进行与加氧酶ycnE协同的还原酶的相关研究。
表5吲哚含量测定
(3)反应产物检测方法如下:前期检测发现无法将吲哚酮与靛红分离,因此,样品加入25mmol/L NaOH使靛红转化为靛红酸,更改步骤(2)中的液相条件为:色谱柱为ZORBAXEclipse XDB-C18(4.6×250mm,5μm),流动相为50%(v/v)乙腈,流速1.0mL/min,温度35℃,在210nm处检测产物;
进一步使用HPLC-HRMS检测反应产物,样品加入等体积的乙酸乙酯抽提除盐,12000rpm离心10分钟,收集上清并使用0.22μm有机相滤膜过滤样品,滤液通过BrukermaXis超高分辨飞行时间质谱仪检测产物,其中液相条件为:Agilent1260系统,色谱柱为Luna 5u C18(2)100A 4.6×250mm 5μm流动相为60%(v/v)乙腈,流速1.0mL/min,温度35℃,在210nm处检测;质谱条件为:Capillary 3800V,End plate offset 500V,Nebulizer1.0bar,Dry Gas 6.0L/min,Dry Temp 180℃,scan range(m/z)100–2000;
经检测发现,加入有活性的ycnE的反应液中可以检测到吲哚、吲哚酮以及靛红(图4和图5),进一步使用吲哚酮作为底物能检测到靛红酸,表明ycnE降解吲哚的途径为吲哚→吲哚酮→靛红,本发明制备的单加氧酶ycnE可以降解吲哚产生靛红。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
序列表
<110> 广东省农业科学院动物科学研究所
<120> 一种利用单加氧酶ycnE降解吲哚和/或吲哚酮产生靛红的方法
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 288
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 单加氧酶ycnE基因序列
<400> 1
atgatcgtta tcaatgcaaa attttcaatc aaaccagaaa aaagaaatga atttcttgcg 60
gaagtcaatg aacttgttgc aagtacaaga aaagaagacg ggtgcttaag ctatcaattg 120
tacgaatcaa tcgatattga aaatgaattt gtcatggttg aaaattggcg tgatcaagca 180
gcaattgaag gacacaatca aagtccgtta cttcaacaat tatttaaaaa tatgagtcag 240
tatagtagta agaagacaga aataaatgtt tctcaaacag taaactaa 288
<210> 2
<211> 95
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 单加氧酶ycnE氨基酸序列
<400> 2
Met Ile Val Ile Asn Ala Lys Phe Ser Ile Lys Pro Glu Lys Arg Asn
1 5 10 15
Glu Phe Leu Ala Glu Val Asn Glu Leu Val Ala Ser Thr Arg Lys Glu
20 25 30
Asp Gly Cys Leu Ser Tyr Gln Leu Tyr Glu Ser Ile Asp Ile Glu Asn
35 40 45
Glu Phe Val Met Val Glu Asn Trp Arg Asp Gln Ala Ala Ile Glu Gly
50 55 60
His Asn Gln Ser Pro Leu Leu Gln Gln Leu Phe Lys Asn Met Ser Gln
65 70 75 80
Tyr Ser Ser Lys Lys Thr Glu Ile Asn Val Ser Gln Thr Val Asn
85 90 95
Claims (10)
1.单加氧酶ycnE在降解吲哚和/或吲哚酮中的应用,其特征在于:所述的单加氧酶ycnE的氨基酸序列如SEQ ID NO.2所示。
2.一种利用单加氧酶ycnE降解吲哚和/或吲哚酮产生靛红的方法,其特征在于:将单加氧酶ycnE添加到含有吲哚和/或吲哚酮的样品中,反应,以实现所述的样品中吲哚和/或吲哚酮的降解;所述的单加氧酶ycnE的氨基酸序列如SEQ ID NO.2所示。
3.根据权利要求2所述的利用单加氧酶ycnE降解吲哚和/或吲哚酮产生靛红的方法,其特征在于:
所述的样品中包含:吲哚0~0.2g/L,和/或吲哚酮0~0.2g/L;
所述的单加氧酶ycnE的添加量,按其在体系中的终浓度为1~125mg/L计。
4.根据权利要求2所述的利用单加氧酶ycnE降解吲哚和/或吲哚酮产生靛红的方法,其特征在于:
所述的样品中包含:吲哚0~100mg/L,和/或吲哚酮0~100mg/L;
所述的单加氧酶ycnE的添加量,按其在体系中的终浓度为25~125mg/L计。
5.根据权利要求2~4任一项所述的利用单加氧酶ycnE降解吲哚和/或吲哚酮产生靛红的方法,其特征在于:
所述的反应的体系中还含有细菌或细菌细胞破碎液,其添加量按5%v/v计。
6.根据权利要求2~4任一项所述的利用单加氧酶ycnE降解吲哚和/或吲哚酮产生靛红的方法,其特征在于:
所述的反应的条件为温度20~30℃,时间1~30h。
7.权利要求1或2中所述的单加氧酶ycnE的制备方法,其特征在于:
先将合成的单加氧酶ycnE的编码序列构建到表达载体pet28a,然后将所得重组质粒转化大肠杆菌(E.coli)BL21(DE3),最后诱导表达及纯化。
8.根据权利要求7所述的单加氧酶ycnE的制备方法,其特征在于:
所述的单加氧酶ycnE的编码序列如SEQ ID NO.1所示。
9.根据权利要求7所述的单加氧酶ycnE的制备方法,其特征在于:
所述的重组质粒的构建过程中的双酶切位点为BamHI和EcoRI;连接体系每10μL含:pet28a为1μL、ycnE为3μL、T4连接酶为0.5μL。
10.根据权利要求7所述的单加氧酶ycnE的制备方法,其特征在于:
所述的转化的条件为42℃热击70秒;
所述的诱导表达的条件为0.5mmol/LIPTG于37℃诱导2.5小时;
所述的纯化通过镍柱亲和层析实现。
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