【作者】 杨慧；

【导师】 朱宏吉；

【作者基本信息】 天津大学 ， 制药工程， 2020， 硕士

【摘要】 苔藓植物可以产生多种具有生物活性的倍半萜化合物,有极大的应用价值。倍半萜合成酶是生物合成倍半萜化合物的关键。本课题组发现一种苔藓植物长萼叶苔(Jungermannia exsertifolia)可以产生多种倍半萜化合物,并在对其转录组进行分析时,筛选到倍半萜合成酶基因MTK。本论文围绕倍半萜合成酶基因MTK进行了如下研究:通过生物信息学分析可知,MTK基因的的开放阅读框为1068 bp,编码355个氨基酸,蛋白分子量为40.4 k Da,具有DDxxx和NSE/DTE两个保守区域,属于微生物类似倍半萜合成酶。构建重组菌株WQ011 p RS426-MTK并进行发酵。GC-MS的产物检测结果为β-花柏烯35.17%、β-雪松烯24.81%、橙花叔醇19.89%、α-花柏烯7.97%、(-)-罗汉柏烯7.82%和(+)-α-长叶蒎烯4.35%。构建了重组菌株E.coli BL21(DE3)pET28a-MTK。在温度16℃,IPTG浓度0.5 m M的最适条件下诱导表达蛋白,通过多级纯化得到纯度较高MTK蛋白进行结晶实验。在结晶条件为蛋白浓度7 mg/m L,池液种类PEG/ION 36,池液添加量0.5-0.7μL,无添加剂,温度16℃,结晶方法油滴法时,得到了晶型较好的晶体。通过同源模建构建MTK蛋白与底物复合物的三维结构,确定构成蛋白活性口袋的18个氨基酸。分析模拟结构选择His71,Asp106,Phe179,Thr214,Asn256和Tyr337定点突变,构建了六个突变菌株WQ011 p RS426-MTK H71P、WQ011p RS426-MTK D106E、WQ011 p RS426-MTK F179L、WQ011 p RS426-MTK T214S、WQ011 p RS426-MTK N256D和WQ011 p RS426-MTK Y337F。根据发酵结果,可知Asp106和Asn256确实为MTK蛋白保守序列上的位点,对酶催化活性十分重要;His71对环化反应有重要作用;Phe179对活性口袋起支撑作用;Thr214对选择性地C1-C6环化反应有影响;Tyr337参与质子转移的反应体系,对于磷酸基团离去有重要影响。本论文初步探究了倍半萜合成酶MTK的功能和结构,分析其催化机制,为未来微生物类似倍半萜合成酶的研究提供了理论依据。更多还原

【Abstract】 Bryophytes can produce a variety of sesquiterpene compounds with biological activity and application value.The key to using synthetic biology methods to obtain sesquiterpene compounds more efficiently is the sesquiterpene synthase.In the previous research,the sesquiterpene synthase gene MTK was screened from the transcriptome analysis of Jungermannia exsertifolia,which are rich of sesquiterpene compounds.In this study,we conducted bioinformatics analysis,functional identification,protein crystallization and catalytic mechanism research on MTK..According to the bioinformatics analysis,the ORF of MTK gene is composed of1068 bp,encoding 355 amino acids.The predicted MTK protein molecular weight is40.4 k Da.It has two conserved metal binding motifs DDxxx and NSE/DTE and belongs to Microbial terpene synthase-like terpene synthase.The recombinant strain WQ011 p RS426-MTK was constructed.The fermention products were 35.17%β-Chamigrene,24.81% β-Himachalene,19.89% Herolidol,7.97% α-Chamigrene,7.82%(-)-Thujopsene and 4.35%(+)-α-Longipinene.The recombinant strain E.coli BL21(DE3)p ET28a-MTK was constructed.The optimal induction condition for inducing protein is 0.5 m M IPTG for 16℃.Through multi-stage purification,MTK protein with higher purity was obtained and protein crystallization experiments were conducted.The optimal condition for protein crystallization by oil drop method is as follow: the protein concentration is 7 mg/m L,adding 0.5-0.7 μL of PEG/ION-36,no additives and for 16℃.The three-dimensional structure of the complex of MTK protein and substrate was constructed by homology modeling.18 amino acids constituted the active pocket of the protein.His71,Asp106,Phe179,Thr214,Asn256 and Tyr337 were selected for the mutation study and six mutant strains WQ011 p RS426-MTK H71 P,WQ011 p RS426-MTK D106 E,WQ011 p RS426-MTK F179 L,WQ011 p RS426-MTK T214 S,WQ011 p RS426-MTK N256 D and WQ011 p RS426-MTK Y337 F were obtained.According to the fermentation results,it can be seen that Asp106 and Asn256 on the conserved sequence of MTK protein are very important for the catalytic activity of the enzyme,His71 plays an important role in the cyclization reaction,Phe179 supports the active pocket,Thr214 slightly affects the C1-C6 cyclization reaction,Tyr337 participates in the proton transfer reaction system,which has an important effect on the departure of the phosphate group.This study preliminary explored the function and structure of sesquiterpene synthase MTK,and the analysis of its catalytic mechanism,which provided a theoretical basis for the future research of the Microbial terpene synthase-like terpene synthase.更多还原