蓝藻硫酯酶催化多肽环化适用性分析  

Analysis of the Applicability of Peptides Cyclization Catalyzed by Cyanophyta Thioesterase

作　　者：何魁芳 高威芳 方剑[2] 王佳琪[1] 李玲钰[1] 朱鹏[1] 

机构地区：[1]宁波大学应用海洋生物技术教育部重点实验室,浙江宁波315211 [2]绍兴文理学院医学院,浙江绍兴312000

出　　处：《中国生物化学与分子生物学报》2017年第1期88-97,共10页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金(No.40906080);海洋公益性行业科研专项(No.201305013);绍兴文理学院项目(No.2015LG1011);浙江省教育厅项目(No.Y201635696)资助~~

摘　　要：硫酯酶(thioesterase,TE)具有区域定向性(regiospecific)、化学定向性(chemospecific)及立体定向性(stereospecific)的特点。这些特性决定了TE作为生物催化剂(biocatalysis)在工业生产中具有较高的应用价值和广阔的应用前景。Mcy C TE(microcystin thioesterase,Mcy C TE)来自铜绿微囊藻(microcystis aeruginosa)NRPS/PKS生物合成基因簇。我们利用正交试验提高Mcy C TE表达量,得到稳定的诱导表达条件,并结合成熟的线性多肽化学合成法对其底物适用性做了进一步研究。得到的最佳诱导表达条件为:诱导时机2 h,诱导剂异丙基-β-D-硫代半乳糖苷(isopropyl-β-Dthiogalactopyranoside,IPTG)浓度0.75 mmol/L,诱导时间6 h,诱导转速210 r/min,诱导温度20℃,使TE的表达量由8.75 mg/L提高至22.15 mg/L,时间缩短了6.5 h。TE表达量的大幅度提升和表达时间的缩短为将来酶的结构及催化机制研究奠定了基础。TE底物适用性研究结果发现:Mcy C TE并不遵循"4 n+2原则";底物中转角过多不仅不利于环肽的形成,更可能形成卷曲影响环化;无D型氨基酸亦可通过加入其它位阻较小较灵活的Gly或者自带天然转角Pro的可弱化肽链的刚性,促进催化反应;含苯环的Phe的引入在一定程度上阻碍了环化;底物无肽链氨基酸数目奇偶性的选择;延长多肽链长度也可环化,Mcy C TE的底物容忍度较大,使天然多肽药物筛选范围增大,也为增强天然多肽药物药效增加了改良方案,为进一步研究Mcy C TE的催化功能提供了实验基础。Thioesterase（ TE） shows impressive stereospecificity,regioselectivity and chemoselectivity during the cyclization of peptide. The biocatalysis property of TE provides high value and extensive application in industrial production. We used the orthogonal experiment to further increase the expression of Mcy C TE and analyzed its substrate suitability by the synthesis of mature linear peptide chemistry.Finally the best induction condition was detected as follows： after incubation for 2 hours,0. 75 mmol / L IPTG was added and the culture was shaked at 210 r / min and 20 ℃ for 6 hours. The protein levels of TEwere improved from 8. 75 mg / L to 22. 15 mg / L and the induction time reduced 6. 5 hours. The improvement of TE expression quantity and induction time would provide clues for the future research of the enzyme structure and catalytic mechanism. The results showed that the Mcy C TE does not follow the principle of ＂ 4 n ＋ 2＂ or parity selection of the number of peptide chains of amino acids. Over-rotation is more likely to form curling which affects cyclization. Amino acids with lower steric hindrance and flexibility,such as Gly,or amino acids providing natural angle,such as Pro,could reduce the rigidity of the peptide chain to promote induction. The introduction of the Phe containing benzene ring inhibited cyclization. The study proved that extended the length of polypeptide chains could also induce cyclization and the Mcy C TE substrate tolerance was broadened. These results expanded the screening scope of natural peptide drugs,which provided the experimental basis for the further research of Mcy C TE catalytic function which may improve the efficacy of natural peptide drug.

关 键 词：硫酯酶 环肽 底物适用性 

分 类 号：Q556[生物学—生物化学] Q71