蓝藻硫酯酶催化多肽环化适用性分析

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。McyC-TE (microcystin thioesterase, McyC 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McyC 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-D-thiogalactopyranoside, 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：McyC 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,McyC-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McyC TE的催化功能提供了实验基础。

Analysis of the Applicability of Peptides Cyclization Catalyzed by Cyanophyta Thioesterase

Abstract TE shows impressive stereospecificity, regioselectivity and chemoselectivity during the cyclization of peptide. This property brings about high value and broad application prospect in industrial production. We use the orthogonal experiment to further increase the expression of McyC TE and have a further research on its substrate suitability combining the mature linear peptide chemistry synthesis. Finally we get the best induced condition : the induction time is 2 h, concentration of inducer tendency for 0.75 mmol/L, induction time of 6 h, induction of 210 r/min, induction temperature 20 ℃, the expression of TE quantity from 8.75 mg/L up to 22.15 mg/L and the time reduced 6.5 h. The greatly enhance of TE expression quantity and expression in a shorter time laid a foundation for the future research of the structure and catalytic mechanism of enzymes . The results of substrate suitability study show that the McyC TE does not follow the principle of "4 n + 2", nor the peptide chains of amino acids number parity sexual selection. Too much rotation is more likely to form curling effecting cyclization. We can join other small steric hindrance, flexible amino acid or these bring their own natural Angle to weak the rigidity of the peptide chain, such as Gly and Pro. The introduction of the Phe containing benzene ring show that the addition of benzene ring is not good for cyclization. The study proves that the more extended polypeptide chain length can also be cyclization, McyC TE substrate tolerance is large. These conclusions provides the experimental basis for the further research of McyC TE catalytic function, increased the screening area of natural peptide drug, and also increased the improved solution to enhance natural peptide drug efficacy.