不同植物源的黄烷酮-3-羟化酶的催化特性

catalytic properties of flavanone-3-hydroxylase from different plants

摘要:

【背景】黄烷酮-3-羟化酶(flavanone-3-hydroxylase，F3H)是黄酮类化合物代谢途径中的关键酶之一，不同植物来源的F3H催化特性可能存在差异，并对黄酮类化合物的生物合成产生重要影响。【目的】比较分析不同植物源F3H的酶学性质、异源催化能力差异，为今后在黄酮类化合物代谢工程中F3H的选用提供参考。【方法】通过系统进化分析选择来自茶树(Camellia sinensis)、银杏(Ginkgo biloba)和大豆(Glycine max)的F3H基因，诱导表达后用亲和层析对F3H进行纯化，以柚皮素为底物表征不同植物来源F3H的酶学性质；采用单因素分析法对F3H在原核宿主Escherichia coli和真核宿主Saccharomyces cerevisiae体内的催化活性进行分析。【结果】酶学性质分析发现，CsF3H、GbF3H和GmF3H的最适反应温度分别为40、40和35 ℃，最适反应pH值分别为7.5、7.0和7.5。催化动力学研究发现，CsF3H的k_cat/K_m为0.36 L/(mmol·s)，高于GbF3H和GmF3H。体内催化实验表明，当底物柚皮素的浓度为500 μmol/L时，CsF3H、GmF3H单基因大肠杆菌工程菌对柚皮素的转化率达80%以上，而GbF3H工程菌对柚皮素的转化率仅为23.8%；3种单基因酵母工程菌对柚皮素的转化率都在40%左右，没有明显差异。【结论】不同植物源的F3H催化活性存在差异，而且同一种F3H在原核底盘细胞与真核底盘细胞中催化能力也存在较大差异，3种F3H中CsF3H具有优良的催化性能，并在原核底盘细胞内有较好的应用潜力。

Abstract:

[Background] Flavanone-3-hydroxylase (F3H) is one of the key enzymes in the metabolic pathway of flavonoids.The catalytic properties of F3H from different plants may be different,which has an important impact on the biosynthesis of flavonoids.[Objective] In order to compare and analyze the enzymatic properties and catalytic abilities of F3H,providing reference for the selection of F3H in the metabolic engineering of flavonoids in the future.[Methods] Three genes,CsF3H(Camellia sinensis),GbF3H(Ginkgo biloba) and GmF3H(Glycine max),were identified by phylogenetic analysis.The three F3H were purified by affinity chromatography,and the enzymatic properties were characterized by naringenin as substrate in vitro.Single-factor culture method was used to analyze the activities of F3H in Escherichia coli and Saccharomyces cerevisiae.[Results] Enzymatic analysis showed that the optimal temperature of CsF3H,GbF3H and GmF3H was 40,40 and 35℃,and the optimal pH was 7.5,7.0 and 7.5,respectively.The k_cat/K_m of CsF3H was 0.36 L/(mmol·s),which was higher than that of GbF3H and GmF3H.When substrate naringenin concentration was 500 μmol/L,the conversion rate was more than 80% of the E.coli contained CsF3H,GmF3H,while the strain contained GbF3H was only 23.8%.As for the S.cerevisiae with different F3H,the conversion efficiency was 40%,with no significant difference.[Conclusion] There were different catalytic activities of F3H from different plants.Moreover,one F3H also showed great differences within prokaryotic and eukaryotic chassis cells.CsF3H has excellent catalytic capacity and showed better development and application potential in prokaryotic chassis cells.