荔枝果皮中4种酚类代谢酶活性的测定方法

Methods for Determination of the Activities of Four Enzymes Involving Phenolic Metabolism in the Pericarp of Litchi

为了改进酚类物质生物合成途径中关键酶酶活性的测定方法,以南方重要常绿果树荔枝Litchi chinensis果皮为材料,采用两点法和酶动力法2种酶活性研究方法,结合分光光度法和高效液相色谱法检测产物浓度变化,优化了4种酚类代谢常见酶[包括苯丙氨酸解氨酶(PAL)、查儿酮异构酶(CHI)、二氢黄酮醇还原酶(DFR)和类黄酮糖基转移酶(UFGT)]的测定方法,指出了酶液的纯化、酶活性的保护以及反应底物的溶解方法等对试验结果的稳定性和可靠性的影响,介绍了试验过程中常遇到的问题和具体的解决方法以及试验过程中的注意事项.应用改进后的方法测定荔枝发育过程中果皮PAL、CHI、DFR和UFGT的酶活性,结果发现活性变化趋势与前人的报道基本一致.改进的PAL、CHI、DFR和UFGT酶活性的测定方法可靠,可为相关的研究提供重要的参考.

The objective of this study was to establish improved methods for determination of the activities of the key phenolic biosynthesis enzymes .The pericarp of litchi （ Litchi chinensis ） , an important ever-green fruit crop cultivated in south China , was used as material .Two-point method and enzymatic kinetic method combined with the use of photospectrometry and high performance liquid chromatography to ana -lyze the formation of end products were applied .The key phenolic biosynthesis enzymes included phenyl-alanine ammonia-lyase （ PAL ） , chalcone flavanone isomerase （ CHI ） , dihydroflavonol 4-reductase （ DFR ） and UDP-glucose： flavonoid 3-O-glucosyltransferase （ UFGT ） .The enzyme purification from crude extracts , enzyme activity protection and methods to dissolve substrates were all critical for reliable enzyme activity determinations .The possible problems during the activity determination of these enzymes and corresponding solutions were mentioned .The changes in PAL, CHI, DFR and UFGT activities meas-ured by the improved methods in the pericarp of litchi during fruit development were generally consistent with previous reports .The improved methods are reliable and will provide important reference for phenol-ic biosynthesis studies .