天然产物研究与开发 ›› 2023, Vol. 35 ›› Issue (5): 819-827.doi: 10.16333/j.1001-6880.2023.5.010

• 开发研究 • 上一篇    下一篇

枸杞过氧化物酶的提取工艺优化及其酶学特性研究

高倩玲,胡龄文,陈鸿平*,刘友平,陈   林,胡   媛   

  1. 成都中医药大学药学院 西南特色中药资源国家重点实验室,成都 611137
  • 出版日期:2023-05-26 发布日期:2023-05-26
  • 基金资助:
    国家自然科学基金青年科学基金(81403098)

Optimization of extraction process of peroxidase from wolfberry and its enzymatic characteristics

GAO Qian-ling,HU Ling-wen, CHEN Hong-ping*,LIU You-ping,CHEN Lin,HU Yuan   

  1. State Key Laboratory of Southwest Characteristic Traditional Chinese Medicine Resources,School of Pharmacy,Chengdu University of Traditional Chinese Medicine,Chengdu 611137,China
  • Online:2023-05-26 Published:2023-05-26

摘要:

枸杞为常用“药食同源”中药材,在加工、贮藏、运输等过程中易发生褐变,严重影响枸杞的外观和质量。前期研究发现枸杞中具有较高的过氧化物酶(peroxidase,POD)活性,而过氧化物酶在植物的酶促褐变中发挥着重要作用。为更好地控制枸杞酶促褐变的发生,本研究采用响应面法对枸杞过氧化物酶提取工艺进行优化,对枸杞过氧化物酶的性质进行了研究。结果显示枸杞过氧化物酶最佳提取工艺为料液比1∶3,浸提时间5 h,缓冲液pH为6.0,枸杞过氧化物酶活力为(5 148.59±50.00)U,与模型预测值接近,拟合性好,表明所得响应面模型可以很好地预测和分析枸杞过氧化物酶提取工艺条件。枸杞过氧化物酶最适温度为50 ℃,最适pH为6.0,金属离子Na+、Ca2+、K+、Zn2+、Cu2+和柠檬酸对枸杞过氧化物酶起激活作用,抗坏血酸和亚硫酸能抑制枸杞过氧化物酶活性;该酶酶促反应动力学符合米氏方程,过氧化氢浓度一定时,酶对愈创木酚的Km=21.52 mmol/L,Vmax=115.47 U/mL;愈创木酚浓度一定时,酶对H2O2的Km=1.41 mmol/L,Vmax=148.81 U/mL。本研究可为后续枸杞加工过程中褐变的控制提供参考。

关键词: 枸杞, 过氧化物酶, 提取, 响应面法, 酶学特性

Abstract:

Wolfberry is a commonly used Chinese medicinal material with “medicine and food homology”,which is prone to browning during processing,storage,and transportation,resulting in seriously affects the appearance and quality of Chinese wolfberry.Our previous study found that wolfberry has high peroxidase activities,and peroxidase (POD) plays an essential part in the enzymatic browning of plants.In order to better control the occurrence of enzymatic browning of wolfberry,the extraction process of peroxidase from wolfberry was optimized by response surface method,and the properties of POD from wolfberry were studied.The results showed that the optimal extraction process of POD was as follows:the solid-liquid ratio was 1∶3,the extraction time was 5 h,and the buffer pH was 6.0,the enzyme activity of POD was (5 148.59±50.00) U,which was similar to the predicted value.The extraction conditions of POD could be predicted and analyzed by the response surface model.The optimum temperature and pH were 50 ℃ and 6.0.The effects of different metal ions on the PPO were studied,and the result showed that Na+,Ca2+,K+,Zn2+,Cu2+ and citric acid could activate the activity of POD; ascorbic acid and sulfite could inhibit the activity of POD.The kinetics of enzymatic reaction was fit to Michaelis-Menten equation,and the Km value of the enzyme to guaiacol was 21.52 mmol/L and the Vmax was 115.47 U/mL.The Km value of H2O2 was 1.41 mmol/L and the Vmax was 148.81 U/mL.This study can provide a reference for the control of browning in the subsequent processing of wolfberry.

Key words: wolfberry, peroxidase, extraction, response surface methodology, enzymatic characteristics

中图分类号:  R284