Fe2+和Fe3+对菠萝蛋白酶活性和稳定性的影响

Effects of Iron (II) (Fe2+) and Iron (III) (Fe3+) ions on the Activity and Stability Bromelain

摘要:

本文研究不同浓度的Fe2+和Fe3+对菠萝蛋白酶活性和60 ℃下酶稳定性的影响，以及通过圆二色谱检测Fe2+和Fe3+对酶构象的变化，并初步探究EDTA-2Na结合超滤膜法在菠萝蛋白酶制备工艺中清除铁离子的应用效果。结果表明：Fe2+浓度在0~0.75 mmol/L范围内，对菠萝蛋白酶活性有促进作用，以0.5 mmol/L促进效果最佳，但浓度大于0.75 mmol/L时，抑制酶活，且随浓度增加，抑制程度增强。Fe3+对菠萝蛋白酶只有抑制作用，抑制程度与Fe3+浓度成正比，且Fe3+对酶的抑制作用强于Fe2+。60 ℃下，Fe2+在浓度为0.5 mmol/L对菠萝蛋白酶的稳定性表现出促进作用，延长了酶的半衰期。Fe3+对酶的稳定性呈现抑制作用。Fe2+和Fe3+浓度大于0.5 mmol/L时，随离子浓度的升高，对酶稳定性的抑制作用增强。采用圆二色谱检测酶的二级结构表明：Fe2+和Fe3+对酶抑制作用表现为α-螺旋含量下降，β-折叠和β-转角含量稍下降，无规卷曲含量明显提高。EDTA-2Na结合超滤膜法除铁的效果很明显，铁含量（干重）从291.63 mg/kg降低到142.99 mg/kg，铁离子去除率达50.97%，其酶活也由597.27 U/mg上升到808.52 U/mg，酶活提高了26.13%。

Abstract:

The effects of different concentrations of iron (II) (Fe2+) and iron (III) (Fe3+) on the activity of bromelain and the thermostability of bromelain at 60 ℃ were studied, and circular dichroism (CD) spectra were used to determine conformational changes of bromelain caused by Fe2+ and Fe3+. In addition, the iron ion removal effect of a method combining ethylenediaminetetraacetic acid disodium salt and ultrafiltration during the preparation of bromelain was preliminarily explored. The results showed that Fe2+ at a concentration range from 0 to 0.75 mmol/L improved the activity of bromelain, and the best Fe2+ concentration was found as 0.5 mmol/L. When the concentration of Fe2+ was higher than 0.75 mmol/L, the activity of the enzyme was inhibited, and the extent of inhibition was increased with increasing Fe2+ concentration. Fe3+ had an inhibitory effect on enzyme activity, and the extent of inhibition was proportional to the concentration of Fe3+. The inhibitory effect of Fe3+ on the enzyme was stronger than that of Fe2+. At 0.5 mmol/L, Fe2+ had a promoting effect on the thermostability of bromelain at 60 ℃ and extended the half-life of the enzyme. However, Fe3+ had an inhibitory effect on the thermostability of bromelain. When the concentrations of Fe2+ and Fe3+ were higher than 0.5 mmol/L, their inhibitory effects on the thermostability of bromelain increased with increasing ion concentration. According to CD spectra, the inhibitory effects of Fe2+ and Fe3+ on bromelain were reflected in a decrease in the contents of α-helices, a slight decrease in the contents of β-sheets and β-turns, and a significant increase in random coil contents. The method combining ethylenediaminetetraacetic acid disodium salt and ultrafiltration was effective for iron ion removal, with a reduction of iron content from 291.63 mg/kg to 142.99 mg/kg (dry weight), and the elimination rate of iron ion was 50.97%. Moreover, the enzyme activity increased from 597.27 U/mg to 808.52 U/mg, and the growth rate of activity was increased by 26.13%.