摘要
本文研究不同浓度的Fe^(2+)和Fe^(3+)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Fe^(2+)和Fe^(3+)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:Fe^(2+)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。Fe^(3+)对菠萝蛋白酶只有抑制作用,抑制程度与Fe^(3+)浓度成正比,且Fe^(3+)对酶的抑制作用强于Fe^(2+)。60℃下,Fe^(2+)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。Fe^(3+)对酶的稳定性呈现抑制作用。Fe^(2+)和Fe^(3+)浓度大于0.5mmol/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:Fe^(2+)和Fe^(3+)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%。
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
出处
《现代食品科技》
EI
CAS
北大核心
2017年第8期176-181,共6页
Modern Food Science and Technology
基金
广东省科技计划项目(2016B090918103)
珠海进出口公共技术服务平台产学研究协同创新计划(IETP201601006)
关键词
菠萝蛋白酶
Fe2+和Fe3+
酶活及稳定性
半衰期
圆二色谱
bromelain
iron（Ⅱ）（Fe2＋） and iron（Ⅲ）（Fe3＋）
the activity and thermostability of bromelain
half-life
circular dichroism spectra
