来自短小克萨酵母HSC6的用于将β-紫罗兰酮转化为二氢-β-紫罗兰酮的新型烯还原酶的纯化及生化特性分析

Purification and biochemical characterization of a novel ene- reductase from Kazachstania exigua HSC6 for dihydro-β-ionone from β-ionone.

作者信息

Long Zhangde, Li Kena, Xue Yun, Sun Yongwei, Li Jigang, Su Zan, Sun Jiansheng, Liu Qibin, Liu Hong, Wei Tao

机构信息

China Tobacco Guangxi Industrial Co., Ltd., Nanning, 530001, China.

School of Food and Biological Engineering, Zhengzhou University of Light Industry, 5 Dongfeng Rd, Zhengzhou, 450002, China.

出版信息

Biotechnol Lett. 2023 Apr;45(4):499-508. doi: 10.1007/s10529-023-03355-1. Epub 2023 Feb 4.

DOI:10.1007/s10529-023-03355-1

PMID:36738355

Abstract

PURPOSE

We purified and characterized a novel ene-reductase (KaDBR1) from Kazachstania exigua HSC6 for the synthesis of dihydro-β-ionone from β-ionone.

METHODS

KaDBR1 was purified to homogeneity by ammonium sulfate precipitation and phenyl-Sepharose Fast Flow and Q-Sepharose chromatography. The purified enzyme was characterized by measuring the amount of dihydro-β-ionone from β-ionone with LC-MS analysis method.

RESULTS

The molecular mass of KaDBR1 was estimated to be 45 kDa by SDS-PAGE. The purified KaDBR1 enzyme had optimal activity at 60 °C and pH 6.0. The addition of 5 mM Mg, Ca, Al, Na, and dithiothreitol increased the activity of KaDBR1 by 25%, 18%, 34%, 20%, and 23%, respectively. KaDBR1 favored NADH over NADPH as a cofactor, and its catalytic efficiency (kcat/Km) toward β-ionone using NADH was 8.1-fold greater than when using NADPH.

CONCLUSION

Owing to its unique properties, KaDBR1 is a potential candidate for the enzymatic biotransformation of β-ionone to dihydro-β-ionone in biotechnology applications.

摘要

目的

我们从嗜盐碱地酵母HSC6中纯化并鉴定了一种新型的烯还原酶（KaDBR1），用于从β-紫罗兰酮合成二氢-β-紫罗兰酮。

方法

通过硫酸铵沉淀、苯基-琼脂糖快速流动柱层析和Q-琼脂糖柱层析将KaDBR1纯化至同质。用LC-MS分析方法通过测量从β-紫罗兰酮生成的二氢-β-紫罗兰酮的量来表征纯化后的酶。

结果

通过SDS-PAGE估计KaDBR1的分子量为45 kDa。纯化后的KaDBR1酶在60°C和pH 6.0时具有最佳活性。添加5 mM的镁、钙、铝、钠和二硫苏糖醇分别使KaDBR1的活性提高了25%、18%、34%、20%和23%。KaDBR1更倾向于以NADH而非NADPH作为辅因子，其使用NADH时对β-紫罗兰酮的催化效率（kcat/Km）比使用NADPH时高8.1倍。