赖氨酸脱羧酶的基于液体的比色测定法及其在酶活性测定中的应用。

A Liquid-Based Colorimetric Assay of Lysine Decarboxylase and Its Application to Enzymatic Assay.

作者信息

Kim Yong Hyun, Sathiyanarayanan Ganesan, Kim Hyun Joong, Bhatia Shashi Kant, Seo Hyung-Min, Kim Jung-Ho, Song Hun-Seok, Kim Yun-Gon, Park Kyungmoon, Yang Yung-Hun

机构信息

Department of Biological Engineering, College of Engineering, Konkuk University, Seoul 143-701, Republic of Korea.

Chemical Engineering, Soongsil University, Seoul 156-743, Republic of Korea.

出版信息

J Microbiol Biotechnol. 2015 Dec 28;25(12):2110-5. doi: 10.4014/jmb.1505.05063.

DOI:10.4014/jmb.1505.05063

PMID:26282689

Abstract

A liquid-based colorimetric assay using a pH indicator was introduced for high-throughput monitoring of lysine decarboxylase activity. The assay is based on the color change of bromocresol purple, measured at 595 nm in liquid reaction mixture, due to an increase of pH by the production of cadaverine. Bromocresol purple was selected as the indicator because it has higher sensitivity than bromothymol blue and pheonol red within a broad range and shows good linearity within the applied pH. We applied this for simple determination of lysine decarboxylase reusability using 96-well plates, and optimization of conditions for enzyme overexpression with different concentrations of IPTG on lysine decarboxylase. This assay is expected to be applied for monitoring and quantifying the liquid-based enzyme reaction in biotransformation of decarboxylase in a high-throughput way.

摘要

引入了一种使用pH指示剂的基于液体的比色测定法，用于赖氨酸脱羧酶活性的高通量监测。该测定基于在液体反应混合物中于595nm处测量的溴甲酚紫的颜色变化，这是由于尸胺的产生导致pH升高所致。选择溴甲酚紫作为指示剂是因为它在广泛范围内比溴百里酚蓝和酚红具有更高的灵敏度，并且在所应用的pH范围内显示出良好的线性。我们将此用于使用96孔板简单测定赖氨酸脱羧酶的可重复使用性，以及用不同浓度的异丙基-β-D-硫代半乳糖苷（IPTG）对赖氨酸脱羧酶进行酶过量表达条件的优化。该测定有望用于以高通量方式监测和定量脱羧酶生物转化中基于液体的酶反应。