UltraPure^TM Tris Hydrochloride

Company: Thermo Fisher Scientific

Catalog#: 15506017

Preparation and Purification of β-1,3-glucan-Linked Candida glabrata Cell Wall Proteases by Ion-Exchange Chromatography, Gel Filtration, and MDPF-Gelatin-Zymography Assay

Pirjo Pärnänen, Timo Sorsa, Taina Tervahartiala and Pirjo Nikula-Ijäs

Published online: 2024-03-20

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Easy and Efficient Permeabilization of Cyanobacteria for in vivo Enzyme Assays Using B-PER

Author:

Simon Matthé Erstad and Yumiko Sakuragi,

Date:

2018-01-05

[Abstract] Cyanobacteria are photosynthetic bacteria that thrive in diverse ecosystems and play major roles in the global carbon cycle. The abilities of cyanobacteria to fix atmospheric CO₂ and to allocate the fixed carbons to chemicals and biofuels have attracted growing attentions as sustainable microbial cell factories. A better understanding of activities of enzymes involved in the central carbon metabolism might lead to increased product yields. Currently, cell-free lysates are widely used for the determination of intracellular enzyme activities. However, due to thick cell walls in cyanobacteria, lysis of cyanobacterial cells is inefficient and often laborious. The present protocol describes an easy and efficient method to permeabilize cyanobacterial cells, without lysing them, and ... [摘要] 蓝细菌是光合细菌，在不同的生态系统中繁衍，在全球碳循环中发挥重要作用。蓝藻固定大气CO 2和将固定碳分配到化学品和生物燃料的能力作为可持续的微生物细胞工厂已经引起越来越多的关注。更好地了解参与中央碳代谢的酶的活性可能导致产物产量增加。目前，无细胞裂解物被广泛用于细胞内酶活性的测定。然而，由于蓝细菌细胞壁较厚，蓝细菌细胞的裂解效率低下且费力。目前的方案描述了一种简单而有效的方法来渗透蓝藻细胞，而不溶解它们，并直接使用透化细胞来测定体内代谢酶活性。

【背景】我们之前已经报道了使用B-PER TM试剂（Thermo Fisher Science）（Rasmussen等人，2016）简单，有效且可扩展的蓝细菌的透化。 B-PER TM TM试剂含有溶解在Tris-HCl缓冲液中的未公开的温和洗涤剂，通常用于裂解细菌细胞如大肠杆菌（Escherichia coli）。偶然地，我们发现B-PER TM TM试剂渗透蓝细菌细胞而不是溶解它们，可能是因为厚的蓝细菌细胞壁（Hoiczyk和Hansel，2000）赋予了试剂中使用的去污剂的抗性。在生物技术感兴趣的蓝细菌中进行通透化。聚球蓝细菌PCC 7002（以下简称“Synechococcus”7002）和“Synechocystis”sp。 PCC ...

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