An easy and efficient permeabilization protocol for in vivo enzyme activity assays in cyanobacteria

Microbial Cell Factories

Technical Notes

Simon Matthé Erstad¹  Erick Miguel Ramos-Martinez¹  Randi Engelberth Rasmussen¹  Yumiko Sakuragi¹  Lorenzo Fimognari¹  Alice Jara De Porcellinis¹ 

[1] Department of Plant and Environmental Sciences, Copenhagen Plant Science Center, University of Copenhagen, Frederiksberg, Denmark;

关键词: B-PER;    Permeabilization;    Cyanobacteria;    Glucose-6-phosphate dehydrogenase;    Rubisco;    SYTOX Green;    Enzyme activities;   

DOI  :  10.1186/s12934-016-0587-3

 received in 2016-06-15, accepted in 2016-10-29,  发布年份 2016

来源: Springer

【 摘 要 】

BackgroundCyanobacteria 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. Better understanding of the activities of enzymes involved in the central carbon metabolism would lead to increasing product yields. Currently cell-free lysates are the most widely used method for determination of intracellular enzyme activities. However, due to thick cell walls, lysis of cyanobacterial cells is inefficient and often laborious. In some cases radioisotope-labeled substrates can be fed directly to intact cells; however, label-free assays are often favored due to safety and practical reasons.ResultsHere we show an easy and highly efficient method for permeabilization of the cyanobacteria Synechococcus sp. PCC 7002 and Synechocystis sp. PCC 6803, and determination of two intracellular enzymes, ribulose-1,5-bisphosphate carboxylase/decarboxylase (Rubisco) and glucose-6-phosphate dehydrogenase (G6PDH), that play pivotal roles in the central carbon metabolism in cyanobacteria. Incubation of the cyanobacterial cells in the commercially available B-PER reagent for 10 min permeabilized the cells, as confirmed by the SYTOX Green staining. There was no significant change in the cell shape and no major loss of intracellular proteins was observed during the treatment. When used directly in the assays, the permeabilized cells exhibited the enzyme activities that are comparable or even higher than those detected for cell-free lysates. Moreover, the permeabilized cells could be stored at −20 °C without losing the enzyme activities. The permeabilization process and subsequent activity assays were successfully adapted to the 96-well plate system.ConclusionsAn easy, efficient and scalable permeabilization protocol was established for cyanobacteria. The permeabilized cells can be directly applied for measurement of G6PDH and Rubisco activities without using radioisotopes and the protocol may be readily adapted to studies of other cyanobacterial species and other intracellular enzymes. The permeabilization and enzyme assays can be performed in 96-well plates in a high-throughput manner.

【 授权许可】

CC BY   
© The Author(s) 2016