After the equilibration, 1?L of AtaA PSD answer (0.1?mg/mL) was added and the rate of recurrence switch was measured for 5?min. at 28?C with shaking at 115?rpm. After incubation for 10?min, the helps were retrieved and lightly washed with BVT 948 100?mM KCl solution. Then, the supports were picked up with tweezers and shaken in 100?mL of 100?mM KCl solution for 1?min. 12934_2017_740_MOESM2_ESM.avi (3.7M) GUID:?04D6D8A8-ACEE-405C-9FE0-2CD8BFE4D8B2 Data Availability StatementAll data generated or analyzed during this study are included in this published article and its additional documents. Abstract Background Immobilization of microbial cells is an important strategy for the efficient use of whole-cell catalysts because it simplifies product separation, enables the cell concentration to be improved, stabilizes enzymatic activity, and enables repeated or continuous biocatalyst use. However, conventional immobilization methods have practical limitations, such as limited mass transfer in the inner portion of a gel, gel fragility, cell leakage from your support matrix, and adverse effects on cell viability and catalytic activity. We previously showed a new method for bacterial cell immobilization using AtaA, a member of the trimeric autotransporter adhesin family found in sp. Tol 5. This approach is definitely expected to solve the drawbacks of standard immobilization methods. However, similar to all other immobilization methods, the use of support materials increases the cost of bioprocesses and subsequent waste materials. Results We found that the stickiness of the AtaA molecule isolated from Tol 5 cells is definitely drastically diminished at ionic advantages lower than 10?mM and that it cannot adhere in deionized water, which also inhibits cell adhesion mediated by AtaA. Cells immobilized on well plates and polyurethane foam inside a salt answer were detached in deionized water by rinsing and shaking, respectively. The detached cells regained their adhesiveness inside a salt answer and could rapidly become re-immobilized. The cells expressing the BVT 948 gene taken care of their adhesiveness throughout four repeated immobilization and detachment cycles and could be repeatedly immobilized to polyurethane foam by a 10-min shake inside a flask. We also shown that both bacterial BVT 948 cells and a support used in a reaction could be reused for any different type of reaction after detachment of the in the beginning immobilized cells from your support and a subsequent immobilization step. Conclusions We developed a unique reversible immobilization method based on the salt-dependent adhesion of the AtaA molecule that allows us to reuse bacterial cells and supports by a simple manipulation including a deionized water wash. This mitigates problems caused by the use of support materials and greatly helps to enhance the effectiveness and productivity of microbial production processes. Electronic supplementary material The online version of this article (doi:10.1186/s12934-017-0740-7) contains supplementary material, which is available to authorized users. sp. Tol 5 [20C22], which belongs to the trimeric autotransporter adhesin (TAA) family . Although AtaA shares a fibrous architecture consisting of an N-terminuspassenger website (PSD) containing head and stalk domainstransmembrane anchor (TM)C-terminus with TAA family members , which usually bind to target biotic surfaces, AtaA distinctively confers nonspecific high adhesiveness to both abiotic and biotic surfaces on bacterial cells transformed with its gene. Large amounts of growing, resting, actually lyophilized Amotl1 transformant cells can be quickly and strongly immobilized onto any material surfaces selected according to the software . Cells immobilized directly on surfaces through AtaA are not inlayed in extracellular polymeric substances with mass transfer limitations, BVT 948 show enhanced tolerance , increase chemical reaction rates, and may become repeatedly used in reactions without inactivation . However, similar to all other immobilization methods, the use of support materials increases the cost of bioprocesses and subsequent waste materials. These might be inevitable problems as long as support materials are used in the immobilization process. A way to minimize these drawbacks should be developed so as to, for example, reduce the amount of support materials, use inexpensive materials or waste materials, and reuse support materials. AtaA is definitely a homotrimer of polypeptides comprising 3630 amino acids. In a earlier study, we developed a method to isolate its PSD, which is definitely secreted to the bacterial cell surface through the TM and is responsible for biological functions, by genetically introducing a recognition site for human rhinovirus 3C (HRV 3C) protease . Specific cleavage by the protease reaps AtaA PSD nanofibers 225?nm in length from the cell surface. This enables biochemical and biophysical analyses of the purified huge AtaA PSD in the native molecular state. Here, we demonstrate a new phenomenon: AtaA PSD cannot adhere to surfaces in deionized water (dH2O). Based on this molecular property of AtaA, we developed a unique method for the reversible immobilization of bacterial cells, which can solve the problems caused by the.
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