Optimization of bacterial cytokine protein production by response surface methodology for environmental bioremediation

Abstract

In natural and engineered systems, most microorganisms would enter a state of dormancy termed as "viable but non-culturable" (VBNC) state when they are exposed to unpredictable environmental stress. One of the major advances in resuscitating from such a state is the discovery of a kind of bacterial cytokine protein called resuscitation-promoting factor (Rpf), which is secreted from Micrococcus luteus. In this study, the optimization of Rpf production was investigated by the response surface methodology (RSM). Results showed that an empirical quadratic model well predicted the Rpf yield, and the highest Rpf protein yield could be obtained at the optimal conditions of 59.56 mg L^-1 IPTG, cell density 0.69, induction temperature 20.82 °C and culture time 7.72 h. Importantly, Phyre2 web portal characterized the structure of the Rpf domain to have a shared homology with lysozymes, and the highest lysozyme activity was at pH 5 and 50 °C. This study broadens the knowledge of Rpf production and provided potential strategies to apply Rpf as a bioactivator for environmental bioremediation.

Fig. 1. Protein profiles and zymograms. Lane 1: molecular weight high range; lane 2: uninduced; lane 3: induced; lane 4: the precipitation of the cell lysate of E. coli BL21 (DE3); lanes 5: the supernatant of the cell lysate of E. coli BL21 (DE3); lane 6: unbound washed fractions; lane 7: eluted fractions; lane 8 and 9: zymograms against M. luteus.

Fig. 2. Contour plots for yield of Rpf. (A) IPTG concentration and induced cell density; (B) IPTG concentration and induction temperature; (C) IPTG concentration and culture time; (D) induced cell density and induction temperature; (E) induced cell density and culture time; (F) induction temperature and culture time. Remaining variables were fixed at coded zero level.

Fig. 3. Response surface plots for yield of Rpf. (A) IPTG concentration and induced cell density; (B) IPTG concentration and induction temperature; (C) IPTG concentration and culture time; (D) induced cell density and induction temperature; (E) induced cell density and culture time; (F) induction temperature and culture time. Remaining variables were fixed at coded zero level.

Fig. 4. Emission fluorescence spectra of the protein yield with and without optimization. Conditions without optimization: IPTG = 100 mg L⁻¹, cell density = 0.4, induction temperature = 20 °C, culture time = 7 h.

Fig. 5. Structure of Rpf and“d1xsfa1” model. (A) Prediction of the Rpf structure generated by the Phyre2 server; (B) “d1xsfa1” model belong to the lysozyme-like superfamily.

Fig. 6. Effects of pH (A) and temperature (B) on the peptidoglycan hydrolases activity of Rpf.