Extended Data Fig. 7: Biocatalysis of heme by CPIII and the stability assessment of HemQ28-M.
From: High-yield porphyrin production through metabolic engineering and biocatalysis
a, b, Quantitative analysis of heme and other chemicals after enzymatic catalysis (n = 3 biologically independent replicates). c, d, Effects of temperature and pH on the enzymatic activity of HemQSa and HemQ28-M. Three biologically independent replicates (n = 3) were conducted for each condition in c and d. e, One-time addition of crude enzyme catalyzed the conversion CPIII to heme in 5-L bioreactor. At the beginning of the reaction, 2 L of CPIII with a concentration of 18 g/L was added all at once. The crude enzyme supernatant obtained by crushing 180 g of wet recombinant E. coli cells was added to the reaction system at the beginning of the reaction (that is, 0 h). The reaction was performed at 37 °C and pH 7.0 with agitation at 100 rpm. f, Heme production rate after fed-batch addition of crude enzyme solution (CES) in 5-L bioreactor. After the enzymatic reaction commenced, the CES obtained by crushing wet recombinant E. coli cells was fed-batch added to the 5-L bioreactor at 1, 3, 5, and 7 h. This study measured the heme production rate within two hours after the addition of CES at 1, 3, and 5 h. The concentration of the target fusion protein in the CES was determined by comparing its band’s grayscale intensity with that of a BSA standard following SDS-PAGE electrophoresis, using a concentration of 0.5 g/L BSA as a reference for quantification. Each addition of CES contains 722 ± 140 mg of target fusion protein (derived from approximately 36 g of wet E. coli cells). After each addition of CES, heme concentration increases were measured at five intervals (specifically, 0–5 min, 5–10 min, 25–30 min, 55–60 min, and 115–120 min) within the subsequent two hours to calculate the heme production rate for each corresponding time interval. Bar graphs with error bars represent mean ± s. d. *P < 0.05.
