Cell-Free Production of Soybean Leghemoglobins and Nonsymbiotic Hemoglobin

Abstract

Hemoglobins are heme proteins and are present in certain microorganisms, higher plants, and mammals. Two types of hemoglobin are found in legume nodules: leghemoglobin (LegH) or symbiotic and nonsymbiotic (nsHb). LegHs occur in high amounts in legume roots, and together with bacteroides, are responsible for the nitrogen fixation process. nsHb Class 1 proteins have very high affinity for O₂ and are found in monocotyledons and legumes. LegH has attracted great interest in the vegetable meat industry owing to its organoleptic and nutritional properties. In this study, soybean LegHs A, C1, C2 and C3 and nsHb were produced via Escherichia coli-based cell-free systems (CFS) and their amino acid sequences were correctly synthesized. In addition, certain post-translational modifications were made, which were confirmed using liquid chromatography-mass spectrometry analysis. All LegHs produced in this system exhibited peroxidase activity and heme binding, which were correlated with their concentrations in the assays. Furthermore, all proteins were readily digested by pepsin within 1 min under analog digestion conditions. Thus, LegHs and nsHb proteins were produced in this study using cell-free systems, maintaining their functionality and digestibility. These findings suggest that they could serve as viable alternative food additives for plant-based meat.

Keywords: cell-free system; hemoglobins; leghemoglobins; nonsymbiotic hemoglobin; plant-based meat; protein production.

1

Production of LegH A, C1, C2, and C3 and nsHb proteins in a CFS. (A) Western blot analysis of cell-free expressed crude extracts of hemoglobins (LegHs, ≈15 kDa; nsHB, ≈18 kDa). (B) Western blot analysis of cell-free expressed purified hemoglobins. (C) Western blot analysis of nsHB protein produced using a medium-scale commercial continuous-exchange cell-free (CECF). (D) A Coomassie stained SDS-PAGE analysis of purified nsHB produced by CECF.

2

LegH A, C1, C2, and C3 and nsHb were produced in a CFS with their correct amino acid sequences. The digested proteins were subjected to LC–MS and analyzed using the PEAKS BD software. The peptides detected from each sample were filtered using an FDR of up to 1% and aligned with the LegH A, C1, C2, and C3 and nsHb amino acid sequences provided by the UniProt database. The blue lines below each amino acid sequence indicate trypsin-generated peptides identified using LC–MS; bold and highlighted letters represent the peptide coverage of original sequences. Amino acids for which PTMs, such as oxidation, have been identified are shown as red squares under the respective amino acid.

3

LegH A, C1, C2, and C3 and nsHb produced from the CFS exhibited peroxidase activity. The area under the curve was determined based on the action of 40, 20, 10, and 5 μg/mL of LegH A (A), C1 (B), C2 (C), and C3 (D) and nsHb (E). Units of enzymatic activity with 40 μg/mL of LegH A, C1, C2, and C3 and nsHb (F). Values shown are means and standard error from four independent experiments performed in duplicate. P-values were calculated using one-way ANOVA, with multiple comparisons corrected using Tukey’s test. Significance was represented by *­(p < 0.05); **­(p < 0.01); ***­(p < 0.001); ****­(p < 0.0001). NS: nonsignificant.

4

LegH A, C1, C2, and C3 and nsHb produced from the CFS have a heme group in their structure. (A) The heme concentration was measured from the absorbance and represented as the mean and standard error from three independent experiments performed in duplicate. P-values were calculated using one-way ANOVA, with multiple comparisons corrected using Tukey’s test. Significance was represented by *­(p < 0.05); **­(p < 0.01); ***­(p < 0.001); ****­(p < 0.0001). NS: nonsignificant. (B) Correlation graphic from heme concentration and Hb protein concentration, showing R ² values for each data set. Only significant comparisons are shown in the graphs; other comparisons were nonsignificant. P-values were calculated using the Pearson linear correlation coefficient. Significance was represented by *­(p < 0.05); **­(p < 0.01); ***­(p < 0.001); ****­(p < 0.0001). NS: nonsignificant.

5

LegH A, C1, C2, and C3 and nsHb produced in CFS are digested using pepsin. Hbs were incubated with pepsin (10 U/μg of LegH) in a simulated gastric fluid (SGL) of pH 2.0 at 37 °C for 0, 1, 5, 25, and 50 min. Digestion products were separated in SDS–PAGE and submitted to Western blot analysis (A–E).