Cell-free synthesis of functional phospholipase A1 from Serratia sp

Abstract

Background: Phospholipase A1 is an enzyme that hydrolyzes phospholipids at the sn-1 position. It has potential applications across diverse fields including food, pharmaceutical, and biofuel industries. Although there has been increasing interest in the use of phospholipase A1 for degumming of plant oils during biodiesel production, production of recombinant phospholipase A1 has been hampered by low efficiency of gene expression and its toxicity to the host cell.

Results: While expression of phospholipase A1 in Escherichia coli resulted in extremely low productivity associated with inhibition of transformed cell growth, drastically higher production of functional phospholipase A1 was achieved in a cell-free protein synthesis system where enzyme expression is decoupled from cell physiology. Compared with expression in E. coli, cell-free synthesis resulted in an over 1000-fold higher titer of functional phospholipase A1. Cell-free produced phospholipase A1 was also used for successfully degumming crude plant oil.

Conclusions: We demonstrate successful production of Serratia sp. phospholipase A1 in a cell-free protein synthesis system. Including the phospholipase A1 investigated in this study, many industrial enzymes can interfere with the regular physiology of cells, making cellular production of them problematic. With the experimental results presented herewith, we believe that cell-free protein synthesis will provide a viable option for rapid production of important industrial biocatalysts.

Keywords: Biodiesel; Cell-free protein synthesis; Enzymatic degumming; Industrial enzymes; Phospholipase A1.

Fig. 1

Expression of recombinant PLA1 in E. coli. Escherichia coli strain BL21 (DE3) was grown in 400 mL of LB media after being transformed with the plasmid pET21a Serr PLA1. Samples of culture broth were taken throughout the culture period to measure optical density at 600 nm (a) and PLA1 activity (b) as described in the “Methods” section. At 16 h after induction with IPTG, the total amount of cellular protein was measured by the BCA assay (c). Arrows indicate the time point for IPTG induction. Results from the E. coli cultures with or without IPTG induction are shown in closed and open circles, respectively. Error bars represent the standard deviation from three independent experiments

Fig. 2

Synthesis of PLA1 from Serratia sp. in a cell-free protein synthesis system. The plasmid pET21a Serr PLA1 was incubated in the reaction mixture for cell-free protein synthesis as described in “Methods” section. After incubation for 3 h, total and soluble amounts of cell-free synthesized PLA1 were quantified by measuring the TCA-insoluble radioactivity of the samples (a). Embedded image of a shows the results of SDS-PAGE and western blot analysis. PLA1 activity produced per mg of endogenous cellular protein in E. coli or cell-free protein synthesis system was compared in b

Fig. 3

Purification of PLA1 expressed in each system. Cell-free synthesized PLA1 from a 3 mL reaction was purified through a Ni-NTA column (a). Turbidity of the crude sesame oil was cleared after incubation with the purified PLA1 (30 µg PLA1 in 10 mL oil) for 12 h at 40 °C, indicating that the cell-free synthesized enzyme catalyzed degumming of crude oil (b)

Fig. 4

Cell-free synthesis of PLA1 in varying reaction volumes. The reaction scale of cell-free PLA1 synthesis was increased sequentially from 150 µL to 3 mL to 500 mL. The total and soluble productivity and titer of functional PLA1 were not significantly affected by the reaction volume. The bars in the graph represent total (blank) and soluble (filled) amounts of cell-free synthesized protein. Circles represent PLA1 activity measured in the reaction mixture. Error bars represent the standard deviation from three independent experiments