Development of highly efficient whole-cell catalysts of cis-epoxysuccinic acid hydrolase by surface display

Abstract

Bacterial cis-epoxysuccinic acid hydrolases (CESHs) are intracellular enzymes used in the industrial production of enantiomeric tartaric acids. The enzymes are mainly used as whole-cell catalysts because of the low stability of purified CESHs. However, the low cell permeability is the major drawback of the whole-cell catalyst. To overcome this problem, we developed whole-cell catalysts using various surface display systems for CESH[L] which produces L(+)-tartaric acid. Considering that the display efficiency depends on both the carrier and the passenger, we screened five different anchoring motifs in Escherichia coli. Display efficiencies are significantly different among these five systems and the InaPbN-CESH[L] system has the highest whole-cell enzymatic activity. Conditions for InaPbN-CESH[L] production were optimized and a maturation step was discovered which can increase the whole-cell activity several times. After optimization, the total activity of the InaPbN-CESH[L] surface display system is higher than the total lysate activity of an intracellular CESH[L] overexpression system, indicating a very high CESH[L] display level. Furthermore, the whole-cell InaPbN-CESH[L] biocatalyst exhibited good storage stability at 4 °C and considerable reusability. Thereby, an efficient whole-cell CESH[L] biocatalyst was developed in this study, which solves the cell permeability problem and provides a valuable system for industrial L(+)-tartaric acid production.

Keywords: cis-Epoxysuccinic acid hydrolase; Surface display; Whole-cell biocatalyst.

Fig. 1

CESH[L] surface display efficiencies of different anchoring motifs. a SDS-PAGE analysis of the whole cells or the cell lysate with and without trypsin treatment. Lanes 1–2, whole cells of intracellular CESH[L] expression system without and with trypsin treatment; Lane 3, trypsin-treated cell lysate of the intracellular CESH[L] expression system. Lanes 4–13, each pair of lanes are trypsin-untreated and treated whole cells expressing LOA-CESH[L], MipAV₁₄₀-CESH[L], YiaTR₂₃₂-CESH[L], InaKN-CESH[L], and InaPbN-CESH[L], respectively. Lanes M, standard protein molecular weight markers. b Whole-cell activities of various CESH[L] expression systems incubated at 4 °C for different days. c Comparison of the surface display systems and intracellular expression system. Intra, LOA, MipA, YiaT, InaKN, and InaPbN represent the cells expressing intracellular CESH[L], LOA-CESH[L], MipAV₁₄₀-CESH[L], YiaTR₂₃₂-CESH[L], InaKN-CESH[L], and InaPbN-CESH[L], respectively. Intra-L, Intra-T, and Intra-T-S represent the whole-cell lysate, the Triton X-100 treated cells, and the supernatant after the Triton X-100 treatment, respectively, of the intracellular expression system. The activities of all surface display systems were measured after 2-day incubation at 4 °C, while the activities of the intracellular expression system were measured without incubation. Symbols ** indicate significant differences with p-values less than 0.01

Fig. 2

SDS-PAGE analysis of the InaPbN-CESH[L] expression. a The effect of the inducer IPTG concentrations. Lanes 1–12, each pair of lanes are trypsin-untreated and treated whole cells induced by IPTG with the concentrations of 0.0 mM, 0.2 mM, 0.4 mM, 0.6 mM, 0.8 mM, and 1.0 mM. Lane M, standard protein molecular weight markers. b The effect of the expression temperature. Lanes 1–10, each pair of lanes are trypsin-untreated and treated whole cells with the expression temperatures 16 °C, 20 °C, 25 °C, 30 °C, and 37 °C. Lane M, standard protein molecular weight markers. c The whole-cell enzymatic activities of InaPbN-CESH[L] with different expression temperatures. d Fraction analysis of intracellular expressed CESH[L] (lanes 1–3) and displayed InaPbN-CESH[L] (lanes 4–6). W, whole-cell lysate; S, supernatant after centrifugation of the cell lysate; P, pellet after centrifugation of the cell lysate. Lane M, standard protein molecular weight markers

Fig. 3

Effects of pH and temperature on surface-displayed InaPbN-CESH[L] enzymatic activity and stability. a Relative enzymatic activities in buffers with different pH values. The activities were measured after incubation at 4 °C for 5 h. The highest whole-cell enzymatic activity in Tris–HCl buffer (pH8.5) was set to 100%. b The long-term stability at 4 °C in Tris–HCl buffers with different pH values. c Trypsin accessibility assays of the InaPbN-CESH[L] displayed whole cells after incubation at 4 °C. Lanes 1–8, each pair of lanes are trypsin-untreated and treated whole cells with different lengths of incubation time. Lane M, standard protein molecular weight markers. d The long-term stability of the InaPbN-CESH[L] displayed whole cells incubated at different temperatures

Fig. 4

Repeated use of the InaPbN-CESH[L] surface display system. The cells after protein expression were incubated at 4 °C for different days and then the whole-cell enzyme activities were repetitively assayed four times. After each enzymatic reaction, the cells were centrifuged and washed with the assay buffer, and then the whole-cell enzyme activities were immediately assayed again as repetitive usage