Enhancing the synthesis of latex clearing protein by different cultivation strategies

In this study, we improved the synthesis of the latex clearing protein from Gordonia polyisoprenivorans VH2 (Lcp1_VH2), a key enzyme for the initial cleavage of the rubber backbone. Cultivations using a recombinant strain of Escherichia coli were optimized to overcome poor solubility of Lcp1_VH2 and improve the production yields. Different cultivation temperatures and agitation rates were evaluated in the process to demonstrate their impact on the solubility of Lcp1_VH2. A specific maximum production rate of 28.3 mg Lcp1_VH2 g^-1 cell dry weight h^-1 was obtained at 25 °C and at agitation rates between 200-300 rpm. The activity of Lcp1_VH2 was strongly influenced by variations in the cultivation temperature with a specific maximum activity of 0.81 U mg^-1 in cultures incubated at 30 °C. Besides cultivation-based optimization, also the strategy of fusion protein expression with NusA was successfully applied. The in vivo solubility of the Lcp1_VH2 fusion protein was calculated to be 73.1%, which means an enhancement of 5.7-fold in comparison to the solubility of the native Lcp1_VH2. The fusion protein of Lcp1_VH2 and NusA still exhibited oxygenase activity with polyisoprene latex as a substrate. In fact, NusA-His-Lcp1_VH2 reached a 4-fold higher volumetric activity in comparison to Lcp1_VH2. Oligo(cis-1,4-isoprene) molecules were produced as degradation products due to the cleavage of the polymer backbone by NusA-His-Lcp1_VH2. The formation of oligo-isoprenoid molecules with molecular weights between 236 and 984 Da were confirmed by electrospray ionization-mass spectrometry analysis.