Enhancing the synthesis of latex clearing protein by different cultivation strategies

•The solubility of Lcp1VH2 played an important role in the production yields.•Variations in temperature and agitation rate affected the solubility of Lcp1VH2.•The activity of Lcp1VH2 was affected by variations in the cultivation conditions.•The use of NusA as fusion partner improved the protein solubility.•NusA-His-Lcp1VH2 showed oxygenase activity in the presence of polyisoprene. In this study, we improved the synthesis of the latex clearing protein from Gordonia polyisoprenivorans VH2 (Lcp1VH2), 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 Lcp1VH2 and improve the production yields. Different cultivation temperatures and agitation rates were evaluated in the process to demonstrate their impact on the solubility of Lcp1VH2. A specific maximum production rate of 28.3 mg Lcp1VH2 g−1 cell dry weight h-1 was obtained at 25 °C and at agitation rates between 200–300 rpm. The activity of Lcp1VH2 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 Lcp1VH2 fusion protein was calculated to be 73.1%, which means an enhancement of 5.7-fold in comparison to the solubility of the native Lcp1VH2. The fusion protein of Lcp1VH2 and NusA still exhibited oxygenase activity with polyisoprene latex as a substrate. In fact, NusA-His-Lcp1VH2 reached a 4-fold higher volumetric activity in comparison to Lcp1VH2. Oligo(cis-1,4-isoprene) molecules were produced as degradation products due to the cleavage of the polymer backbone by NusA-His-Lcp1VH2. The formation of oligo-isoprenoid molecules with molecular weights between 236 and 984 Da were confirmed by electrospray ionization-mass spectrometry analysis.