Enzymatic production of trans-4-hydroxy-l-proline by proline 4-hydroxylase

Abstract

Trans-4-hydroxy-l-proline (Hyp) is a useful chiral building block for production of many nutritional supplements and pharmaceuticals. However, it is still challenging for industrial production of Hyp due to heavy environmental pollution and low production efficiency. To establish a green and efficient process for Hyp production, the proline 4-hydroxylase (DsP4H) from Dactylosporangium sp. RH1 was overexpressed and functionally characterized in Escherichia coli BL21(DE3). The recombinant DsP4H with l-proline as a substrate exhibited K_m , k_cat and k_cat /K_m values up to 0.80 mM, 0.52 s^-1 and 0.65 s^-1 ·mM^-1 respectively. Furthermore, DsP4H showed the highest activity at 35°C and pH 6.5 towards l-proline. The highest enzyme activity of 175.6 U mg^-1 was achieved by optimizing culture parameters. Under the optimal transformation conditions in a 5-l fermenter, Hyp titre, conversion rate and productivity were up to 99.9 g l^-1 , 99.9% and 2.77 g l^-1 h^-1 respectively. This strategy described here provides an efficient method for production of Hyp and thus has a great potential in industrial application.