High-level Production And Directed Evolution Study Of Pyrroloquinoline Quinone-dependent Glucose Dehydrogenase In Escherichia Coli

Soluble pyrroloquinoline quinone-dependent glucose dehydrogenase (EC1.1.5.2, sPQQGDH) is an important kind of oxidoreductase, and has attracted wide attention for its application of glucose detection in the field of clinic diagnosis and industrial glucose sensor. However, low expression level and inferior thermal stability limit its mass industrial production and application scope. The main purpose of our work here was to realize the high-level production of sPQQGDH and its excellent thermostability.Firstly, three different expression vectors pET28-gdh, pTrc99a-gdh and pMALc5E-gdh were constructed, and then transformed into E.coli BL21(DE3). After induction, recombinant sPQQGDH could be efficiently overexpressed by the above three vectors, and pET28-gdh showed the highest production yield, which was chosen as the final expression vector. Then different induction conditions, the types and concentrations of carbon source were investigated for over-expression of soluble sPQQGDH. The results indicated that the optimal medium was MMBL with20g/L of glucose, and the target protein displayed the best solubility when cells were induced with0.05mM IPTG at the middle stage of exponential phase and then cultured at25℃. Moreover, the highest-level expression (1530kU/L) of sPQQGDH was achieved by optimized cultivation strategy in the10L fermentor, which was about7-fold improvement over the reported value. Our work provided an alternative way to over-express sPQQGDH and revealed its promising potential for the large-scale production of this important glucose dehydrogenase.Recombinant sPQQGDH was expressed as fusion protein with His tag, which could be purified by affinity chromatography. After the Ni-NTA affinity chromatography purification, high-purity enzyme with the specific activity of5811U/mg was obtained with a good recovery rate of55%. Then enzyme kinetics parameters, thermostability and substrate specificity of sPQQGDH were investigated:Km value was21mM, the half-life period at55℃was calculated as12.1min, and substrate specificity was wide. These characteristics indicated that His tag didn’t affect the structure and function of target protein, and the purified recombinant sPQQGDH presented similarly as the native one.Owning to the thermal instability of sPQQGDH, two strageties were adopted to improve its thermal stability. On the one hand, a kind of composite protectants (1%sucrose,10%glycerol and lmg/ml BSA) selected from usual protective agents was used, and the half-life period at55℃was doubled. On the other hand, directed evolution strategy was employed. Random mutation was obtained by erro-prone PCR. Then the efficiencies of electricity competence cells and ligation were optimized to establish a large mutation library with capacity of104. Our work here created a new High Throughput Screening method——overlap filter paper with reaction substrate on the plates after heat treatment, and then screen positive clones according to the color change on filter paper. Based on this method, five mutations Gly263Cys, Thr416Ile, Gln270His, Asp111Glu and Thr331Met were selected out.The thermal stability of G1y263Cys was the strongest, and its half-life period at55℃was as much as130min, which was evenly comparable to the reported value. In conclusion, the thermal stability of sPQQGDH was improved greatly by protective agents and directed evolution, which lay the foundation for its further application and industrial production.