Journal
ENGINEERING IN LIFE SCIENCES
Volume 12, Issue 5, Pages 574-582Publisher
WILEY-BLACKWELL
DOI: 10.1002/elsc.201100224
Keywords
Glucose dehydrogenase; Optimization; Recombinant expression; PQQGDH
Categories
Funding
- National High Technology Research and Development Program of China [2011AA02A114]
- National Natural Science Foundation of China [20736008, 20928006, 20876140]
- Outstanding Young Scientist Group of Zhejiang Natural Science Foundation [R4090041]
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Pyrroloquinoline quinone-dependent glucose dehydrogenase (EC1.1.5.2, PQQGDH) has attracted progressive attention due to its application in glucose detection in clinic diagnosis and industrial bioprocess controls. To satisfy its increasing demand, improvement of PQQGDH production derived from Acinetobacter calcoaceticus L.M.D. 79.41 in recombinant Escherichia coli is necessary and is therefore the focus of the current study. Different carbon sources as well as induction conditions were investigated for overexpression of soluble PQQGDH. The results indicate that the target protein was optimally produced with 20 g/L glucose as the substrate. Moreover, the highest expression level (1530 kU/L) was achieved by a novel two-temperature cultivation strategy in the 10-L fermentor. This presents a sixfold improvement over previously reported values. After Ni-NTA affinity chromatography purification, high-purity enzyme with the specific activity of 5811 U/mg was obtained with a purification yield of 55%. The purified recombinant PQQGDH showed thermal stability and substrate specificity as the native enzyme. In summary, this work provides an alternative production process to overexpress PQQGDH and shows high applicability for large-scale production of this important glucose dehydrogenase.
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