Abstract: The objective of this work is to obtain high-yield FAD-dependent glucose dehydrogenase（FAD-GDH）. First,by optimizing the codons of FAD-GDH gene according to the codon preference of Pichia pastoris,the gene fragment was artificially synthesized,then the recombinant vector pMD-GDH containing FAD-GDH e gene was constructed and transferred into P. pastoris（X33）,and the secretory expression by methanol induction was achieved. Results showed that a stable recombinant strain with high enzymatic activity was obtained by screening positive transformants at test-tube level. In the 10 L fermenter after 136 h induction culture,enzyme activity reached 257 600 U/L. The analysis of enzymatic characterization demonstrated that the optimal pH and temperature while using glucose as substrate were 7.0 and 55℃,respectively. The initial enzyme activity still remained 70% after 150 min treatment at 50℃. In the range of pH 4-7,FAD-GDH still retained over 50% activity after incubated at 37℃ for 4 h. Cu²⁺ presented relatively large inhibition to enzyme activity. FAD-GDH harbored a relative high substrate specificity and D-glucose was the optimal substrate. The efficient expression of FAD-GDH in P. pastoris is achieved by optimizing the codon preference of P. pastoris,which provides a theoretical basis for the detection of blood glucose.

Key words: FAD-dependent glucose dehydrogenase, codon optimization, recombinant Pichia pastoris, enzymatic characterization