Engineering a chimeric pyrroloquinoline quinone glucose dehydrogenase: improvement of EDTA tolerance, thermal stability and substrate specificity

An engineered Escherichia coli PQQ glucose dehydrogenase (PQQGDH)with improved enzymatic characteristics was constructed by substitutingand combining the gene-encoding protein regions responsiblefor EDTA tolerance, thermal stability and substrate specificity.The protein region responsible for complete EDTA tolerance inAcinetobacter calcoaceticus, which is recognized as the indicatorof high stability in co-factor binding, was elucidated. Theregion is located between 32 and 59% from the N-terminus ofA.calcoaceticus PQQGDH(A27 region) and also corressponds tothe same position from 32 to 59% from the N-terminus in E.coliPQQGDH, though E.coli PQQGDH is EDTA sensitive. We previouslyreported that the C-terminal 3% region of A.calcoaceticus (A3region) played an important role in the increase of thermalstability, and that His775Asn substitution in E.coli PQQGDHresulted in an increase in the substrate specificity of E.coliPQQGDH towards glucose. Based on these findings, chimeric and/ormutated PQQGDHs, E97A3 H775N, E32A27E41 H782N, E32A27E38A3 andE32A27E38A3 H782N were constructed to investigate the compatibilityof two protein regions and one amino acid substitution. His775substitution to Asn corresponded to His782 substitution to Asn(H782N) in chimeric enzymes harbouring the A27 region. Sinceall the chimeric PQQGDHs harbouring the A27 region were EDTAtolerant, the A27 region was found to be compatible with theother region and substituted amino acid responsible for theimprovement of enzymatic properties. The contribution of theA3 region to thermal stability complemented the decrease inthe thermal stability due to the His775 or His782 substitutionto Asn. E32A27E38A3 H782N, which harbours all the above mentionedthree regions, showed improved EDTA tolerance, thermal stabilityand substrate specificity. These results suggested a strategyfor the construction of a semi-artificial enzyme by substitutingand combining the gene-encoding protein regions responsiblefor the improvement of enzyme characteristics. The characteristicsof constructed chimeric PQQGDH are discussed based on the predictedmodel, ß-propeller structure.