Characterization and Engineering of a Novel Pyrroloquinoline Quinone Dependent Glucose Dehydrogenase from Sorangium cellulosum So ce56

Abstract

A novel pyrroloquinoline quinone dependent glucose dehydrogenase like enzyme (PQQ GDH) was isolated from Sorangium cellulosum So ce56. The putative coding region was cloned, over expressed in E. coli and the resulting enzyme was characterized. The recombinant protein has a relative molecular mass of 63 kDa and shows 43% homology to PQQ GDH-B from Acinetobacter calcoaceticus. In the presence of PQQ and CaCl₂ the enzyme has dehydrogenase activity with the substrate glucose as well as with other mono- and disaccharides. The thermal stability and its pH activity profile mark the enzyme as a potential glucose biosensor enzyme. In order to decrease the activity on maltose, which is unwanted for a potential application in biosensors, the protein was rationally modified at three specified positions. The best variant showed a 59% reduction in activity on maltose compared to the wild type enzyme. The catalytic efficiency (k(cat)/K(M)) was reduced fivefold but the specific activity still amounted to 63% of the wild type activity.

The cooperativity effect in the reaction of soluble quinoprotein (PQQ‐containing) glucose dehydrogenase is not due to subunit interaction but to substrate‐assisted catalysis

“…sGDH has first been isolated and characterized from a strain of the bacterium Acinetobacter calcoaceticus . Later, similar enzymes have been found: glucose dehydrogenase from Sorangium cellulosum ; aldose dehydrogenase from Escherichia coli and from the thermophilic archaeon Pyrobaculum aerophilum ; l‐ sorbosone dehydrogenase from Ketogulonicigenium vulgare . Moreover, genes encoding homologous sequences have been detected in a large variety of prokaryotes .…”

“…Sorangium cellulosum [6]; aldose dehydrogenase from Escherichia coli [7] and from the thermophilic archaeon Pyrobaculum aerophilum [8]; L-sorbosone dehydrogenase from Ketogulonicigenium vulgare [9]. Moreover, genes encoding homologous sequences have been detected in a large variety of prokaryotes [10].…”

Recombinant expression of insoluble enzymes in Escherichia coli: a systematic review of experimental design and its manufacturing implications

“…The remaining 12% utilized K12 derivates. Commercially available K12 strains used include JM109 [ 25 , 51 , 55 ], DH5α [ 30 , 56 ], NovaBlue [ 57 ], XL1 Blue [ 58 ], M15 [ 31 , 59 , 60 ], and Top10 [ 61 , 62 ]; alternatively non-commercial K12 strains W3110 [ 63 ] and MG1655 [ 62 , 64 ] were used in a limited number of cases. K12 strains serve as useful tools when plasmid instability is encountered resulting in plasmid loss from the host [ 54 ]; this may explain its use in limited cases for expression.…”

“…High transformation efficiency [ 78 ] Fructose-1,6-bisphosphate aldolase DH5α (K12) Various Suppliers (Thermo Fisher Scientific, New England Biolabs, Gold Biotechnology Ltd.) Generally, a strain used for cloning and blue/white screening. (recA) mutation allows for better insert stability [ 30 ] Chitobiase M15 (K12) Qiagen Generally used in conjunction with plasmid (pQE) found within a expression kit from Qiagen [ 59 ] Cellobiose phosphorylase JM109 (K12) Promega Corporation Generally, a strain used for cloning and blue/white screening [ 55 ] Quinoprotein glucose dehydrogenase B NovaBlue (DE3) (K12) Merck KGaA Generally, a strain used for cloning and blue/white screening [ 57 ] Fuculose-1-phosphate aldolase XL-1 Blue (K12) Promega Corporation Generally, a strain used for cloning and blue/white screening [ 58 ] Trehalose transferase TOP10 (K12) Thermo Fisher Scientific Generally, a strain used for cloning and plasmid propagation [ 61 ] …”

“…We observed a twofold stronger preference for N-terminal allocation of the His-tag than for C-terminal allocation in the reviewed literature [ 58 , 104 , 114 – 116 ]. C-terminal fusions were found in a limited number of cases [ 35 , 40 , 42 , 51 , 57 , 117 – 119 ]. However, the discussion on the factors contributing to this predilection was limited; our analysis of the reviewed literature did not indicate a strong benefit received from either choice in terms of the physical condition of the final product.…”