Recombinant Expression, Purification and Characterization of Pyridoxal 5ʹ-phosphate Synthase from Geobacillus sp. H6a, Thermophilic Bacterium Producing Extracellular Vitamin B6

Document Type : Research Paper

Authors

1 Department of Biochemistry, Faculty of Science, Khon Kaen University, Khon Kaen, 40002, Thailand

2 Department of Biochemistry, Faculty of Science, Khon Kaen University, Thailand, 40002, Thailand.

Abstract

Background: Pyridoxal 5ʹ -phosphate synthase (PLPS) is present in deoxyxylose 5ʹ-phosphate-independent of the de novo vitamin B6 biosynthesis pathway. This enzyme complex consists of PdxS and PdxT, which function as synthase and glutamine amidotranferase respectively to produce PLP.
Objectives: This study aimed to clone, express, and purify PLPS of Geobacillus sp. H6a, followed by its characterization.
Material and Methods: The PdxS and PdxT genes were amplified from Geobacillus (Gh) sp. H6a. Recombinant vectors pET28a-GhpdxS and pET28a-GhpdxT were constructed and the resulting His-tagged proteins were expressed in E. coli BL21(DE3). The soluble rGhpdxS and rGhpdxT were purified via nickel-affinity chromatography and cation-exchange chromatography. The mixture of rGhpdxS and rGhpdxT was further characterized.
Results: The molecular weights of rGhpdxS and rGhpdxT were estimated to be 35 and 23 kDa by SDS-PAGE, respectively. The native form of rGhpdxS showed hexamer and dodecamer, whereas those of rGhpdxT were a monomer upon detection with non-denaturing gel electrophoresis and gel filtration. A molar ratio of 1:1 of rGhpdxS:rGhpdxT showed the highest PLP synthesis activity (4.16 U.mg-1) and was used for analyzing the biochemical properties. The kinetic values were obtained by using glyceraldehyde 3-phosphate, ribose 5-phosphate, and glutamine as the substrates. The rGhPLPS showed pentose phosphate isomerization without triose phosphate isomerase activity. The metal ions affected PLP synthesis activity. The optimum pH and optimum temperature of rGhPLPS were 9 and 70 °C, respectively. The rGhPLPS was active over a broad range of temperatures and pH values.
Conclusions: These results support the potential of rGhPLPS as a candidate for industrial application.

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