Molecular cloning, expression and characterisation of glucokinase gene from the mixotrophic green alga Chlorella kessleri

摘要

Chlorella kessleri is a green alga that grows photoautotrophically, heterotrophically and mixotrophically. To explore the mechanism of glucose metabolism at the molecular level, cDNA (GenBank ID KF011248) encoding glucokinase (named CkeGK) from C. kessleri was cloned and sequenced. The Ckegk cDNA was 1167 base pairs (bp) long, with an open reading frame of 1089 bp encoding 362 amino acids with a calculated molecular mass of 38.98 kDa and an estimated isoelectric point of 4.78. Multiple alignment analysis revealed that the deduced amino acid sequence of Ckegk shared a high identity of 65%-78% with corresponding glucokinase from other eukaryotes. The catalytic motifs of glucokinase were detected in the amino acid sequence of Ckegk. Under different trophic modes, the transcriptional profiles of Ckegk showed that it could be up-regulated under a heterotrophic culture condition but not under a mixotrophic culture condition. Furthermore, the gene was differentially expressed in Escherichia coli, and biochemical characterisation was determined. According to SDS-PAGE, the purified recombinant CkeGK showed a single band about 43.0 kDa, which correlated with the theoretical molecular weight predictions. Biochemical analysis showed that CkeGK was stable at 40 degrees C and 50 degrees C, but it lost 60% of its activity after incubation for 40 min at 60 degrees C. The optimal pH for the activity of CkeGK was 9.0, and the K-m and V-max for ATP were 0.1168 mM and 5.346 nmol ml(-1) min(-1) and for glucose were 0.1465 mM and 6.022 nmol ml(-1) min(-1).