Molecular engineering of transketolase from Escherichia coli and tartaric semialdehyde biosynthesis / 生物工程学报

Transketolase (EC 2.2.1.1, TK) is a thiamine diphosphate-dependent enzyme that catalyzes the transfer of a two-carbon hydroxyacetyl unit with reversible C-C bond cleavage and formation. It is widely used in the production of chemicals, drug precursors, and asymmetric synthesis by cascade enzyme catalysis. In this paper, the activity of transketolase TKTA from Escherichia coli K12 on non-phosphorylated substrates was enhanced through site-directed saturation mutation and combined mutation. On this basis, the synthesis of tartaric semialdehyde was explored. The results showed that the optimal reaction temperature and pH of TKTA_M (R358I/H461S/R520Q) were 32 ℃ and 7.0, respectively. The specific activity on d-glyceraldehyde was (6.57±0.14) U/mg, which was 9.25 times higher than that of the wild type ((0.71±0.02) U/mg). Based on the characterization of TKTA_M, tartaric acid semialdehyde was synthesized with 50 mmol/L 5-keto-d-gluconate and 50 mmol/L non-phosphorylated ethanolaldehyde. The final yield of tartaric acid semialdehyde was 3.71 g with a molar conversion rate of 55.34%. Hence, the results may facilitate the preparation of l-(+)-tartaric acid from biomass, and provide an example for transketolase-catalyzed non-phosphorylated substrates.

isolation of enzyme transketolase from human erythrocytes: the characterization of its quaternary structure

Human erythrocyte transketolase [sedoheptulose-7-phosphate: D-glyceraldehyde-3-phosphate, glycolaldehyde transferase, E. C. 2.2.1.1.] has been isolated from erythrocytes with a specific activity of 59.84 U/mg. SDS-PAGE and SE-.HPLC were used both as a measure of purity and as a preparative mean to obtain a higher degree of purity. Four protein bands corresponding to molecular weights of 32,000, 39,000, 43,000 and 60,000 were obtained in electrophoresis and SE-HPLC preparations. Activity measurements on the two fractions obtained from SE-HPLC that contained a monomer with the molecular weight of 32,000 and a dimeric fraction with the molecular weight of 60,000 showed that the monomeric form of the enzyme displays activity in the presence and absence of the TPP and Mg[II]. This activity was measured to be 14.76 U/mg in the absence of TPP and Mg[II], and 40.24 U/mg in the presence of the cofactors. The dimeric form showed an activity of 58.84 U/mg in the presence of the cofactors