大肠杆菌转酮醇酶分子改造及催化酒石酸半醛合成

Molecular engineering of transketolase from Escherichia coli and tartaric semialdehyde biosynthesis

摘要:

转酮醇酶(transketolase,TK,EC. 2.2.1.1)是一种焦磷酸硫胺素和二价阳离子依赖性酶，可催化二碳单位的转移，可逆形成C–C键，在多酶催化生产化学品、药物前体和不对称合成方面有广泛应用。文中以大肠杆菌(Escherichia coli)K12转酮醇酶TKTA为研究对象，通过定点饱和突变和组合突变提升对非磷酸化底物的反应活性，并探索突变酶TKTA_M催化合成酒石酸半醛。结果表明：突变酶TKTA_M(R358I/H461S/R520Q)最适反应温度为32℃，最适反应pH为7.0，以d-甘油醛为受体底物的比酶活为(6.57±0.14)U/mg，是野生型比酶活((0.71±0.02)U/mg)的9.25倍。在酶学性质研究的基础上，设计20mL的反应体系，以50mmol/L5-酮基-d-葡萄糖酸和50mmol/L非磷酸化乙醇醛为底物，TKTA_M催化合成酒石酸半醛，最终酒石酸半醛的产量为3.71g，摩尔转化率为55.34%。研究结果为生物质制备l-(+)-酒石酸提供数据支撑，同时为转酮醇酶催化非磷酸化底物提供了借鉴。

关键词:转酮醇酶;定点突变;酶学性质;生物催化

Abstract:

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.

Key words:transketolase;site-directed mutation;enzyme characteristics;biocatalysis