Expression, characterization and thermostability improvement of low-specificity L-threonine aldolase from Pseudomonas putida KT2440

Abstract:

[Objective] To construct a recombinant Escherichia coli BL21 (DE3)/pET-KT2440, a low-specificity L-threonine aldolase gene (ltaE) was cloned from the genome of Pseudomonas putida KT2440. The enzyme characterization and the effects of key amino acid mutations on the enzyme activity and thermo-stability were investigated.[Methods] The ltaE gene was amplified by PCR with the genome of P. putida KT2440 as template. The recombinant plasmid pET28a-KT2440 was constructed and transformed into E. coli BL21 (DE3). The low-specificity L-threonine aldolase (LTA) was purified by Ni²⁺ affinity chromatography and then was characterized. The key amino acids Thr206 and lys207 were mutated by site-directed mutagenesis. [Results] SDS-PAGE analysis showed that LTA were highly expressed in E. coli BL21 (DE3) with a molecular weight of about 40 kDa, consistent with the theoretical value. A single band was observed through Ni²⁺ affinity chromatography. LTA had a specific activity of 5577 U/mg by two coupled-enzyme assay method. The optimal temperature and pH were 50℃ and 8.0, respectively, and was stable below 40℃ and pH between 5.0 and 9.0. LTA exhibited K_m and k_cat values of 23.95 mmol/L and 19216.6 s^-1 under the optimal conditions. Mg²⁺ and Ca²⁺ obviously stimulated enzyme activity, whereas Ni²⁺, Cu²⁺, Zn²⁺ and Fe²⁺ obviously inhibited it. LTA enzyme presented good resistance in tert-butyl methyl ether. The residual activity was retained over 90% after pre-incubation of the enzyme in TBME for 1 h. Site-directed mutation indicated that Thr206Ser significantly increased the thermal stability. Lys207 is essential for enzymatic function. Any mutation of K207 was lethal for enzyme activity. [Conclusion] The thermostability was improved by site-directed mutation. The work provides a solid foundation for the efficient and stable biosynthesis of β-hydroxy-α-amino acids by LTA.