Pseudomonas putida KT2440低特异性L-苏氨酸醛缩酶的表达、酶学性质及温度稳定性提高

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

摘要:

[目的]从Pseudomonas putida KT2440基因组中，钓取低特异性L-苏氨酸醛缩酶基因（ltaE），构建重组大肠杆菌。研究目标酶的酶学性质，和关键氨基酸位点突变对酶活和温度稳定性的影响。[方法]以P.putida KT2440基因组DNA为模板，PCR扩增出ltaE基因，构建重组表达质粒pET28a-KT2440并转化Escherichia coli BL21（DE3），获得重组菌E.coli BL21（DE3）/pET-KT2440，利用Ni²⁺柱亲和层析纯化低特异性L-苏氨酸醛缩酶（LTA），对关键氨基酸位点Thr206和Lys207实施定点突变。[结果]SDS-PAGE结果表明LTA在大肠杆菌中获得高效表达，分子量为40 kDa左右，与理论值大小相符。Ni²⁺柱亲和层析纯化LTA，获得单一条带。利用双酶耦联法测得LTA酶活为5577.3 U/mg，最适反应温度为50℃，最适pH为8.0。在温度低于45℃，pH 5.0-9.0时，重组酶较稳定。LTA酶的K_m和k_cat值为23.95 mmol/L和19216.6 s^-1。Mg²⁺、Ca²⁺金属离子对LTA有明显的促进作用，而Ni²⁺、Cu²⁺、Zn²⁺、Fe²⁺等对酶有明显的抑制作用。该酶在叔丁基甲基醚溶剂中具有良好的耐受性，在叔丁基甲基醚中保存1 h后仍保留90%以上的酶活。Thr206Ser突变明显提高了酶对温度的稳定性。Lys207对酶催化功能是必需的，该位点突变对酶活都是致死的。[结论]克隆并表达P.putida KT2440的LTA酶，研究了酶学性质，通过定点改造提高了酶的温度稳定性，筛选获得一种酶耐受性好的有机溶剂，为LTA酶在有机溶剂中高效稳定催化β-羟基-α-氨基酸奠定了较坚实的研究基础。

关键词:恶臭假单胞菌;低特异性L-苏氨酸醛缩酶;酶学性质;致死突变;温度稳定性

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.

Key words:Pseudomonas putida;low-specificity L-threonine aldolase;characterization;lethal mutation;thermal stability