Mn2+浓度对地衣芽孢杆菌产不同构型聚-γ-谷氨酸机理分析

Regulation of stereochemical composition of poly-γ-glutamic acids produced by Bacillus licheniformis within various Mn2+ concentration

摘要:

【目的】分析Mn2+对地衣芽孢杆菌(Bacillus licheniformis) WBL-3产不同比例聚-γ-谷氨酸(γ-PGA)的机理。【方法】以枯草芽孢杆菌(B. subtilis) KH-2为对照菌株，克隆表达L-谷氨酸和D-谷氨酸代谢关键酶，分别为谷氨酸消旋酶(GR)、D-丙氨酸转氨酶(D-DDT)和谷氨酰胺合成酶(GS)。通过体外酶活实验与体内转录水平分析，初步探讨Mn2+浓度对不同比例γ-PGA的生成机理。【结果】当Mn2+浓度为0与0.6 mmol/L时，γ-D-PGA所占的比例分别为22%和67%。在0.6 mmol/L Mn2+浓度下，B. licheniformis WBL-3中GR的催化活性(kcat/Km值)比未添加Mn2+时高，GS只有在Mn2+存在下才具有催化活性。实时荧光定量PCR结果表明，Mn2+提高了GR、D-DDT和GS的转录水平，提高倍数分别为2.16、4.44和1.84倍。【结论】Mn2+激活了GR、D-DDT与GS的表达，促进L-谷氨酸的代谢，使得菌体内D-谷氨酸比例升高，从而提高了γ-D-PGA的比例。

Abstract:

[Objective] The mechanism of the generation of various kinds of poly-γ-glutamic acids (γ-PGA) from Bacillus licheniformis WBL-3 affected by Mn2+ was investigated in this study. [Methods] Three L- and D-glutamic metabolism related enzymes, glutamate racemase (GR; encoded by racE), D-alanine aminotransferase (D-DDT; encoded by dat) and glutamine synthetase (GS; encoded by gltA), were cloned and expressed meanwhile B. subtilis KH-2 was used as the control strain. The mechanism synthesis of γ-PGA by different concentration of Mn2+ was studied using the methods of enzyme activity, combined with transcriptional analysis. [Results] When the concentration of Mn2+ varied from 0 to 0.6 mmol/L, the proportion of D- isomer increased from 22% to 67%. The catalytic activity (kcat/Km) of GR in Bacillus licheniformis WBL-3 with 0.6 mmol/L Mn2+ is higher than that without Mn2+. The GS activity was measured only in the presence of Mn2+. RT-PCR study showed that the transcription ratio of racE, dat and gltA with 0.6 mmol/L Mn2+ to those without Mn2+ was 2.16, 4.44 and 1.84-fold, respectively. [Conclusion] Mn2+ activated the expression of GR, D-DDT and GS, promoted the metabolism of L-glutamic acid, increased the proportion of D-glutamic acid, and increased the proportion of γ-D-PGA.