The Mechanism Of Mn²⁺ Concentration On Different Configurations Of ?-polyglutamic Acid Produced By Bacillus Licheniformis

Bacillus licheniformis is a productive bacterium to efficiently synthesize poly-?-glutamic acid by fermentation.The concentrations of Mn²⁺ can affect the configuration of poly-?-glutamic acid produced by Bacillus licheniformis.Due to there is no distinction of configuration existing in ?-PGA synthetase,therefore,the ratio of ?-L-PGA and ?-D-PGA in ?-PGA is dependent on the ratio of L-glutamic acid and D-glutamic acid in the substrate.Three key enzymes from L-and D-glutamic metabolism,i.e.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 in this study.Also,B.subtilis KH-2 served as the control strain.The synthesis mechanism of ?-D-PGA produced by various concentrations of Mn²⁺ was investigated in this work by the means of functional genomics analysis,including enzymatic properties in vitro and in vitro and gene transcription.The configuration transformation of ?-PGA produced from B.licheniformis and S.subtilis within various Mn²⁺ concentrations was explored firstly.The proportion of ?-D-PGA was stable?84%-86%?in the fermentation broth produced from B.subtilis KH-2 within different concentrations of Mn²⁺?0,0.6,1.2,1.8 and 2.4 mmol/L?.However,the ratio of ?-L-PGA and ?-D-PGA produced from B.licheniformis WBL-3 was significantly different within the same range of Mn²⁺ concentration.The proportion of ?-D-PGA was only 22% without the existence of Mn²⁺.With the increase of Mn²⁺ concentration,the proportion of ?-D-PGA configuration increased,reaching to its highest level at 67% when the concentration of Mn²⁺ was 0.6 mmol/L.Afterwards,with the increase of Mn²⁺ concentration,the proportion of ?-D-PGA configuration decreased.The in vitro enzyme activities of key metabolic enzymes in both L-glutamic acid and D-glutamic acid from two strains were compared in two different Mn²⁺ concentrations at 0 and 0.6 mmol/L respectively.It can be seen that Mn²⁺ had no activated effect on the three key metabolic enzymes in B.subtilis KH-2.However,the catalytic activity?Kcat/Km?of GR in Bacillus licheniformis WBL-3 with 0.6 mmol/L Mn²⁺ was higher than that without Mn²⁺.Also,the GS activity was detected only within the presence of Mn²⁺.L-glutamine was produced from L-glutamic acid through the catalysis of GS,while D-glutamic acid was produced from L-glutamic through the catalysis of GR,therefore,GR catalyzes the conversion of to D-glutamic acid.Therefore,the decrease of L-glutamic acid and the accumulation of D-glutamic acid within the presence of Mn²⁺ in the system led to the proportion increase of ?-D-PGA in the fermentation broth.The in vivo enzyme activity of glutamine synthetase was investigated by HPLC,which detected GS in enzyme solution from bacterium cell disruption in B.licheniformis WBL-3.Glutamine was not detected without addition of Mn²⁺ in the reaction solution.However,0.71 mmol/L of glutamine was detected within the presence of Mn²⁺.The results above were in an agreement with the results of enzymatic activity in vivo,which further demonstrated that the reduce of the L-glutamic acid concentration in B.licheniformis WBL-3 resulted in an increase of the proportion of ?-D-PGA.Real-time quantitative PCR was used to measure the transcriptional levels in vivo of related genes synthesizing ?-D-PGA in B.licheniformis,including racE,dat,and gltA.The transcription levels of racE,dat and gltA with 0.6 mmol/L Mn²⁺ were 2.16,4.44 and 1.84 times respectively the levels of those without Mn²⁺.In the light of above,the transcriptional levels of metabolizing gene of L-glutamic acid and synthesis gene of D-glutamic acid in B.licheniformis WBL-3 were higher with the existence of Mn²⁺ than those without Mn²⁺.This further indicated that Mn²⁺ can facilitate the accumulation of D-glutamic acid,leading to the increase of ?-D-PGA proportion in the fermentation broth.