The Study On Properties And Regulation Of Key Enzyme In Synthesis For Gentamicin

Gentamicin was produced by Micromonospora strain, but its yield was low sinceit was discovered. A new method with low cost and high efficiency was invented toproduce gentamicin in our laboratory, and it has been awarded a national patent. Theaddition of several amino acids could shorten fermentation time from120~140hoursto70~80hours, and the producing rate of gentamicin increased by80%~90%. But themechanism of amino acids remains unknown. So the effect of amino acids on themembrane and key enzymes in the glycolytic pathway was explored in this paper.The New Method was established to determine the activity of hexokinase (HK)by Reversed-Phase High Performance Liquid Chromatography (RP-HPLC). Thismethod was reported firstly in this paper. The HK activity was determined bymeasuring the amount of ADP which was one of products from a reaction thathexokinase catalyzed glucose to produce glucose-6-phosphate. The concentration ofADP in the range of0.01～0.40mmol/L has a good linear relationship with its peakareas, and the correlation coefficient was0.999. When the signal-to-noise ratio ofHPLC was3, the minimum detectable concentration of ADP was0.7μg/mL. Theoptimal catalytic conditions for HK were confirmed: the reaction temperature was30℃, the reaction pH was7.0and the reaction time was30minutes. Comparing withenzyme-coupled assay, the new method was accurate and had low cost relativelywhich could determine the activity of HK accurately.The effect of amino acids on HK activity was studied. The crude enzyme of HKwas extracted from Micromonospora purpurea which was cultured to logarithmicphase and prepared into crude enzyme solution. The best conditions for cell brokenwere ultrasonic power350W and ultrasonic time35min (ultrasonic treatment for2swith interval of3s). Activity of hexokinase was measured by RP-HPLC, the optimalreaction temperature was30℃, and the optimal reaction pH was7.0. The recovery ofHK was between95.3%and97.6%. The impact of amino acids on HK activity wasexplored in the study. The results showed that Lys, Arg and Met made activity of HKincrease by13.1%,6.8%and9.6%respectively. The result of orthogonal experimentshown that the impact of three amino acids on HK activity was Lys>Met>Arg. The concentration of amino acids that made HK activity improved sharpest was Lys0.02%, Arg0.0001%and Met0.03%, which made HK activity increase by17.1%comparing with the control.The effect of amino acids on phosphofructokinase (PFK) activity was studied.The crude enzyme of PFK was extracted from Micromonospora purpurea which wascultured to logarithmic phase, and prepared into crude enzyme solution. The bestconditions for cell broken were same as HK’s. The optimal reaction temperature ofPFK was30℃, and the optimal reaction pH was7.5. The recovery of PFK wasbetween89.5%and93.1%. The impact of amino acids on PFK activity was exploredin the study. The results shown that Lys, Arg and Met made activity of PFK increaseby10.4%、6.5%、9.38%respectively. The result of orthogonal experiment shown thatthe impact of three amino acids on PFK activity was Lys>Met>Arg. Theconcentration of amino acids that made PFK activity improved sharpest was Lys0.02%，Arg0.001%and Met0.03%, which made HK activity increase by14.4%comparing with the control.The effects of amino acids on cell membrane of Micromonospora purpurea werestudied. The results show that: gentamicin secretion rate of new method was higherthan that of original technology, the effect was best at84h and the former than thelatter increased by40.3%. After adding penicillin, gentamicin secretion rate of newmethod was higher than that of the original technology too, the effect was best at84h,and the former than the latter increased by33.7%.To some extent, the mechanism that amino acids could increase the producingrate of gentamicin was explained in the new method.