Study On Fermentation And Conversion Conditions Of L-carnitine Dehydratase

L-Carnitine is an essential cofactor in the transport of long-chain fatty acids across the inner mitochondrial membrane for subsequent fat degradation and energy production.It is an effective method to obtain L-carnitine by enzymatic biotransformation from substrate at present, however,the relative research in China was later and less.The work of my study was to optimize the process for L-carnitine dehydratase production by microbiology,analyse the fermentation dynamic models and conditions for the bioconversion,in order to develop an efficient biotransformation technology for L- carnitine production.Initially,a new method for quantitative determination of L-carnitine dehydratase activity (LCD) was proposed.The enzymatic activity was determined either as the amount of substrate crotonobetaine consumed by UV spectrophotometry,or as the amount of L-carnitine formed by enzymatic DTNB method. One linear and two non-linear curve fits were employed to the data on quantization of LCD analyzed by UV spectrophotometer and DTNB method . The formula where x was the LCD analyzed by UV spectrophotometer and y was the LCD analyzed by DTNB method was the most appreciated.Result showed that UV spectrophotometer combined with regression formula was a quick,accurate and low cost method to analyzed LCD.In order to find out the crucial factors influencing the microbial information of L-carnitine dehydratese,firstly,the culture medium has been tested,in which the influences of carbon source, nitrogen source,and enzyme induction were carried out by using single factor design.Then the experiment for the optimization of fermentation medium was evaluated through the rotation-regression-orthogonal combination design and the model equation is Y=5.222+0.197X₁-0.068X₂+0.022X₃+0.016X₄-0.176X₁²-0.111X₂²-0.086X₃²-0.127X₄²+0.090X₁X₂+0.048X₁X₃+0.040X₁X₄The optimized concentrations of culture medium (g/100 ml) were determined by multinomial regression techniques and response surface analysis as follows:0.678 peptone,0.961 yeast extract,0.335 crotonbetaine and 2.093 fumaric acid salt.The L-carnitine dehydratase activity increased from 1.90U to 5.32 U after the optimization of the fermentation medium,the experimental data had validated the theoretical values.Then,single factor design and orthogonal design were adopted to optimize the fermentation condition.A satisfactory activity of L-carnitine dehydratase was obtained under the condition: initial pH7.0,1ml/100ml (quantity of inoculation), 30℃,120r/min stirring,100 ml/flask (quantity of culture medium).The sequences of primary and secondary influence factor were stirring speed,quantity of inoculation,quantity of culture medium,culture temperature and initial pH.When the optimal culture was used,the activity of L-carnitine dehydratase was achieved 5.78±0.12 U.The batch cultivation conditions and dynamic models of Proteus mirabilis in 5-L bioreactor were studied.The cell growth and the enzyme production reached a maximum at pH 7.5, compared to the natural fermentation,the biomass,enzyme productivity and enzyme activity increased significantly.Increasing the stirring speed at some extent can improve cell biomass, and the enzymatic activity with 400r/min stirring was maximum.Then,the studies discussed the fermentation kinetics of Proteus mirabilis using Logistic equation for growth,the Luedeking-Piret equation for L-carnitine dehydratase production and Luedeking-Piret-Like equation for crotonobetaine consumption. The models were as follows: the kinetic equation of biomass growth was ;the kinetic equation of L-carnitine dehydratase production was p = 1.4697 A( t ) + 0.001506 B ( t), where the kinetic equation of crotonobetaine consumption was ,in which .The conditions for the bioconversion of L-carnitine from crotonobetaine using L-carnitine dehydratase were optimized.The optimal reaction conditions were: 40℃of reaction temperature, pH7.0,2% crotonobetaine,2.0 g/100ml L-carnitine dehydratase (wet weight),fumarate as electron acceptor,5-8 h of reaction time.The medal ion including Zn²⁺,K⁺,Cu²⁺,Li⁺,Ba²⁺ and Fe²⁺ could increase the activity of L-carnitine dehydratase,and all of these,Fe²⁺ was the most effective to improve the enzymatic activity,Mg²⁺ could decrease the enzymatic activity.