Study On The Formation And Catalytic Conditions Of L-carnitine Dehydrase In Escherichia Coli

L-carnitine（β-hydroxy-γ-trimethylaminobutyric acid）,also known as L-carnitine,botulinum toxin,vitamin BT,clinical also known as left carnitine,is a kind of nutritional safety,medical fine chemicals.In recent years,it has been found that L-carnitine and its derivatives have strong physiological functions,making L-carnitine widely used in food,medicine,feed,health care products and other fields.Some scholars even put forward that L-carnitine can be as important as vitamin and included in daily nutritious diet.It can be seen that L-carnitine has a bright prospect and inestimable market value.L-carnitine can be prepared by direct extraction,chemical synthesis,enzyme transformation,gene recombination and so on.L-carnitine,which relies on enzymes contained in a variety of microorganisms in nature to transform corresponding substrates to prepare L-carnitine,is currently recognized at home and abroad as an efficient and environmentally friendly preparation method with low cost.The research on this in China is not in-depth enough,and the main source is still imported from abroad.Based on the above background,the fermentation conditions and catalytic reaction conditions of L-carnitine dehydrase produced by laboratory-preserved E.coli strain E.coli.F171622 were optimized and explored in this paper,which provided a theoretical basis for later amplification production.HPLC method for the determination of substrate croton betaine and DTNB method for the determination of L-carnitine dehydrase were established.The enzyme activity was determined by the biomass and the yield of L-carnitine.These methods were simple,rapid,reproducible and specific.Through the single factor investigation of carbon source,nitrogen source,initial PH value,oxygen,temperature and culture time for the growth and fermentation of the strain,the culture medium components（mass fraction,%）were determined as peptone1.0,yeast extract 0.5,fumaric acid 2.0,KH₂PO₄ 0.2,（NH₄）₂SO₄ 0.5.Mg SO₄·7H₂O0.12,Mn SO₄·4H₂O 0.001,Fe SO₄·7H₂O 0.001,Na₂HPO₄ 0.28,PH7.5±0.2（25℃）,60 m L liquid,120 rpm,35℃for 24 hours of shaking culture,the best bacterial biomass and enzyme production activity were 2.62 g/L and 4.6 U,respectively.Through the single factor investigation of temperature,PH value,reaction time and resting cell concentration,the optimal catalytic conditions were determined as resting cell concentration of 50-60 g/L,PH value of phosphate 8.0,40℃reaction for10 hours,the highest yield of L-carnitine could reach 13.6 g/L.