Synthesis Of L-carnitine And Its Acylate Catalyzed In Series By Two Enzymes In Ionic Liquid Medium

L-carnitine and acyl-L-carnitine are widely useful in pharmaceutical, cosmetic, and food industries at present. L-carnitine can be biosynthesized through the hydration of crotonobetaine under the action of carnitine dehydratase. However, the conversion is relatively low, and crotonobetaine can not be recoveried in the process of L-carnitine extraction and separation. Acetonitrile is by far the best reaction medium for the enzymatic synthesis of acyl-L-carnitine, but the product yield is low and the lipase activity loss is serious in acetonitrile. In adition, compared to the foreign countries, there are only a few varieties of acyl-L-carnitine in china. In order to solve these problems, the synthesis of L-carnitine and its acylate catalyzed in series by two enzymes in ionic liquid medium was studied in the paper.The methods for determination of carnitine dehydratase and acyl-L-carnitine were established. The activity of carnitine dehydratase can be defined as the total mmols of crotonobetaine consumed per hour, or that of L-carnitine produced per hour. The former could be determined by UV. But the value (x) must be modified through curve fit by the latter, which could be accurately determined by DTNB (y). The curve fit formula was y=7.286-5.45/[1+e(x-8.746)/ 2318]. The detection limit was 1.87 U. Acyl-L-carnitine could be measured by colorimetry method. Color reaction was performed in organic solution-salt solution biphasic system. The organic solution was 1, 2-dichloroethane-isoamyl alcohol mixture (V/V=96:4) added bromophenol blue (per 100 ml added 0.05 g). The salt solution was 55% K2HPO4 aqueous solution added Na2CO3 (per 100 ml added 14 g). There was a linear relationship (R2>0.99) between OD value and acyl-L-carnitine content which range was 20-130μmol in the biphasic system. To analyze the system of L-arnitine esterification, the recovery was 99.53-103.33%, the RSD was below 4.30%, and the detection limit was 1.85μmol. The method was not disturbed by L-carnitine.The conditions for the bioconversion of L-carnitine from crotonobetaine were optimized. The optimal reaction conditions were: 40°C of reaction temperature, pH7.0, 2.0% crotonobetaine, 2.0 g/100ml carnitine dehydratase (wet weight), 0.2% fumarate, 5-8 h of reaction time. The medal ion including Zn²⁺, K⁺, Cu²⁺, Li⁺, Ba²⁺ and Fe²⁺ could increase the activity of carnitine dehydratase, and all of these, Fe²⁺ was the most effective to improve the enzymatic activity. Mg²⁺ could decrease the enzymatic activity.The conditions of lipase-catalyzed acyl-L-carnitine synthesis in ionic liquid were optimized. The optimal reaction conditions were: for isovaleryl-L-carnitine, 0.4 mmol L-carnitine, 2.0 ml [Bmim]PF₆, 50 mg Novozyme 435 (10 000 PLU/g), 0.22 a_W, 200 mg molecular sieves, 4:1 molar ratio of fatty acid and L-carnitine, 60 oC, 150 r/min and 60 h; for octanoyl-L-carnitine and palmitoyl-L-carnitine, 0.4 mmol L-carnitine, 2.0 ml [Bmim]PF₆, 40 mg Novozyme 435, 0.22 aW, 250 mg molecular sieves, 5:1 molar ratio of fatty acid and L-carnitine, 60 oC (octanoyl-L-carnitine) and 65 oC (palmitoyl-L-carnitine), 200 r/min, 48 h. Their overall yields could reach 59.14%, 90.79% and 98.03%, respectively. The activation energy of isovaleryl-L-carnitine (163.08 KJ/mol) was higher than that of octanoyl-L-carnitine (130.74 KJ/mol) and palmitoyl-L-carnitine (127.16 KJ/mol). The higher yield and operational stability were obtained in [Bmim]PF₆ than in acetonitrile. The optimal conditions for acyl-L-carnitine synthesis by transesterification were: for isovaleryl-L-carnitine, 0.4 mmol L-carnitine, 2.0 ml [Bmim]PF₆, 40 mg Novozyme 435, 0.22 aW, 6:1 molar ratio of Allylisovalerate and L-carnitine, 60oC, 120 r/min and 48 h; for octanoyl-L-carnitine and palmitoyl-L-carnitine, 0.4 mmol L-carnitine, 2.0 ml [Bmim]PF₆, 30 mg Novozyme 435, 0.22 aW, 3:1 molar ratio of fatty acid vinyl ester and L-carnitine, 60 oC, 150 r/min, 32 h. Their overall yields could reach 73.61%, 96.76% and 98.82%, respectively.The route"synthesis of L-carnitine and its acylate catalyzed in series by two enzymes"was established. Using [Bmim]PF₆ ionic liquid as the reaction medium, L-carnitine was biosynthesized through the hydration of crotonobetaine under the action of carnitine dehydratase, which was transformed into palmitoyl-L-carnitine by lipase. Palmitoyl-L-carnitine was extracted with 2-methyl-2-butano. With the removal of L-carnitine, the biotransformation of crotonobetaine into L-carnitine was continued in the reversible hydration reaction. The molar yield of total L-carnitine could attain 87.2% after the five times intermittent extraction. Vacuum-rotary evaporator was a suitable reactor to realize the strategy. Through the route, the conversion was improved, the key technology of L-carnitine separation and extraction was solved, and acyl-L-carnitine was synthesized in the meantime.The key technology of acyl-L-carnitine separation and extraction was studied. Acetone could precipitate the three acyl-L-carnitine from [Bmim]PF₆ ionic liquid, with 6-7:1 volume ritio of acetone and [Bmim]PF₆, and their precipitation rates could reach 90%. The centrifugation after refrigeration could lead to the complete precipitation and decrease the time of precipitation. Water could extract isovaleryl-L-carnitine and octanoyl-L-carnitine from [Bmim]PF₆. Their partition ratio between water and the ionic liquid were 1.35 and 1.16, respectively. Palmitoyl-L-carnitine could be extracted from the ionic liquid by 2-Methyl-2-butano, and partition ratio between 2-Methyl-2-butano and the ionic liquid was 5.31. The extraction time was 15-30 min. The decrease of temperature was favourable to the extraction. Emulsification easily occurred during the extraction, which could be eradicated by centrifugation at 1000 r/min for 10 min.