Immobilization Of Lipase And Its Application On The Resolution Of Dl-menthol

Menthol is one of the three major flavor chemicals in the world.It exists in eight stereoisomeric forms but only L-menthol has great commercial application value,which is widely used in production of food,medicine,cosmetics,tea,cigar and so on.In this paper,the lipase from Thermomyces lanuginosus?TLL?is immobilized to improve the activity and stability of TLL for chiral resolution of DL-menthol in non aqueous.The commercial lipase Lipozyme TL100L from Thermomyces lanuginosus was immobilized on the styrene,non-polar macroporous resin?D101?and its best stability and activity of the immobilized enzyme was optimized.The optimum conditions were:immobilization temperature 30?,resin and lipase by 1 g:2.5 mL,adding 2%trehalose,oscillation speed 150 rpm,8 h.In this study,an efficient resolution of DL-menthol in non-aqueous organic solvent was described.The optimum reaction conditions were found to be transesterification with vinyl acetate at 40?in methyl tert-butyl ether?MTBE?with a mole ratio?vinyl acetate:DL-menthol?of3.8:1,molecular sieve of 0.12 g,oscillation speed 150 rpm and an enzyme concentration of 20 g/L.At optimum reaction conditions,L-menthyl acetate with 99%enantiomeric excess?e.e._p?was obtained when the conversion was 41.23%with the reaction time of 24 h at the substrate concentration of 65 mM.Finally,for large-scale production in industry,a packed-bed reactor for the continuous enzymatic esterification was adopted.The influence of the substrate concentration,the superficial residence time and flow rate of the substrate solution on continuous production were studied.The optimum conversion of the reactor was 43.46%under the condition of 5%substrate concentration and 0.01 mL/min flow rate.More than 89.5%of the initial activity could be recovered with e.e._p 99.9%after the immobilized lipase had been continuously used for at least 22 cycles.Through quick screening and GC detection rescreening and Aspergillus oryzae WZ007 was screened from soil and laboratory strains.Then,by doing single factor experiment,the fermentation and enzyme producing conditions for Aspergillus oryzae WZ007 were investigated and the optimal formula of the fermentation medium obtained is as follows:lactose 1.6%,yeast extract 1.2%,yeast extract 0.1%,Fe³⁺2mmol/L;The optimal conditions for its incubation is at pH 7.0,30oC for24 h.After fermentation,high activity strains?65 U/g?was obtained.The strain keep a good operational stability in MTBE,indicating that it was a promising biocatalyst in the resolution of DL-menthol in organic solvent.