Method for preparing L-menthol
A material and dissociation technology, applied in the field of preparation of L-menthol, can solve the problem of low enantioselectivity of L-menthol enzyme
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Embodiment 1
[0041] Feed-batch fermentation of Pichia pastoris
[0042] Vehicle: pGAP (Invitrogen)
[0043] Plasmid: Lip 1 (lipase from Candida rugosa)
[0044] Expression: Constitutive
[0045] fermentation
[0046] Feed-batch fermentations were carried out in complex media at 30° C. and pH 6 in a 42 liter bioreactor (Bioengineering). The medium contained 1% yeast extract, 2% peptone, 1% glycerol and 0.1M K phosphate buffer pH 6. The feed solution consisted of 20% glucose for the pure glucose feed and 20% glucose / 5% glycerol for the mixed feed. The bioreactor adopts OD 600 Inoculate 2-3 500 ml shaken overnight cultures in the above medium. The stirring speed was 400 rpm and the aeration rate was 15 liters (STP) / minute. During the fermentation, optical density at 600 nm, biomass wet matter (BWM), biomass dry matter (BDM) and lipolytic activity of the supernatant were determined. The first feed was made after 24 hours and thereafter 300-600 ml of feed solution was added every 12 hou...
Embodiment 2
[0061] Preliminary Experiment on the Extraction of Menthyl Benzoate
[0062] D,L-Menyl benzoate was hydrolyzed in sodium phosphate buffer (pH 7.2, 100 mM) containing gum arabic (0.2% (m / v)) as a solubilizer. Then, the reaction mixture needs to be extracted with a solvent suitable for gas chromatographic analysis.
[0063] To determine a suitable solvent, equimolar amounts of D,L-menthyl benzoate and D,L-menthol were dissolved in isooctane in order to obtain a basis for signal area ratio (menthyl benzoate / menthol) . The signal area ratio [menthyl benzoate / menthol] obtained by gas chromatography was 1.7.
[0064] The solvent was then removed with a rotary evaporator, the residue was dissolved in 10 ml of sodium phosphate buffer (pH 7.2, 100 mM), gum arabic (0.2% (m / v)) was added, the mixture was homogenized for 10 min, and in each case A 1 ml aliquot was added to the plastic reactor. After incubation for 1 hour at 40° C., the mixture was covered with a layer of different sol...
Embodiment 3
[0069] Hydrolysis of Menthyl D,L-benzoate Using Recombinant Lipase from Candida rugosa (rec.CRL)
[0070] In each case, the reactions were carried out in 1 ml of sodium phosphate buffer (pH 7.2, 100 mM) at 40° C. with 0.2% (m / v) gum arabic as solubilizer. In each case, the amount of enzyme used was 400 U of purified Candida rugosa lipase per 0.01 mmol D, L-menthyl benzoate. The enantiomeric excess of menthol was determined by gas chromatography, and the enantiomeric excess of menthyl benzoate was calculated based on the signal area.
[0071] table 3
[0072] Response time [h]
[0073] Table 3 shows that the recombinant lipase from Candida rugosa exhibits very high enantioselectivity (E > 100) for the hydrolysis of menthyl D,L-benzoate under the selected reaction conditions.
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