| Enzyme catalysis applied to preparation of natural flavor and chiral compounds has become the hot spot nowadays. Compared to chemical method, enzyme catalysis has the characteristics of little pollution and low energy consumption, and its products are green and natural. Enzyme catalysis also has the advantages of high stereoselectivity and good specificity when it was applied to the chiral resolution of racemic compound. Enzyme catalysis has wide application prospect. Based on microbial transformation of cinnamic acid with Mucor sp. JX23 and chiral resolution of 1-benzene ethanol with JX13, cinnamic acid degradation enzyme of Mucor sp. JX23 and (S)-1-benzene ethanol oxidase of JX13 were investigated. The high sensitive, stable and precise activity determination methods for the two enzymes were established which can lay the foundation of methodology for the further research of enzymatic preparation of natural spices acetophenone and single optical activity 1-benzene ethanol. The research contents and results were as follows:1. The methods for the activity determination of cinnamic acid degradation enzyme and (S)-1-benzene ethanol oxidase were set up by HPLC respectively. The optimal chromatographic condition for cinnamic acid degradation enzyme were the chromatographic column was Hypersil ODS2 at 30℃, acetonitrile-water-glacial acetic acid (50:50:0.5) as mobile phase at 1.0 mL·min-1, the detection wavelength was 247 nm. The optimal chromatographic condition for (S)-1-benzene ethanol oxidase were the analytical column was chiralcel OJ-RH at 30℃,30:70 acetonitrile and aqueous solution as mobile phase at 0.7 mL·min-1, the detection wavelength was 205 nm. HPLC used for the determination of enzyme was proved to be reliable, high sensitive and anti-interference, which provide a reliable method for enzyme catalysis system analysis.2. Fermentation conditions were optimized for cinnamic acid degradation enzyme produced by Mucor sp. JX23 using one-single factor, including carbon source, nitrogen source, medium initial pH, fermentation time and volume of liquid. The results showed the optimal medium:yeast extract and mannitol were used as nitrogen source and carbon source, the volume of liquid was 200 mL in the 500 mL conical flask, the initial pH was 5.0 and fermentation time was 96 h. After optimization, the enzyme activity was increased from 0.0036 to 0.0092 U·mL-1.3. The enzyme properties of cinnamic acid degradation enzyme were studied. The results showed that the optimal temperature of cinnamic acid degradation enzyme was 30℃, the optimal pH was 7.0, the enzyme had high temperature stability between 30℃ and 50℃, the cinnamic acid degradation enzyme had high pH stability when the pH was between 6.0 and 7.5. The catalytic kinetics properties of cinnamic acid degradation enzyme were researched, and the results showed the catalytic kinetic parameters of cinnamic acid degradation enzyme Km=0.07 g-L-1, Vmax=1.62 μmol-L-1-min-1。4. Fermentation conditions were optimized for (S)-1-benzene ethanol oxidase produced by JX13 using one-single factor, including carbon source and nitrogen source, medium initial pH, fermentation time and volume of liquid. The results showed the optimal medium:starch and yeast extract were used as carbon source and nitrogen source, the volume of liquid was 150 mL in the 500 mL conical flask, the initial pH was 6.0, fermentation time was 72 h. After optimization, the enzyme activity was increased from 1.32×10-6 to 4.14×10-6 U·mL-1.5. The enzyme properties of (S)-1-benzene ethanol oxidase were researched. The results showed that the optimal temperature of (S)-1-benzene ethanol oxidase was 35℃, the optimal pH was 5.5, the enzyme had high temperature stability between 30 and 40℃, the (S)-1-benzene ethanol oxidase had high pH stability when the pH was between 5.0 and 6.5. The catalytic kinetics properties of (S)-1-benzene ethanol oxidase were researched, the results showed the catalytic kinetic parameters of (S)-1-benzene ethanol oxidase Km=2.9 g·L-1, Vmax=0.0039 μmol·L-1·min-1. |
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