| The project is about the bioconversion in hydrophilic organic solvent/salt/water two-phase system and hydrophobic organic solvent single-phase system.Hydrophilic organic solvent (such as methanol, ethanol, acetone and acetonitrile) and inorganic salt (such as NaCl, NaH2PO4, K2HPO4 and Na2SO4) dissolved in water can form hydrophilic organic solvent/salt/water two-phase system. Its top phase mainly contains organic solvent and its bottom phase mainly contains salt and water. The two-phase system has some advantages, such as easy phase separation, cheap and low-toxic raw material, which has been applied in sample enrichment and separation of metal and biomolecules, and has got some good results. But up to now, there are no any reports about the application of this two-phase system in bioconversion.Enzymatic hydrolysis of casein catalyzed by papain to produce amino acids was performed in the novel two-phase system, which was composed of n-propanol, NaCl and water. Papain and casein were mainly distributed in bottom phase, and aromatic amino acids produced by enzymatic hydrolysis were mainly in top phase. It was beneficial to the bioconversion. n-Propanol/NaCl/water two-phase system got a higher bioconversion than n-propanol/water single-phase system, aqueous single-phase system, n-hexane/water two-phase system and PEG/phosphate aqueous two-phase system. It was investigated that the effects of temperature, pH, n-propanol concentration, NaCl concentration, enzyme concentration and substrate concentration on bioconversion yield. When the two-phase system consisted of 44% n-propanol, 60 g/l NaCl, 0.15 g/l papain and 13 g/l casein at 55℃and pH 5.6, the transformation yield was 99.5% after 5 h reaction. The result showed that the two-phase system is suitable for the bioconversion by papain.The research also included the biotransformation of L-phenylpyruvic acid to L-phenylalanine by ATCC 11250 and the biotransformation of penicillin G to 6-aminopenicillanic acid (6-APA) by ATCC 9637 and immobilized penicillin acylase in hydrophilic organic solvent/salt/water two-phase systems. It was found that hydrophilic organic solvents and inorganic salts could inhibit the enzymatic activities. The synthesis of ethyl caproate from caproic acid and anhydrous ethanol catalyzed by pocine pancreas lipase in hydrophilic organic solvent/salt/water two-phase systems was investigated. It was found that water and hydrophilic organic solvents could inhibit the activity of pocine pancreas lipase. So the novel two-phase systems were not suitable to above bioconversion. The hydrophilic organic solvent/salt/water two-phase systems are only applicable for some special enzymes.Hydrophobic organic solvents are other common media for bioconversion. In the system, there is no water or little water. It was investigated that synthesis of ethyl caproate from caproic acid and anhydrous ethanol catalyzed by pocine pancreas lipase in hydrophobic organic solvent. The effects of reaction media, substrate concentration, mole ratio of substrates, enzyme amount and reaction time on bioconversion yield were studied. It was found that the influences of organic solvent on enzymatic reaction with low concentration of substrate were different from those with high concentration of substrate. Middle concentration of substrate and moderate lg P value of solvent were suitable. The appropriate conditions for biocatalysis were: 0.6 M caproic acid in cyclohexane, mole ratio of caproic acid and anhydrous ethanol with 1:1.25, 15 g/l lipase, 37℃. After 24 h reaction, the conversion yield was 90.5%. When the substrate concentration was 1.5-3.0 times as high as reports from the references, the similar conversion yield was obtained. |
PDF Full Text Request