Enzymatic Hydrolysis of Penicillin in an Integrated System Containing Ionic Liquids

Abstract

Ionic liquids were employed as novel green solvents to develop an integrated process involving ionic liquids aqueous two-phase system (ILATPS) and mixed ionic liquids/water two-phase system (MILWS) for separation and enzymatic catalysis of penicillin. First, hydrophilic [C(4)mim]BF4 (1-butyl-3-methylimidazolium tetrafluoroborate) and NaH2PO4 salt form an ionic liquid aqueous two-phase system (ILATPS), which could extract penicillin from its fermentation broth efficiently. Second, hydrophobic [C(4)mim]PF6 (1-butyl-3-methylimidazolium hexafluorophosphate) is introduced into the ionic liquid-rich phase of ILATPS containing penicillin and converses it into MILWS. Penicillin is hydrolyzed by penicillin acylase in the water phase of MILWS at pH 5. The byproduct phenylacetic acid (PAA) is partitioned into the ionic liquids mixture phase, while the intended product 6-aminopenicillanic acid (6-APA) is precipitated at this pH. In comparison with a similar butyl acetate/water system (BAWS) at pH 4, MILWS exhibits two advantages: (1) The selectivity between PAA and penicillin is greatly optimized at pH 5 by varying the mole ratio of [C(4)mim]PF6/[C(4)mim]BF4 in MILWS, whereas in BAWS the unalterable nature of the organic solvent restricts the optimized pH for maximum selectivity between PAA and penicillin at pH 4. (2) The pH for 6-APA precipitation in BAWS is 4, whereas it shifts to pH 5 in MILWS due to the complexation between negatively charged 6-APA and the cationic surface of the ionic liquids micelle. As a result, the removal of the two products from the enzyme sphere at relatively high pH is permitted in MILWS, which is beneficial for enzymatic activity and stability in comparison with the acidic pH 4 environment in BAWS.