Method for separating amoxicillin and phenylacetic acid from reaction solution in one-step enzymatic synthesis of amoxicillin

A method for separating amoxicillin and phenylacetic acid from a reaction solution obtained by a one-step enzymatic synthesis of amoxicillin, comprising catalyzing a one-step synthesis of amoxicillin from penicillin potassium using an immobilized penicillin acylase mutant to obtain a reaction suspension;

• and subjecting the reaction suspension to a separation process comprising the following steps:
• (1) adding deionized water to the reaction suspension, and performing vacuum filtration to obtain an amoxicillin filtrate and a retained immobilized penicillin acylase mutant;
• (2) washing the retained immobilized penicillin acylase mutant with deionized water, and combining a wash solution with the amoxicillin filtrate from step (1) to obtain a mixed solution;
• (3) adjusting a pH of the mixed solution obtained in step (2) to 2 with hydrochloric acid to obtain a separation-ready mixture;
• (4) adding a NaOH solution dropwise to the separation-ready mixture until reaching a pH between 3.5 and 5.5, followed by static crystallization at 4° C.;
• and
• (5) after the static crystallization is complete, filtering to obtain amoxicillin crystals and a liquid phase containing phenylacetic acid;
• wherein a method for catalyzing the one-step synthesis of amoxicillin from the penicillin potassium using the immobilized penicillin acylase mutant comprises:
• using only one immobilized penicillin acylase mutant as a sole enzyme in a reaction system, with penicillin or a salt thereof and D-p-hydroxyphenylglycine methyl ester as substrates, and carrying out a reaction in a reaction buffer system of pH 4-8;
• wherein compared with the amino acid sequence shown in SEQ ID NO:
• 1, the immobilized penicillin acylase has mutations:
• F146αK and F24βR and F71βY and N241βY and G385βR.