Cephalexin is one of the most widely used β-lactam antibiotics, which possesses a broad spectrum against infectious microorganisms, but the industrial production of cephalexin using chemical scheme suffers from many problems, so enzymatic methods are regarded as a superior method that can produce cephalexin in one-step reaction and cut down the pollution. However, the enzymatic method still has remaining challenges in low productivity due to hydrolysis of the product causing by the characteristic of the enzyme. In this study, the enzymes for cephalexin synthesis (also called cephalexin-synthesizing enzyme, CSE) from different Gluconobacter species have been amplified by PCR using the primers designed based on the genome sequences including α-amino acid ester hydrolase (AEH) from G. oxydans BCRC 10683, glutaryl-7-aminocephalosporanic acid acylase (GL-7-ACA acylase) from Gluconobacter frateurii BCRC 10383, and penicillin acylase from G. oxydans BCRC 10384. Both genes encoding AEH from G. oxydans BCRC 10683 and GL-7-ACA acylase from G. frateurii BCRC 10383 were successfully cloned and the cloned DNA fragments were sequenced and blast in GenBank to confirm to be correct. The AEH gene from G. oxydans BCRC 10683 was further subcloned into pET21b and pET28a in Escherichia coli to overexpress AEH (named GoAEH). After purification by one-step Ni-NTA column, the purified protein fractions were conducted cephalexin-synthesizing reactions from phenylglycine methyl ester (PGME) and 7-aminodeacetoxycephalosporanic acid (7-ADCA), and examined by TLC and HPLC analysis. The results showed that the one-step purified GoAEH did possess good activity to catalyze the condensing reaction of PGME and 7-ADCA to synthesize cephalexin, which suggested that AEH is the CSE in G. oxydans. There are several proteins have very high homology with GoAEH in Gluconobacter species according to the phylogeny and multiple sequence alignment analyses, which was expected that all of them have the same catalyzing ability to producing cephalexin, but needs to further study in gene cloning, protein expression and activity assay to validate it.
