Purification and Characterization of Inducible Cephalexin Synthesizing Enzyme inGluconobacter oxydans

Abstract

Cephalexin synthesizing enzyme (CSE) of Gluconobacter oxydans ATCC 9324 was purified up to about 940-fold at a yield of 12%. CSE biosynthesis in G. oxydans was found inducible in the presence of D-phenylglycine but not its substrate phenylglycine methyl ester. The purified enzyme was shown homogeneous on SDS-PAGE and exhibited a specific activity of 440 U per mg protein. The apparent molecular mass of the native enzyme was estimated to be 70 kDa over a Superdex 200 gel filtration column and 68 kDa on SDS-PAGE, indicating that the native enzyme is a monomer. Its isoelectric focusing point is 7.1, indicating a neutral character. The enzyme had maximal activity around pH 6.0 to 6.5, and this activity was thermally stable up to 40 degrees C. Synthesis of cephalexin from D-phenylglycine methyl ester and 7-amino-3-deacetoxycephalosporanic acid (7-ADCA) by the purified CSE was demonstrated. Its L-enantiomer was not accepted by CSE. Apart from cephalexin, ampicillin was also synthesized by the purified CSE from its acyl precursors and 6-aminopenicillanic acid (6-APA). Substrate specificity studies indicated that the enzyme required a free alpha amino group and an activated carboxyl group as a methyl ester of D-form phenylglycine. Interestingly, the purified enzyme did not catalyze hydrolysis of its products, e.g., cephalexin, cephradine, and ampicillin, in contrast to enzymes from other strains of Pseudomonadaceae.

Genetic engineering approaches for the fermentative production of phenylglycines

“…and cephalosporins (cefalexin, cefradine, cefaclor, cefaloglycine, etc. ) (Stevenazzi et al 2014;Shiau et al 2005;Schmid et al 2001;Müller et al 2013). Currently, D-Phg is produced in a scale > 5000 tons per year worldwide (Vedha-Peters et al 2006).…”

Alkenylation of ortho-palladated phenylglycine: synthesis of stilbene derivatives and 3-aryl-isoquinoline-1-carboxylates

“…The α-amino acid phenylglycine (Figure 1, left) can be considered as the simplest representative of the aromatic series and, although it is not one of the proteinogenic amino acids, it shows remarkable pharmacological activity. [1][2][3][4][5][6] For example, this scaffold is present in many of the top 200 pharmaceutical products by retail sales in 2018 in the USA, for instance, in drugs such as Clopidrogrel or Augmentin (Amoxicillin) (middle and right in Figure 1). [7,8] The phenylglycine motif can also be found in the core of Vancomycin, Teicoplanin and Sulperazon, which are used as antiplatelet, antibiotic and bacteriostatic drugs, respectively.…”