| Penicillin G acylase (PGA, E.C. 3.5.1.11) plays a central role in semi-synthetic β-lactam antibiotics industry. PGA catalyze the hydrolysis of benzylpenicillin to phenylacetic acid and 6-aminopenicillanic acid-one of the key intermediates for the production of semisynthetic β-lactam antibiotics.PGA can also catalyze the synthesis of these antibiotics.The sequence determined penicillin G acylases are those from gram-positive bacteria family members (Esherichia coli, Kluyvera citrophila, Providencia rettgeri, Alcaligenes faecalis), and those from gram-positive bacteria family members (Bacillus megaterium Arthrobacter viscosus). The PGA was synthesized as a protein precursor molecule which is proteolytically processed to generate the active enzyme consisting of two non-identical peptide chains (α and β). From the N-terminus the precursor consists of four structural regions:signal peptide, α-peptide,linker peptide and β-peptide.The processing starts with removal of signal peptide to direct translocation to periplasmic space. Subsequently, there is a second highly specific cleavage between the C-terminus of the linker peptide and the N-terminus of the β-peptide. The further maturation of active PGA is characterized by a sequential removal of the linker peptide from the α-peptide. In particular due to this unusual processing,PGA are also of great fundamental interest. There is some evidence that the PGA Psubstrate specificity is mainly associated with the α-peptide whereas theβ-peptide contains residues involved in catalysis,as well as structural elements responsible for amine moiety binging.Recent studies have identified N-terminal Ser290 of the β-peptide as the residue which is acylated during the catalytic reaction.PGA from gram-positive bacteria family members possess similar amino acid sequences, amino acids of subunit interface and that of substrate interface.The homology between PGA from E.coli, K.citrophila was 83％ in α-subunit,86% in β-subunit,and that between PGA from P.rettgeri and E.coli, K.citrophila respectively are higher than 60%.But that between PGA from A.faecalis and E.coli, K.citrophila ,P.rettgeri,respectively are lower than 50%,and PGA from A.faecalis and P.rettgeri are share less evolutionarily relative .The subunit interface are more conserved in sequence than the rest of the protein surface.The amino acids in substrate interface are very conserved.PGA from E.coli, K.citrophila ,P.rettgeri have the same amino acids in substrate interface,and three amino acids differ with PGA from A.faecalis.Twelve cross-species penicillin G acylase genes coding for an signal peptide- α-peptide-linker peptide from four gram-positive bacteria and a β-peptide from four gram-positive bacteria ,respectively,has been constructed and cloned in E.coli.The highly-conserved processing pathway,as well as a certain homology on protein level should make it possible to combineα-andβ-peptide from different sources to produce a hybrid PGA. The activity of hybrid enzymes reduced with amino acid (aa)identity of wild type PGA decreasing。Though the PGA of P.rettgeri and A.faecalis share 40% aa identity,the hybrid enzyme of them was active against 6-nitro-3- phenylacet amido benzoic acid (NIPAB).It implies that the subunits of PGA may evolve independently.
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