In experimental tests of enzyme evolution, a gene encoding an enzyme was subjected to multiple cycles of random mutagenesis and selection for altered substrate specificity. The resulting enzyme had altered substrate specificity and multiple amino acid changes. Where in the enzyme would you expect these amino acid changes to be located?

Using directed evolution, we have selected an adipyl acylase enzyme that can be used for a one-step bioconversion of adipyl-7-aminodesacetoxycephalosporanic acid (adipyl-7-ADCA) to 7-ADCA, an important compound for the synthesis of semisynthetic cephalosporins. The starting point for the directed evolution was the glutaryl acylase from Pseudomonas SY-77. The gene fragment encoding the β-subunit was divided into five overlapping parts that were mutagenized separately using error-prone PCR. Mutants were selected in a leucine-deficient host using adipyl-leucine as the sole leucine source. In total, 24 out of 41 plate-selected mutants were found to have a significantly improved ratio of adipyl-7-ADCAversus glutaryl-7-ACA hydrolysis. Several mutations around the substrate-binding site were isolated, especially in two hot spot positions: residues Phe-375 and Asn-266. Five mutants were further characterized by determination of their Michaelis-Menten parameters. Strikingly, mutant SY-77N266H shows a nearly 10-fold improved catalytic efficiency (k cat/K m) on adipyl-7-ADCA, resulting from a 50% increase in k cat and a 6-fold decrease in K m, without decreasing the catalytic efficiency on glutaryl-7-ACA. In contrast, the improved adipyl/glutaryl activity ratio of mutant SY-77F375L mainly is a consequence of a decreased catalytic efficiency toward glutaryl-7-ACA. These results are discussed in the light of a structural model of SY-77 glutaryl acylase.

Semisynthetic cephalosporins and penicillins are the most widely used antibiotics. All clinically important semisynthetic cephalosporins are made from 7-aminocephalosporanic acid (7-ACA)1 or 7-aminodesacetoxycephalosporanic acid (7-ADCA). 7-ACA is derived from cephalosporin C (aminoadipyl-7-ACA), which is obtained by fermentation of the fungus Cephalosporium acremonium. Deacylation is performed either chemically or by a two-step enzymatic process using aD-amino acid oxidase and a glutaryl acylase. The latter enzyme can be found in several Pseudomonas andAcinetobacter species (1-7) as well as in some Gram-positive bacteria (8, 9). 7-ADCA is produced from penicillin G made by Penicillium chrysogenum involving several polluting chemical steps followed by enzymatic deacylation by penicillin acylase (10). A first step toward the introduction of a simplified, more environmentally friendly production of 7-ADCA was the development of a genetically modified P. chrysogenum that produces adipyl-7-ADCA (AD-7-ADCA) (11). For the deacylation of this novel β-lactam, an adipyl acylase is needed. Since the presently identified acylases show little or no activity toward AD-7-ADCA, it is of interest to investigate whether a glutaryl acylase can be converted into an adipyl acylase.

In the past few years, directed evolution has been successfully implemented in changing the substrate specificity of several enzymes (12, 13), resulting in biocatalysts with novel activities. It has become clear that the success of a directed evolution experiment greatly depends on the availability of a good selective substrate, which unfortunately is absent for most bioconversions (12). Artificial substrates that mimic one of the desired catalytic steps may be used for selection; however, it is not clear to what extent the resulting mutants will have lost activity on their natural substrate.

Here we describe a strategy to evolve the glutaryl acylase ofPseudomonas SY-77 into an adipyl acylase with an improved activity toward AD-7-ADCA. The glutaryl acylase fromPseudomonas SY-77 has proven to be particularly suitable for developing an industrial process for deacylation (14). The natural action of the enzyme seems to be directed at hydrolyzing diamino acids with a glutaryl side chain as judged from its high activity on glutaryl-7-A(D)CA. It appears that the enzyme also has a low activity on AD-7-ADCA but no activity on cephalosporin C (2).

Explanation: I know my stuff I get B's in school.