The catalytic activity of isopenicillin N synthase (IPNS), a crucial enzyme which converts delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine to isopenicillin N in the beta-lactam antibiotic biosynthetic pathway, is known to be dependent upon the ligation of two histidines and an aspartate to the iron active centre. Recent studies have ruled out the suggested requirement of the penultimate glutamine, Q330 and Q328 in Aspergillus nidulans and Streptomyces jumonjinensis IPNS respectively, for catalysis. As a counter proposal, glutamine-230 from S. jumonjinensis IPNS was presented to be crucial for activity. However, we report differing results from the site-directed mutagenesis of the corresponding glutamine-234 in Cephalosporium acremonium IPNS. Based on IPNS enzymatic assays, we conclude that glutamine-234 is not essential for catalysis in cIPNS. Furthermore, we advocate the use of soluble proteins over solubilized proteins especially for studies which involve enzymatic catalysis.