Cecchini Davide A, Serra Immacolata, Ubiali Daniela, Terreni Marco, Albertini Alessandra M
Dipartimento di Genetica e Microbiologia, via Ferrata 1, Università degli Studi di Pavia, 27100 Pavia, Italy.
BMC Biotechnol. 2007 Sep 10;7:54. doi: 10.1186/1472-6750-7-54.
Immobilized Penicillin G Acylase (PGA) derivatives are biocatalysts that are industrially used for the hydrolysis of Penicillin G by fermentation and for the kinetically controlled synthesis of semi-synthetic beta-lactam antibiotics. One of the most used supports for immobilization is glyoxyl-activated agarose, which binds the protein by reacting through its superficial Lys residues. Since in E. coli PGA Lys are also present near the active site, an immobilization that occurs through these residues may negatively affect the performance of the biocatalyst due to the difficult diffusion of the substrate into the active site. A preferential orientation of the enzyme with the active site far from the support surface would be desirable to avoid this problem.
Here we report how it is possible to induce a preferential orientation of the protein during the binding process on aldehyde activated supports. A superficial region of PGA, which is located on the opposite side of the active site, is enriched in its Lys content. The binding of the enzyme onto the support is consequently forced through the Lys rich region, thus leaving the active site fully accessible to the substrate. Different mutants with an increasing number of Lys have been designed and, when active, immobilized onto glyoxyl agarose. The synthetic performances of these new catalysts were compared with those of the immobilized wild-type (wt) PGA. Our results show that, while the synthetic performance of the wt PGA sensitively decreases after immobilization, the Lys enriched mutants have similar performances to the free enzyme even after immobilization. We also report the observations made with other mutants which were unable to undergo a successful maturation process for the production of active enzymes or which resulted toxic for the host cell.
The desired orientation of immobilized PGA with the active site freely accessible can be obtained by increasing the density of Lys residues on a predetermined region of the enzyme. The newly designed biocatalysts display improved synthetic performances and are able to maintain a similar activity to the free enzymes. Finally, we found that the activity of the immobilized enzyme proportionally improves with the number of introduced Lys.
固定化青霉素G酰化酶（PGA）衍生物是生物催化剂，在工业上用于通过发酵水解青霉素G以及动力学控制合成半合成β-内酰胺抗生素。最常用的固定化载体之一是乙醛酸活化琼脂糖，它通过与表面的赖氨酸残基反应来结合蛋白质。由于在大肠杆菌PGA中，赖氨酸也存在于活性位点附近，通过这些残基进行的固定化可能会因底物难以扩散到活性位点而对生物催化剂的性能产生负面影响。为避免此问题，希望酶的活性位点远离载体表面呈优先取向。
在此我们报告了如何在醛活化载体上的结合过程中诱导蛋白质的优先取向。PGA位于活性位点另一侧的表面区域富含赖氨酸。因此，酶通过富含赖氨酸的区域被迫与载体结合，从而使活性位点完全可被底物接触。设计了具有越来越多赖氨酸的不同突变体，并在其具有活性时固定在乙醛酸琼脂糖上。将这些新催化剂的合成性能与固定化野生型（wt）PGA的性能进行了比较。我们的结果表明，虽然wt PGA固定后的合成性能显著下降，但富含赖氨酸的突变体即使在固定后仍具有与游离酶相似的性能。我们还报告了对其他突变体的观察结果，这些突变体无法成功经历产生活性酶的成熟过程，或者对宿主细胞有毒性。
通过增加酶预定区域上赖氨酸残基的密度，可以获得活性位点可自由接触的固定化PGA的理想取向。新设计的生物催化剂显示出改善的合成性能，并且能够保持与游离酶相似的活性。最后，我们发现固定化酶的活性随引入的赖氨酸数量成比例提高。
