RESUMO
Commercial interest in plant cell wall degrading enzymes (PCWDE) is motivated by their potential for energy or bioproduct generation that reduced dependency on non-renewable (fossil-derived) feedstock. Therefore, underlying work analysed the Penicillium chrysogenum isolate for PCWDE production by employing different biomass as a carbon source. Among the produced enzymes, three xylanase isoforms were observed in the culture filtrate containing sugarcane bagasse. Xylanase (PcX1) presenting 35â¯kDa molecular mass was purified by gel filtration and anion exchange chromatography. Unfolding was probed and analysed using fluorescence, circular dichroism and enzyme assay methods. Secondary structure contents were estimated by circular dichroism 45% α-helix and 10% ß-sheet, consistent with the 3D structure predicted by homology. PcX1 optimally active at pHâ¯5.0 and 30⯰C, presenting t1/2 19â¯h at 30⯰C and 6â¯h at 40⯰C. Thermodynamic parameters/melting temperature 51.4⯰C confirmed the PcX1 stability at pHâ¯5.0. PcX1 have a higher affinity for oat spelt xylan, KM 1.2â¯mg·mL-1, in comparison to birchwood xylan KM 29.86â¯mg·mL-1, activity was inhibited by Cu+2 and activated by Zn+2. PcX1 exhibited significant tolerance for vanillin, trans-ferulic acid, ρ-coumaric acid, syringaldehyde and 4-hydroxybenzoic acid, activity slightly inhibited (17%) by gallic and tannic acid.
Assuntos
Endo-1,4-beta-Xilanases/química , Endo-1,4-beta-Xilanases/isolamento & purificação , Proteínas Fúngicas/química , Proteínas Fúngicas/isolamento & purificação , Penicillium chrysogenum/enzimologia , Agricultura , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Resíduos de Serviços de Saúde , Estrutura Secundária de Proteína , Desdobramento de ProteínaRESUMO
In the last step of penicillin biosynthesis, acyl-CoA:isopenicillin N acyltransferase (IAT) (E.C. 2.3.1.164) catalyzes the conversion of isopenicillin N (IPN) to penicillin G. IAT substitutes the α-aminoadipic acid side chain of IPN by a phenylacetic acid phenolate group (from phenylacetyl-CoA). Having a three-dimensional (3D) structure of IAT helps to determine the steps involved in side chain exchange by identifying the atomic details of substrate recognition. We predicted the IAT 3-D structure (α- and ß-subunits), as well as the manner of IPN and phenylacetyl-CoA bind to the mature enzyme (ß-subunit). The 3D IAT prediction was achieved by homology modeling and molecular docking in different snapshots, and refined by molecular dynamic simulations. Our model can reasonably interpret the results of a number of experiments, where key residues for IAT processing as well as strictly conserved residues most probably involved with enzymatic activity were mutated. Based on the results of docking studies, energies associated with the complexes, and binding constants calculated, we identified a site located in the region generated by ß1, ß2 and ß5 strands, which forms part of the central structure of ß-subunit, as the potential binding site of IPN. The site comprises the amino acid residues Cys103, Asp121, Phe122, Phe123, Ala168, Leu169, His170, Gln172, Phe212, Arg241, Leu262, Asp264, Arg302, Ser309, and Arg310. Through hydrogen bonds, the IPN binding site establishes interactions with Cys103, Leu169, Gln172, Asp264 and Arg310. Our model is also validated by a recently revealed crystal structure of the mature enzyme.