RESUMO
A comprehensive taxonomic re-evaluation was performed on the marine, zeaxanthin-producing bacterium formerly classified as [Favobacterium] sp. strain R-1 512 (ATCC 21588). This strain, together with two other previously described marine isolates, [Flavobacterium] strain R-1506 and Paracoccus sp. strain MBIC 3966, were found to comprise a new species of the genus Paracoccus. The name Paracoccus zeaxanthinifaciens sp. nov. is proposed, with ATCC 21588T (= R-1512T =LMG 21293T) designated as the type strain.
Assuntos
Paracoccus/classificação , Paracoccus/metabolismo , beta Caroteno/análogos & derivados , beta Caroteno/biossíntese , Composição de Bases , Sequência de Bases , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/genética , Ácidos Graxos/análise , Dados de Sequência Molecular , Paracoccus/genética , Fenótipo , Filogenia , RNA Bacteriano/genética , RNA Ribossômico 16S/genética , Especificidade da Espécie , Xantofilas , ZeaxantinasRESUMO
Previously, we calculated a consensus amino acid sequence from 13 homologous fungal phytases. A synthetic gene was constructed and recombinantly expressed. Surprisingly, consensus phytase-1 was 15-26 degrees C more thermostable than all parent phytases used in its design [Lehmann et al. (2000)Protein Eng., 13, 49-57]. In the present study, inclusion of six further phytase sequences in the amino acid sequence alignment resulted in the replacement of 38 amino acid residues in either one or both of the new consensus phytases-10 and -11. Since consensus phytase-10, again, was 7.4 degrees C more thermostable than consensus phytase-1, the thermostability effects of most of the 38 amino acid substitutions were tested by site-directed mutagenesis. Both stabilizing and destabilizing mutations were identified, but all affected the stability of the enzyme by <3 degrees C. The combination of all stabilizing amino acid exchanges in a multiple mutant of consensus phytase-1 increased the unfolding temperature from 78.0 to 88.5 degrees C. Likewise, back-mutation of four destabilizing amino acids and introduction of an additional stabilizing amino acid in consensus phytase-10 further increased the unfolding temperature from 85.4 to 90.4 degrees C. The thermostabilization achieved is the result of a combination of slight improvements from multiple amino acid exchanges rather than being the effect of a single or of just a few dominating mutations that have been introduced by chance. The present findings support the general validity of the consensus concept for thermostability engineering of proteins.
Assuntos
6-Fitase/química , Proteínas Fúngicas/química , Temperatura Alta , Engenharia de Proteínas , Proteínas de Saccharomyces cerevisiae/química , 6-Fitase/genética , 6-Fitase/metabolismo , Sequência de Aminoácidos , Estabilidade Enzimática , Proteínas Fúngicas/metabolismo , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Pichia/enzimologia , Alinhamento de Sequência , Análise de Sequência de ProteínaRESUMO
For industrial applications in animal feed, a phytase of interest must be optimally active in the pH range prevalent in the digestive tract. Therefore, the present investigation describes approaches to rationally engineer the pH activity profiles of Aspergillus fumigatus and consensus phytases. Decreasing the negative surface charge of the A. fumigatus Q27L phytase mutant by glycinamidylation of the surface carboxy groups (of Asp and Glu residues) lowered the pH optimum by ca. 0.5 unit but also resulted in 70 to 75% inactivation of the enzyme. Alternatively, detailed inspection of amino acid sequence alignments and of experimentally determined or homology modeled three-dimensional structures led to the identification of active-site amino acids that were considered to correlate with the activity maxima at low pH of A. niger NRRL 3135 phytase, A. niger pH 2.5 acid phosphatase, and Peniophora lycii phytase. Site-directed mutagenesis confirmed that, in A. fumigatus wild-type phytase, replacement of Gly-277 and Tyr-282 with the corresponding residues of A. niger phytase (Lys and His, respectively) gives rise to a second pH optimum at 2.8 to 3.4. In addition, the K68A single mutation (in both A. fumigatus and consensus phytase backbones), as well as the S140Y D141G double mutation (in A. fumigatus phytase backbones), decreased the pH optima with phytic acid as substrate by 0.5 to 1.0 unit, with either no change or even a slight increase in maximum specific activity. These findings significantly extend our tools for rationally designing an optimal phytase for a given purpose.
Assuntos
6-Fitase , Aspergillus fumigatus/enzimologia , Engenharia Genética/métodos , 6-Fitase/química , 6-Fitase/genética , 6-Fitase/metabolismo , Sequência de Aminoácidos , Aspergillus fumigatus/genética , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Alinhamento de SequênciaRESUMO
Phytases catalyse the hydrolysis of phytate (myo-inositol hexakisphosphate) to myo-inositol and inorganic phosphate. In this study genes encoding novel phytases from two different filamentous fungi, Aspergillus terreus strain 9A-1 and Myceliophthora thermophila were isolated. The encoded PhyA phytase proteins show 60% (A. terreus) and 48% (M. thermophila) identity, respectively, to the PhyA of Aspergillus niger and have 21-29% identity compared to other histidine acid phosphatases. All three PhyA proteins, in contrast to the A. niger pH 2.5-optimum acid phosphatase, prefer phytic acid as substrate and show enzyme activity at a broad range of acidic pH values. Based on their enzyme characteristics and protein sequence homology, the phytases form a novel subclass of the histidine acid phosphatase family.
