Luis Pasamontes

The phytase subfamily of histidine acid phosphatases: isolation of genes for two novel phytases from the fungi Aspergillus terreus and Myceliophthora thermophila.

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.

Engineering of Phytase for Improved Activity at Low pH

ABSTRACT 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.

Cloning of the phytases from Emericella nidulans and the thermophilic fungus Talaromyces thermophilus.

Phytases (EC 3.1.3.8) belong to the family of histidine acid phosphatases. We have cloned the phytases of the fungi Emericella nidulans and Talaromyces thermophilus. The putative enzyme encoded by the E. nidulans sequence consists of 463 amino acids and has a Mr of 51785. The protein deduced from the T. thermophilus sequence consists of 466 amino acids corresponding to a Mr of 51450. Both predicted amino acid sequences exhibited high identity (48% to 67%) to known phytases. This high level of identity allowed the modelling of all available fungal phytases based on the three-dimensional structure coordinates of the Aspergillus niger phytase. By this approach we identified 21 amino acids which are conserved in fungal phyA phytases and are part of the residues forming the substrate pocket. Furthermore, potential glycosylation sites were identified and compared between the aforementioned phytases and the A. niger phytase.

Isolation and characterization of the carotenoid biosynthesis genes of Flavobacterium sp. strain R1534.

The Gram-negative bacterium Flavobacterium sp. strain R1534 is a natural producer of zeaxanthin. A 14 kb genomic DNA fragment of this organism has been cloned and a 5.1 kb piece containing the carotenoid biosynthesis genes sequenced. The carotenoid biosynthesis cluster consists of five genes arranged in at least two operons. The five genes are necessary and sufficient for the synthesis of zeaxanthin. The encoded proteins have significant homology to the crtE, crtB, crtY, crtI and crtZ gene products of other carotenogenic organisms. Biochemical assignment of the individual gene products was done by HPLC analysis of the carotenoid accumulation in Escherichia coli host strains transformed with plasmids carrying deletions of the Flavobacterium sp. strain R1534 carotenoid biosynthesis cluster.

Active site residue 297 of Aspergillus niger phytase critically affects the catalytic properties.

The wild‐type phytases from the Aspergillus niger strains NRRL 3135 and T213 display a three‐fold difference in specific activity (103 versus 32 U/mg protein), despite only 12 amino acid differences that are distributed all over the sequence of the protein. Of the 12 divergent positions, three are located in or close to the substrate binding site. Site‐directed mutagenesis of these residues in A. niger T213 phytase showed that the R297Q mutation (R in T213, Q in NRRL 3135) fully accounts for the differences in catalytic properties observed. Molecular modelling revealed that R297 may directly interact with a phosphate group of phytic acid. The fact that this presumed ionic interaction – causing stronger binding of substrates and products – correlates with a lower specific activity indicates that product (myo‐inositol pentakisphosphate) release is the rate‐limiting step of the reaction.

Structure-based chimeric enzymes as an alternative to directed enzyme evolution: phytase as a test case

Thermostability is a key feature for commercially attractive variants of the fungal enzyme phytase. In an initial set of experiments, we restored ionic interactions and hydrogen bonds on the surface of Aspergillus terreus phytase, which are present in the homologous but more thermostable enzyme from A. niger. Since these mutations turned out to be neutral, we replaced-in the same region and based on the crystal structure of A. niger phytase-entire secondary structure elements. The replacement of one alpha-helix on the surface of A. terreus phytase by the corresponding stretch of A. niger phytase resulted in an enzyme with improved thermostability and unaltered enzymatic activity. Surprisingly, the thermostability of this hybrid protein was very similar to that of A. niger phytase, although the fusion protein contained only a 31 amino acid stretch of the more stable parent enzyme. This report provides evidence that structure-based chimeric enzymes can be used to exploit the evolutionary information within a sequence alignment. We propose this method as an alternative to directed enzyme evolution if due to expression constraints the screening of large mutant populations is not feasible.

Cloning and characterization of a surface antigen of Eimeria tenella merozoites and expression using a recombinant vaccinia virus.

A rabbit serum raised against Eimeria tenella merozoites was used to screen a lambda gt11 cDNA library made from merozoite mRNA of E. tenella. The insert of the phage clone lambda Mz 5-7 revealed an open reading frame consisting of 945 nucleotides, encoding a 33-kDa protein. This size is consistent with the size of a protein translated in vitro from merozoite mRNA and immunoprecipitated with monospecific anti-Mzp 5-7 antibodies. A smaller protein of 24 kDa, located on the surface of the parasite, also reacted with the monospecific antiserum and is the potential processed form of the Mzp 5-7. Furthermore, a recombinant vaccinia virus expressing the Mzp 5-7 antigen was constructed and used to immunize chickens.

Characteristics of fungal phytases from Aspergillus fumigatus and Sartorya fumigata

Aspergillus fumigatus phytase has previously been identified as a phytase with a series of favourable properties that may be relevant in animal and human nutrition, both for maximising phytic acid degradation and for increasing mineral and amino acid availability. To study the natural variability in amino acid sequence and its impact on the catalytic properties of the enzyme, we cloned and overexpressed the phytase genes and proteins from six new purported A. fumigatus isolates. Five of these phytases displayed ≤2 amino acid substitutions and had virtually identical stability and catalytic properties when compared with the previously described A. fumigatus ATCC 13073 phytase. In contrast, the phytase from isolate ATCC 32239 (Sartorya fumigata, the anamorph of which was identified as A. fumigatus) was more divergent (only 86% amino acid sequence identity), had a higher specific activity with phytic acid, and displayed distinct differences in substrate specificity and pH-activity profile. Finally, comparative experiments confirmed the favourable stability and catalytic properties of A. fumigatus phytase.