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

Pasamontes, Luis; Haiker, Monika; Henriquez-Huecas, Maria; Mitchell, David B.; Loon, A. P. G. M. Van

doi:10.1016/s0167-4781(97)00107-3

Cited by 53 publications

(26 citation statements)

References 17 publications

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“…Thus, phytases are used as a cereal feed additive that enhances the phosphorus and mineral uptake in monogastric animals and reduces the level of phosphate output in their manure. Recently, there have been several reports on the cloning of fungal PhyA phytases from Aspergillus niger (16,20,28), Aspergillus fumigatus (19), Aspergillus terreus, Myceliophthora thermophila (14), Emericella nidulans, Talaromyces thermophilus (18), and Thermomyces lanuginosus (2). Based on their characteristics and on their sequence similarity, the PhyA phytases form a subclass within the histidine acid phosphatase family (14).…”

Section: Phytases (Myo-inositol Hexakisphosphate Phosphohydrolases) Bmentioning

confidence: 99%

Expression, Gene Cloning, and Characterization of Five Novel Phytases from Four Basidiomycete Fungi: Peniophora lycii, Agrocybe pediades , a Ceriporia sp., and Trametes pubescens

Lassen

Breinholt

Østergaard

et al. 2001

Appl Environ Microbiol

127

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Phytases catalyze the hydrolysis of phosphomonoester bonds of phytate (myo-inositol hexakisphosphate), thereby creating lower forms of myo-inositol phosphates and inorganic phosphate. In this study, cDNA expression libraries were constructed from four basidiomycete fungi (Peniophora lycii, Agrocybe pediades, a Ceriporia sp., and Trametes pubescens) and screened for phytase activity in yeast. One full-length phytaseencoding cDNA was isolated from each library, except for the Ceriporia sp. library where two different phytase-encoding cDNAs were found. All five phytases were expressed in Aspergillus oryzae, purified, and characterized. The phytases revealed temperature optima between 40 and 60°C and pH optima at 5.0 to 6.0, except for the P. lycii phytase, which has a pH optimum at 4.0 to 5.0. They exhibited specific activities in the range of 400 to 1,200 U ⅐ mg, of protein ؊1 and were capable of hydrolyzing phytate down to myo-inositol monophosphate. Surprisingly, 1 H nuclear magnetic resonance analysis of the hydrolysis of phytate by all five basidiomycete phytases showed a preference for initial attack at the 6-phosphate group of phytic acid, a characteristic that was believed so far not to be seen with fungal phytases. Accordingly, the basidiomycete phytases described here should be grouped as 6-phytases (EC 3.1.3.26).Phytases (myo-inositol hexakisphosphate phosphohydrolases) belong to the family of histidine acid phosphatases sharing the sequence consensus pattern(27; http://www.expasy.ch /cgi-bin/get-prodoc-entry?PDOC00538). They are capable of catalyzing the hydrolysis of phosphomonoester bonds of phytate (salts of myo-inositol hexakisphosphate or myo-inositol 1,2,3,4,5,6-hexakis dihydrogen phosphate), thereby creating lower forms of myo-inositol phosphates and inorganic phosphate (22,24). Phytases are grouped according to the specific position of the phosphate ester group on the phytate molecule at which hydrolysis is initiated, i.e., as 3-phytases (EC 3.1.3.8) or as 6-phytases (EC 3.1.3.26).Phytate is the primary source of inositol and the primary storage form of phosphate in plant seeds (23). Seeds, cereal grains, and legumes are important components of food and, in particular, of animal feed preparations. However, monogastric animals such as poultry and swine are incapable of utilizing the phosphorus bound in phytate due to low levels of phytase activity in the digestive tract. Furthermore, phytate acts as an antinutrient by chelating divalent cations and preventing the uptake of minerals, e.g., Zn (9). Thus, phytases are used as a cereal feed additive that enhances the phosphorus and mineral uptake in monogastric animals and reduces the level of phosphate output in their manure. Recently, there have been several reports on the cloning of fungal PhyA phytases from Aspergillus niger (16,20, 28), Aspergillus fumigatus (19), Aspergillus terreus, Myceliophthora thermophila (14), Emericella nidulans, Talaromyces thermophilus (18), and Thermomyces lanuginosus (2). Based on their characteristics and on their s...

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Section: Phytases (Myo-inositol Hexakisphosphate Phosphohydrolases) Bmentioning

confidence: 99%

Expression, Gene Cloning, and Characterization of Five Novel Phytases from Four Basidiomycete Fungi: Peniophora lycii, Agrocybe pediades , a Ceriporia sp., and Trametes pubescens

Lassen

Breinholt

Østergaard

et al. 2001

Appl Environ Microbiol

127

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“…General approach. Over the last few years, we have reported the cloning, sequencing, and biochemical, as well as biophysical, characterization of a series of homologous fungal phytases (12,14,15,(28)(29)(30). In addition, we have determined the crystal structure of A. niger phytase at a 2.5-Å resolution (4).…”

Section: Resultsmentioning

confidence: 99%

Engineering of Phytase for Improved Activity at Low pH

Tomschy

Brugger

Lehmann

et al. 2002

Appl Environ Microbiol

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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.Active sites typically contain ionizable groups (Arg, Lys, His, Glu, and Asp) that are involved in substrate or product binding and/or catalysis and that determine the pH activity profile of an enzyme (2). Both for physiological constraints and for industrial applications, it is crucial that an enzyme works properly at the appropriate pH value(s). However, no truly reliable methods for modifying the pH activity profile of an enzyme are yet available. In order to understand more clearly how nature masters catalysis at physiological pH values and to rationally tailor enzymes for industrial use at a predefined pH value, it is important to gain more experimental experience on the pH optimum engineering of enzymes.Different strategies can be chosen to modify the pH activity profile of an enzyme. (i) The first is the replacement of ionizable groups that are directly involved in substrate or product binding and/or catalysis by nonionizable ones or by amino acids with different charge or pK values (i.e., "A residues" [13,23]).(ii) The second is the replacement of residues that are in direct contact with A residues by forming hydrogen bonds and/or salt bridges. Substitution of such residues may disturb the hydrogen-bonding network in the active site or alter the electronic environment of A residues (1,11,19,27). The effects on the pH activity profile caused by this type of mutations are particularly difficult to predict. (iii) The third is the alteration of...

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“…Thermoascus aurantiacus TUB F43 synthesized phytase in a medium containing glucose and starch as carbon sources and peptone as a nitrogen source at 45°C, 150 rpm and pH 5.5 after 72 h of fermentation (Nampoothiri et al, 2004). Aspergillus fumigatus secreted a heat-stable phytase that was able to withstand temperatures up to 100°C for 20 min (Pasamontes et al, 1997a). Pasamontes et al (1997b) reported the cloning of a phytase gene from Talaromyces thermophilus that showed 61% sequence homology with that of A. niger.…”

Section: Production Of Phytasesmentioning

confidence: 99%

Fungal phytases: characteristics and amelioration of nutritional quality and growth of non‐ruminants

Singh

Satyanarayana

2014

Animal Physiology Nutrition

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SummaryFungal phytases are histidine acid phosphatases, a subclass of acid phosphatases, which catalyse the hydrolysis of phytic acid resulting in the release of phosphate moieties and thus mitigate its antinutritional properties. The supplementation of feed with phytases increases the bioavailability of phosphorus and minerals in non-ruminant animals and reduces the phosphorus pollution due to phosphorus excretion in the areas of intensive livestock production. Although phytases are reported in plants, animals and micro-organisms, fungal sources are used extensively for the production of phytases on a commercial scale. Phytases have been produced by fungi in both solid-state fermentation (SSF) and submerged fermentation (SmF). The fungal phytases are high molecular weight proteins ranging from 35 to 500 kDa. They are optimally active within pH and temperature ranges between 4.5 and 6.0, and 45 and 70°C respectively. Phytate degradation leads to amelioration in the nutritional status of foods and feeds by improving the availability of minerals, phosphorus and proteins in non-ruminant animals and human beings and thus mitigates the environmental phosphorus pollution. Our article focuses on the role of fungal phytases in improving nutritional value of foods and feeds with concomitant increase in growth of non-ruminant animals and mitigating environmental phosphorus pollution.

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

Cited by 53 publications

References 17 publications

Expression, Gene Cloning, and Characterization of Five Novel Phytases from Four Basidiomycete Fungi: Peniophora lycii, Agrocybe pediades , a Ceriporia sp., and Trametes pubescens

Expression, Gene Cloning, and Characterization of Five Novel Phytases from Four Basidiomycete Fungi: Peniophora lycii, Agrocybe pediades , a Ceriporia sp., and Trametes pubescens

Engineering of Phytase for Improved Activity at Low pH

Fungal phytases: characteristics and amelioration of nutritional quality and growth of non‐ruminants

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