Phytases: crystal structures, protein engineering and potential biotechnological applications

Phytases are a group of enzymes capable of releasing phosphates from phytates, one of the major forms of phosphorus (P) in animal feeds of plant origin. These enzymes have been widely used in animal feed to improve phosphorus nutrition and to reduce phosphorus pollution in animal waste. This review covers the basic nomenclature and crystal structures of phytases and emphasizes both the protein engineering strategies used for the development of new, effective phytases with improved properties and the potential biotechnological applications of phytases.     

Characterization of Arabidopsis thaliana Plants Expressing Bacterial Phytase

Russian Journal of Plant Physiology - Transgenic plants containing genes of bacterial phytases represent one of the promising ways to solve the problem of phosphorus deficiency in the nutrition of...     

Biotechnological development of effective phytases for mineral nutrition and environmental protection

Phytases are hydrolytic enzymes that initiate the release of phosphate from phytate (myo-inositol hexakisphosphate), the major phosphorus (P) form in animal feeds of plant origin. These enzymes can be supplemented in diets for food animals to improve P nutrition and to reduce P pollution of animal excreta. This mini-review provides a synopsis of the concept of "ideal phytase" and the biotechnological approaches for developing such an enzyme. Examples of Escherichia coli AppA and Aspergillus fumigatus PhyA are presented to illustrate how new phytases are identified from microorganisms and developed by genetic engineering based on the gene sequences and protein structures of these enzymes. We also discuss the characteristics of different heterologous phytase expression systems, including those of plants, bacteria, fungi, and yeast.     

Biotechnological production and applications of phytases

Phytases decompose phytate, which is the primary storage form of phosphate in plants. More than 10 years ago, the first commercial phytase product became available on the market. It offered to help farmers reduce phosphorus excretion of monogastric animals by replacing inorganic phosphates by microbial phytase in the animal diet. Phytase application can reduce phosphorus excretion by up to 50%, a feat that would contribute significantly toward environmental protection. Furthermore, phytase supplementation leads to improved availability of minerals and trace elements. In addition to its major application in animal nutrition, phytase is also used for processing of human food. Research in this field focuses on better mineral absorption and technical improvement of food processing. All commercial phytase preparations contain microbial enzymes produced by fermentation. A wide variety of phytases were discovered and characterized in the last 10 years. Initial steps to produce phytase in transgenic plants were also undertaken. A crucial role for its commercial success relates to the formulation of the enzyme solution delivered from fermentation. For liquid enzyme products, a long shelf life is achieved by the addition of stabilizing agents. More comfortable for many customers is the use of dry enzyme preparations. Different formulation technologies are used to produce enzyme powders that retain enzyme activity, are stable in application, resistant against high temperatures, dust-free, and easy to handle.     

In silico structural,functional and phylogenetic analysis of <Emphasis Type="Italic">Klebsiella</Emphasis> phytases

Phytic acid or phytate (myo-inositol hexakisphosphate) is the principal storage indigestible form of phosphorus in different crops. It is considered as an antinutrient in human as well as animal (including fish, poultry, pig, chicken etc.) diet due to its chelating behavior of certain essential divalent minerals (Fe²⁺, Mg²⁺, Zn²⁺, Ca²⁺ etc.). The unabsorbed, indigested form of phosphorus also causes phosphate pollution in the soil by animal wastes. Phytate degrading enzymes like phytases (myo-inositol hexakisphosphate phosphohydrolase) in this regard can be very useful and also economically feasible to reduce the risk of phosphate pollution and increase the nutrient value in animal feeds at the same time. The Klebsiella phytases are suitable to use in the food industries of plant origin for their excellent thermal stability and high pH tolerance. From the present in silico investigation, it was found that Klebsiella phytases were 46–47 kDa molecular weight protein of histidine phosphatase superfamily having thermostability and alkalinity nature. This thermostability can be achieved due to possession of higher percentage of α helices and β sheets at the same time; the presence of higher aliphatic indices (range in between 88 and 91) etc. Interestingly, a strong correlation was found to be pertinent from phylogenetic studies of proteins with their cDNA among both species and strain level. Hence, the present study would be beneficial for future researchers (3D model available in Protein Model Database with acc. no.: PM0080562) to meet the demand of agricultural and industrial production of bacterial phytases particularly for agricultural farming.     

Effect of Ca2+ on beta-propeller phytases

Beta-propeller phytases (BPPs) are a special class of enzyme that are mainly isolated from Bacillus and are widely used in animal nutrition, human health and environmental protection. BPPs class exhibits both unique Ca2+-dependent catalytic property and highly strict substrate specificity for the calcium-phytate complex. This review describes the effect of Ca2+ on the catalytic activity, thermal stability, and structural conformation of BPPs.     

Molecular advancements in the development of thermostable phytases

Since the discovery of phytic acid in 1903 and phytase in 1907, extensive research has been carried out in the field of phytases, the phytic acid degradatory enzymes. Apart from forming backbone enzyme in the multimillion dollar-based feed industry, phytases extend a multifaceted role in animal nutrition, industries, human physiology, and agriculture. The utilization of phytases in industries is not effectively achieved most often due to the loss of its activity at high temperatures. The growing demand of thermostable phytases with high residual activity could be addressed by the combinatorial use of efficient phytase sources, protein engineering techniques, heterologous expression hosts, or thermoprotective coatings. The progress in phytase research can contribute to its economized production with a simultaneous reduction of various environmental problems such as eutrophication, greenhouse gas emission, and global warming. In the current review, we address the recent advances in the field of various natural as well as recombinant thermotolerant phytases, their significance, and the factors contributing to their thermotolerance.

     

A novel phytase from Yersinia rohdei with high phytate hydrolysis activity under low pH and strong pepsin conditions

Two novel phytase genes belonging to the histidine acid phosphatase family were cloned from Yersinia rohdei and Y. pestis and expressed in Pichia pastoris. Both the recombinant phytases had high activity at pH 1.5-6.0 (optimum pH 4.5) with an optimum temperature of 55 degrees C. Compared with the major commercial phytases from Aspergillus niger, Escherichia coli, and a potential commercial phytase from Y. intermedia, the Y. rohdei phytase was more resistant to pepsin, retained more activity under gastric conditions, and released more inorganic phosphorus (two to ten times) from soybean meal under simulated gastric conditions. These superior properties suggest that the Y. rohdei phytase is an attractive additive to animal feed. Our study indicated that, in order to better hydrolyze the phytate and release more inorganic phosphorus in the gastric passage, phytase should have high activity and stability, simultaneously, at low pH and high protease concentration.     

Production of two Highly Active Bacterial Phytases with Broad pH Optima in Germinated Transgenic Rice Seeds

Phytate is the main storage form of phosphorus in many plant seeds, but phosphate bound in this form is not available to monogastric animals. Phytase, an enzyme that hydrolyzes phosphate from phytate, has the potential to enhance phosphorus availability in animal diets when engineered in rice seeds as a feed additive. Two genes, derived from a ruminal bacterium Selenomonas ruminantium (SrPf6) and Escherichia coli (appA), encoding highly active phytases were expressed in germinated transgenic rice seeds. Phytase expression was controlled by a germination inducible alpha-amylase gene (alphaAmy8) promoter, and extracellular phytase secretion directed by an betaAmy8 signal peptide sequence. The two phytases were expressed in germinated transgenic rice seeds transiently and in a temporally controlled and tissue-specific manner. No adverse effect on plant development or seed formation was observed. Up to 0.6 and 1.4 U of phytase activity per mg of total extracted cellular proteins were obtained in germinated transgenic rice seeds expressing appA and SrPf6 phytases, respectively, which represent 46-60 times of phytase activities compared to the non-transformant. The appA and SrPf6 phytases produced in germinated transgenic rice seeds had high activity over broad pH ranges of 3.0-5.5 and 2.0-6.0, respectively. Phytase levels and inheritance of transgenes in one highly expressing plant were stable over four generations. Germinated transgenic rice seeds, which produce a highly active recombinant phytase and are rich in hydrolytic enzymes, nutrients and minerals, could potentially be an ideal feed additive for improving the phytate-phosphorus digestibility in monogastric animals.     

Phytases: microbial sources,production, purification,and potential biotechnological applications

The review deals with phytase-producing microorganisms along with optimum conditions for its production. Various methods used for purifying phytases and their characteristics are discussed. Heterologous gene expression, cost-effective large-scale phytase production, and various biotechnological applications of the enzyme in animal feed and food industries are also discussed.