Mapping the Catalytic Landscape of a Novel Phytase

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Phytic acid is the major source of inorganic phosphate needed for animal growth and is found in the grains, oil seeds and beans of common animal feeds. However, animals do not produce the enzymes necessary to digest phytic acid, relying instead on enzymes (generically termed phytases) produced by the trillions of bacteria that reside in the animal's gut. In non-ruminants such as pigs and chickens, the capacity of these enzymes to digest the phytic acid of their diet is limited. As a result their feed is supplemented with inorganic phosphorous, most commonly applied in the form of a non-renewable resource, dicalcium phosphate (DCP), which is mined at great cost and environmental expense. To increase the extent of conversion of phytic acid into available dietary phosphate, phytases are routinely added to animal feeds. Efficient breakdown of phytic acid by added phytase is cost effective and enables a reduction in the supplementation of feed with DCP. It is estimated that addition of phytase saves 4-5 £/tonne of feed in the cost of DCP supplement. As a result, approximately 90% of the poultry and 85% of the pig feeds manufactured world-wide contain an added phytases. All phytases currently in use as animal feed supplements are modified derivatives of enzymes from either bacteria or fungi belonging to a class called histidine acid phosphatases (HAP). These HAP enzymes are specific in the way they attack phytic acid: they are either 3-phytases which remove the phosphate from position 3 first, or are 6-phytases which first remove that in the 6-position. The former are found in fungi while the latter are found in bacteria. While both classes of enzyme are efficient, they are susceptible to the build up of concentrations of partially-digested phytic acid and therefore other less susceptible types of enzymes are sought by the many industrial producers of animal feed enzymes.We have recently discovered a new HAP phytase, MINPP, which is secreted by a major human gut bacterium, Bacteroides thetaiotaomicron. This enzyme is the first representative of a new class of HAPs which are characterized by a subtle change in the arrangement of the amino acid residues which endow it with its catalytic activity. Accordingly, the change in position and identity of a catalytic residue combined with an unusual active site character has marked consequences for the properties of the enzyme. The enzyme active site is more open leading to greater flexibility in how it digests phytic acid, i.e. in the position from which it removes the first phosphate. Building on this discovery and in collaboration with a major producer of animal feed enzymes, we propose a programme of research which seeks to develop MINPP as the first of a new generation of phytases with enhanced catalytic flexibility for use in the animal feed industry. To achieve this objective we will firstly map out a complete picture of the biochemical and enzymological properties of the enzyme including a detailed analysis of the role(s) of active site residues on the digestion of phytic acid. We will then go on to engineer the amino acid sequence of the enzyme to imbue it with enhanced thermal stability and other commercially attractive properties. In the second part of the project, we will perform an evaluation of the effects on their catalytic properties of substituting MINPP-like active site residues into the active sites of fungal 3- and bacterial 6-phytases already employed as industrial enzymes. This results of this analysis may prove attractive to feed enzyme manufacturers (who have already invested substantial funds in optimizing the enzymes they have in the market) as a cost-effective means of extending the catalytic flexibility, and therefore utility, of their respective enzymes.