Mapping the Catalytic Landscape of a Novel Phytase

植酸是动物生长所需的无机磷的主要来源，存在于常见动物饲料的谷物、油籽和豆类中。然而，动物并不产生消化植酸所需的酶，而是依赖于寄生在动物肠道中的数万亿细菌产生的酶(一般称为植酸酶)。在非反刍动物中，如猪和鸡，这些酶消化其饮食中植酸的能力是有限的。因此，他们的饲料中添加了无机磷，最常见的是以不可再生资源磷酸氢钙(DCP)的形式使用，开采这种资源的成本和环境代价都很高。为了增加植酸转化为可利用的膳食磷酸盐的程度，通常在动物饲料中添加植酸酶。通过添加植酸酶有效地分解植酸是经济有效的，并能够减少饲料中添加DCP。据估计，添加植酸酶每吨饲料可节省DCP成本4-5 GB。因此，全世界生产的大约90%的家禽和85%的猪饲料含有添加的植酸酶。目前用作动物饲料补充剂的所有植酸酶都是来自细菌或真菌的酶的修饰衍生物，属于一类称为组氨酸酸性磷酸酶(HAP)。这些HAP酶在攻击植酸的方式上是特定的：它们要么是3-植酸酶，首先从第3位去除磷酸盐，要么是6-植酸酶，首先去除6-位的磷酸盐。前者存在于真菌中，而后者存在于细菌中。虽然这两类酶都是有效的，但它们对部分消化的植酸浓度的积累很敏感，因此许多动物饲料酶的工业生产者都在寻找其他不太敏感的酶类型。我们最近发现了一种新的HAP植酸酶，MINPP，它是由人类肠道主要细菌thetaiotaomicron分泌的。这种酶是一类新的HAPS的第一个代表，其特征是氨基酸残基的排列发生了微妙的变化，使其具有催化活性。因此，催化残基的位置和特性的变化与不寻常的活性部位特征相结合，对酶的性质有显著的影响。酶活性部位更加开放，从而在如何消化植酸方面有更大的灵活性，即在去除第一个磷酸盐的位置。在这一发现的基础上，并与动物饲料酶的主要生产商合作，我们提出了一个研究计划，旨在将MINPP开发为新一代植酸酶中的第一个，具有增强的催化灵活性，用于动物饲料行业。为了实现这一目标，我们将首先绘制出该酶的生化和酶学性质的完整图景，包括详细分析活性中心残基(S)在植酸消化中的作用。然后，我们将继续设计这种酶的氨基酸序列，使其具有增强的热稳定性和其他具有商业吸引力的特性。在项目的第二部分，我们将评估将MINPP类活性中心残基替换到已用作工业酶的真菌3-植酸酶和细菌6-植酸酶的活性中心对其催化性能的影响。这一分析结果可能会被证明对饲料酶制造商(他们已经投入大量资金优化他们在市场上的酶)具有吸引力，作为一种经济有效的手段来扩大其各自酶的催化灵活性，从而提高其实用性。

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.