Mapping the Catalytic Landscape of a Novel Phytase

绘制新型植酸酶的催化​​图谱

• 批准号: BB/M022978/1
• 负责人: Andrew Hemmings
• 金额: $ 58.56万
• 依托单位: University of East Anglia
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2015
• 资助国家: 英国
• 起止时间: 2015 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FM022978%2F1
• 关键词: Mapping; Catalytic; Landscape; Novel; Phytase

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.

植酸是动物生长所需的无机磷酸盐的主要来源，存在于常见动物饲料的谷物、油料种子和豆类中。然而，动物不产生消化植酸所需的酶，而是依赖于由驻留在动物肠道中的数万亿细菌产生的酶（一般称为植酸酶）。在非反刍动物如猪和鸡中，这些酶消化其饮食中植酸的能力是有限的。因此，它们的饲料补充了无机磷，最常见的是以不可再生资源磷酸二钙（DCP）的形式使用，磷酸二钙的开采成本很高，环境代价也很大。为了增加植酸转化为可利用的膳食磷酸盐的程度，植酸酶通常被添加到动物饲料中。通过添加植酸酶有效分解植酸具有成本效益，并且能够减少饲料中DCP的补充。据估计，添加植酸酶可以节省4-5英镑/吨饲料的DCP补充成本。因此，全世界生产的约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，属于称为组氨酸酸性磷酸酶（HAP）的类别。这些HAP酶在攻击植酸的方式上是特异性的：它们或者是首先从3位去除磷酸的3-植酸酶，或者是首先去除6位磷酸的6-植酸酶。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敏感，因此许多动物饲料酶的工业生产者正在寻找其他不太敏感的酶类型。这种酶是一类新的HAP的第一个代表，其特征在于赋予其催化活性的氨基酸残基排列的细微变化。因此，催化残基的位置和身份的变化与不寻常的活性位点特征相结合，对酶的性质具有显着的后果。酶活性位点更开放，导致其如何消化植酸的灵活性更大，即在其去除第一个磷酸的位置。基于这一发现并与动物饲料酶的主要生产商合作，我们提出了一项研究计划，旨在开发MINPP作为新一代植酸酶中的第一种，具有增强的催化灵活性，用于动物饲料工业。为了实现这一目标，我们将首先绘制出酶的生化和酶学性质的全貌，包括活性位点残基对植酸消化的作用的详细分析。然后，我们将继续设计酶的氨基酸序列，使其具有增强的热稳定性和其他商业上有吸引力的特性。在该项目的第二部分中，我们将评估将MINPP样活性位点残基取代到已经用作工业酶的真菌3-和细菌6-植酸酶的活性位点中对其催化性质的影响。该分析的结果可能证明对饲料酶制造商（他们已经投入大量资金来优化他们在市场上的酶）具有吸引力，作为延长其各自酶的催化灵活性和因此效用的成本有效的手段。

项目成果

Characterisation of a soil MINPP phytase with remarkable long-term stability and activity from Acinetobacter sp.

具有显着的长期稳定性和活性的土壤MINPP植物酶的表征。

• DOI: 10.1371/journal.pone.0272015
• 发表时间: 2022
• 期刊: PLOS ONE
• 影响因子: 3.7
• 作者: Rix, Gregory D.;Sprigg, Colleen;Whitfield, Hayley;Hemmings, Andrew M.;Todd, Jonathan D.;Brearley, Charles A.
• 通讯作者: Brearley, Charles A.

Structure of a cereal purple acid phytase provides new insights to phytate degradation in plants.

• DOI: 10.1016/j.xplc.2022.100305
• 发表时间: 2022-03-14
• 期刊: PLANT COMMUNICATIONS
• 影响因子: 10.5
• 作者: Faba-Rodriguez, Raquel;Gu, Yinghong;Salmon, Melissa;Dionisio, Giuseppe;Brinch-Pedersen, Henrik;Brearley, Charles A.;Hemmings, Andrew M.
• 通讯作者: Hemmings, Andrew M.

Organic phosphorus in the terrestrial environment: a perspective on the state of the art and future priorities

• DOI: 10.1007/s11104-017-3391-x
• 发表时间: 2018-06-01
• 期刊: PLANT AND SOIL
• 影响因子: 4.9
• 作者: George, T. S.;Giles, C. D.;Haygarth, P. M.
• 通讯作者: Haygarth, P. M.

Insights to the Structural Basis for the Stereospecificity of the Escherichia coli Phytase, AppA.

• DOI: 10.3390/ijms23116346
• 发表时间: 2022-06-06
• 期刊: INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
• 影响因子: 5.6
• 作者: Acquistapace, Isabella M.;Thompson, Emma J.;Kuhn, Imke;Bedford, Mike R.;Brearley, Charles A.;Hemmings, Andrew M.
• 通讯作者: Hemmings, Andrew M.

The crystal structure of mammalian inositol 1,3,4,5,6-pentakisphosphate 2-kinase reveals a new zinc-binding site and key features for protein function.

• DOI: 10.1074/jbc.m117.780395
• 发表时间: 2017-06-23
• 期刊: The Journal of biological chemistry
• 作者: Franco-Echevarría E;Sanz-Aparicio J;Brearley CA;González-Rubio JM;González B
• 通讯作者: González B