豆壳过氧化物酶的分离纯化及其性质研究

从豆壳抽提液经硫酸铵分级沉淀,ＤＥＡＥ－ＳｅｐｈａｄｅｘＡ－５０离子交换层析,ＣｏｎＡ－Ｓｅｐｈａｒｏｓｅ４Ｂ亲合层析和Ｂｉｏ－ＧｅｌＰ－６０凝胶过滤,纯化了豆壳过氧化物酶（ｓｏｙｂｅａｎｈｕｌｐｅｒ－ｏｘｉｄａｓｅ,ＳｈＰ）．纯化酶的比活力为７０７７Ｕ／ｍｇ,在ＳＤＳ－ＰＡＧＥ上显示出一条蛋白质带．ＳｈＰ分子量为３８０００,等电点为３．９;ＳｈＰ为一含血红素的糖蛋白,含糖量为１８．７％,光谱学分析揭示,在４０６ｎｍ处有一典型的Ｓｏｒｅｔ带,在５１０ｎｍ和６４０ｎｍ处有特征吸收峰．酶反应的最适ｐＨ在４．０附近,最适温度为４５℃;在ｐＨ２．５～１２．０之间较稳定,７５℃,保温６０ｍｉｎ,酶活力残余６８％,ＳｈＰ是一种良好的耐酸碱、耐热过氧化物酶．动力学分析求得ＳｈＰ的表观Ｋｍ（愈创木酚）为１．６２ｍｍｏｌ／Ｌ,表现Ｋｍ（Ｈ２Ｏ２）为０．３４ｍｍｏｌ／Ｌ．在所测定的化学试剂中,Ｎ－３、ＣＮ－、Ｆｅ３＋、Ｆｅ２＋和Ｓｎ２＋对酶有较强烈的抑制作用,而重金属离子Ａｇ＋、Ｈｇ２＋、Ｐｂ２＋、Ｃｕ２＋、Ｃｒ３＋以及ＳＤＳ和ＥＤＴＡ对酶活力无显著影响     

夏威环毛蚓纤溶酶的分离纯化及部分性质研究

以夏威环毛蚓（Ｐｈｅｒｅｔｉｍａ ｈａｗａｙａｎａ）为材料,采用磷酸盐缓冲液抽提、（ＮＨ４）２ＳＯ４分段盐析,离子交换树脂 Ｄ２９０、 Ｓｅｐｈａｄｅｘ Ｇ－１００和 ＤＥＡＥ－ｓｅｐｈａｄｅｘ Ａ－５０三种连续柱层析方法得到一种在 ＰＡＧＥ上显示单一区带的纤溶酶组份。采用凝胶柱层析和 ＳＤＳ－ＰＡＧＥ测其分子量为 １２ ０００和 １２３００,由一条肽链组成。该酶具有强烈的纤溶活力和水解ＢＡＥＥ的活力,能直接作用纤维蛋白和间接激活纤溶酶原。其最适反应温度为４５℃,最适反应ｐＨ为８．０。该酶水解ＢＡＥＥ的活力可被Ｎａ＋、Ｋ＋、Ｍｇ２＋、Ｈｇ２＋、金属离子和ＥＤＴＡ、巯基乙醇抑制,Ｃａ＋则有激活作用。该酶中性糖含量为５％,氨基酸组成中Ａｒｇ、Ｌｅｎ含量较多．     

海枣曲霉β—木糖苷酶的提纯和性质

从海枣曲霉麦麸培养物抽提液中,通过聚乙二醇６０００－磷酸钾缓冲液双水相分离,相继用Ｓｅｐｈａｄｅｘ Ｇ－１００凝胶过滤、ＤＥＡＥ－Ｓｅｐｈａｄｅｘ Ａ－５０离子交换柱层析、羟基磷石吸附层析、ＤＥＡＥ－Ｓｅｐｈａｄｅｘ Ａ－５０离子交换层析、ＳＥ－Ｓｅｐｈａｄｅｘ Ｃ－５０离子交换层析以及最适温度为６５℃,在ｐＨ３．５－６．５之间稳定,酶保温３０分钟时的半失活温度（ｔ１／２）为６８℃。酶的分子量为９     

圆弧青霉碱性脂肪酶的分离纯化和特性

圆弧青霉突变株ＰＧ３７ 发酵液经离心、硫酸铵盐析、疏水层析、阴离子交换层析和凝胶过滤分离纯化得到了比活性为每毫克蛋白质５ ２００ ｕ 的碱性脂肪酶, 纯化倍数１６ ．５ , 得率３３．２％ , 在聚丙烯酰胺凝胶电泳（ＰＡＧＥ）和ＳＤＳ聚丙烯酰胺凝胶电泳（ＳＤＳＰＡＧＥ）上均呈现单一蛋白质条带。ＳＤＳＰＡＧＥ 和凝胶过滤分别测得酶的分子量为２７．５ ｋＤ和２９ ｋＤ, 表明该酶以单体形式存在。Ｎ末端１０ 个氨基酸的序列测定结果为ＡＴＡＤＡＡＡＦＰＤ, 与已知的碱性脂肪酶的Ｎ 末端序列没有同源性。酶学特性研究结果表明, 该酶的最适作用温度为２５ ℃, 在３０ ℃以下稳定,４０ ℃处理２０ ｍｉｎ 仅残留３０ ％ 酶活性;ｐＨ 稳定范围在６．５～１０．５ , 最适ｐＨ 为１０ ．０ 。低浓度的碱性蛋白酶对ＰＧ３７ 碱性脂肪酶活性的影响较小, 可同时添加在洗涤剂中。     

长白山白眉蝮蛇蛇毒磷脂酶A2的分离和初步表征

东北长白山白眉蝮蛇（ＡｇｋｉｓｔｒｏｄｏｎｂｌｏｍｈｏｆｆｉＵｓｕｒｅｎｓｉｓ）蛇毒经ＤＥＡＥＳｅｐｈａｄｅｘＡ５０离子交换层析柱,连续３步ＳｅｐｈａｄｅｘＧ７５凝胶过滤柱得到了磷脂酶Ａ２（ＰＬＡ２）的纯品。ＳＤＳ聚丙烯酰胺凝胶电泳（ＳＤＳＰＡＧＥ）以及基质辅助激光解析电离飞行时质谱（ＭＡＬＤＩ／ＴＯＦ／ＭＳ）表征为单一蛋白,其准确分子量为（１４．００８±０．００７）ｋＤ。最适ｐＨ范围８．０～９．０,最适的反应温度为４５℃。在溶液中有多聚体的存在。     

南方鲇碱性磷酸酶的分离纯化及部分性质的研究

用正丁醇抽提,硫酸铵分级沉淀,ＤＥＡＥ－纤维素和ＳｅｐｈａｃｒｙｌＳ－２００柱层析,从南方鲇（Ｓｉｌｕｒｕｓ　ｍｅｒｉｄｉｏｎａｌｉｓ　Ｃｈｅｎ）肠粘膜中提取出碱性磷酸酶（ＡＫＰ）。提纯倍数为３９．５０倍,比活为６８．３５μ／ｍｇ蛋白,提取酶液经ＰＡＧＥ和ＳＤＳ－ＰＡＧＥ只呈现一条区带。该酶的分子量为１３２１４０,Ｎ末端氨基酸为门冬氨酸,最适ｐＨ为１０．１０,７．５＞ｐＨ＞１１．５时不稳定,最适温度为４０℃左右,对热不很稳定,以磷酸苯二钠为底物其Ｋ_ｍ值为１．７２×１０~（－３）ｍｏｌ／Ｌ。Ｍｇ~（２＋）、Ｍｎ~（２＋）为该酶的激活剂,ＫＨ_２ＰＯ_４、Ｌ－ＣｙＳ、ＭＥ、ＤＦＰ、ＥＤＴＡ－Ｎａ_２为抑制剂。选用ＫＨ_２ＰＯ_４和ＤＦＰ作抑制类型的判断,结果表明,ＫＨ_２ＰＯ_４属竞争性掏剂,其抑制常数为２．３ｍｍｏｌ／Ｌ;ＤＦＰ为非竞争性抑制剂,抑制常数为１．０５ｍｍｏｌ／Ｌ。     

地衣芽孢杆菌β—甘露聚糖酶的纯化及酶学性质

地衣芽孢杆菌（Ｂａｃｉｌｌｕｓ ｌｉｃｈｅｎｉｆｏｒｍｉｓ）ＮＫ－２７菌株发酵产生的β－甘露聚糖酶（β－ｍａｎｎａｎａｓｅ）经硫酸铵盐析沉淀,两次ＤＥＡＥ纤维素和Ｓｅｐｈａｄｅｘ Ｇ－１００离子交换柱层析以及制备ＰＡＧＥ等步骤,获得了凝胶电泳均一的样品。用ＳＤＳ－凝胶电泳测得纯化后的β－甘露聚糖酶分子量为２６ｋＤ,用凝胶聚焦电泳测得等电点Ｐ１为５．０。酶反应的最适ｐＨ为９．０,最适温度为６０℃,稳     

系统感染TMV的番茄叶胞外β-1,3-葡聚糖酶

系统感染ＴＭＶ （ｔｏｂａｃｃｏ ｍ ｏｓａｉｃ ｖｉｒｕｓ）的番茄（Ｌｙｃｏｐｅｒｓｉｃｏｎ ｅｓｃｕｌｅｎｔｕｍ Ｍｉｌｌ．）叶胞外蛋白提取液经冰冻干燥浓缩、－ ２０℃丙酮沉淀、ＣＭ－Ｓｅｐｈａｄｅｘ Ｃ－２５离子交换层析、ＤＥＡＥ－Ｓｅｐｈａｄｅｘ Ａ－２５离子交换层析和Ｓｅｐｈａｄｅｘ Ｇ－７５凝胶层析纯化,获得ＰＡＧＥ均一的β－１,３－葡聚糖酶．ＳＤＳ－ＰＡＧＥ证明,它包含分子量为３６ ｋＤ 和２７ ｋＤ的两个同工酶．以昆布多糖为底物,酶的最适ｐＨ 在４．８—５．２之间,在ｐＨ ４—８稳定;酶的最适温度在３０—４０℃之间,在４０℃保温１ｈ 后酶活性不变;Ｋｍ 值为９．２ ｍ ｇ／ｍ Ｌ．在系统感染ＴＭＶ 的番茄叶胞外蛋白提取液中,有分子量为２２ ｋＤ、２７ ｋＤ和３６ ｋＤ的３个β－１,３－葡聚糖酶同工酶     

单宁酶的固定化及其在酯型儿茶素水解反应中的应用

采用自制的壳聚糖为载体对单宁酶（ＴＡ）固定化,ＴＡ与壳聚糖配比１：２．５,３０℃固定２ｈ,活力回收达２３．６％～３３．１％;偶联效率为８４．９％～８８．０％。固定化单宁酶（ＩＴＡ）的表观Ｋｍ值（以没食子酸丙酯为底物）为２２．２×１０－６ｍｏｌ／Ｌ,ＴＡ的Ｋｍ值（以没食子酸丙酯为底物）为１０×１０－６ｍｏｌ／Ｌ,ＴＡ和ＩＴＡ的最适反应温度分别为４０℃和５０℃;６０℃处理１５ｍｉｎ,残存活性分别为１３．６％和６０．３％。ＴＡ和ＩＴＡ的最适ｐＨ值分别为５．８和６．４;ＴＡ在ｐＨ４．８～７．８活力稳定,而ＩＴＡ活力稳定范围在ｐＨ４．８～６．８．ＩＴＡ作用于ＥＧＣＧ的半衰期为７８．７ｈ,ＥＧＣＧ水解率达９０．３％。对茶多酚提取物进行水解,其所含的酯型儿茶素ＥＧＣＧ和ＥＣＧ水解率分别为９６．４％和９６．８％,非酯型儿茶素ＥＧＣ和ＥＣ的含量显著增加。     

湖南尖吻蝮蛇毒两个出血毒素的纯化和理化性质

经ＳｅｐｈａｄｅｘＧ－７５凝胶过滤,ＱＡＥ－ＳｅｐｈａｄｅｘＡ－５０和ＣＭ－ＳｅｐｈａｄｅｘＣ－２５离子交换层析的步骤,从湖南产尖吻蝮（Ｄｉｅｎａｇｋｉｓｔｒｏｄｏｎａｃｕｔｕｓ）蛇毒中纯化出两个出血毒素（ＤａＨＴ－１和ＤａＨＴ－２）．ＳＤＳ－ＰＡＧＥ测得分子量均为２３．５ｋＤ,ＩＥＦ－ＰＡＧＥ测得等电点分别为５．６和５．２,两者具有相似的氨基酸组成,其中酸性氨基酸（Ａｓｘ,Ｇｌｘ）分别占２３％和２４％,ＤａＨＴ－１和ＤａＨＴ－２的最小出血剂量（ＭＨＤ）分别为０．５μｇ和０．８μｇ。都具蛋白水解酶活性,无对ＴＡＭＥ,ＢＡＥＥ的水解活性和ＰＬＡ２酶活性．两者的蛋白水解酶活力与出血活性并非正相关．ＤａＨＴ－１和ＤａＨＴ－２的最适温度分别为３５℃和４０℃,最适ｐＨ为６－９,对热均不稳定,温度高于６０℃活性完全丧失。金属离子的分析显示每摩尔毒素蛋白约含０．５ｍｏｌ的Ｚｎ,１ｍｏｌ的Ｃａ,较多的Ｎａ、Ｋ、Ｍｇ,不含Ｃｏ。     

耐热碱性磷酸酯酶的功能结构域的定位

 为了确定耐热碱性磷酸酯酶 (TAPND2 7)发挥活性所必需的功能结构域 ,通过 PCR介导的诱变缺失 ,得到了 N端分别缺失 8、1 6、2 5个氨基酸的 3个缺失体 p TAPN8、p TAPN1 6和p TAPN2 5以及 C端分别缺失 1 0和 30个氨基酸的两个缺失体 p TAPC1 0和 p TAPC30 .经表达和活性测定 ,发现 p TAPN8和 TAPC1 0保持了较高的活性而其余 3个缺失体则失去酶活性 .据此 ,TAPND2 7的活性区域被定位在 8～ 465氨基酸之间 .在分离纯化的基础上测定了一些酶学性质 .发现 TAPN8和 TAPC1 0的比活没有大的改变 ,Tm 下降了 5.5℃ ;TAPN8的最适反应温度上升了1 0℃ .结果提示了 N端和 C端的这些氨基酸残基对热稳定性有一定的贡献 ,N端氨基酸残基还对酶的亲热性有贡献 .     

An additional aromatic interaction improves the thermostability and thermophilicity of a mesophilic family 11 xylanase: structural basis and molecular study

In a general approach to the understanding of protein adaptation to high temperature, molecular models of the closely related mesophilic Streptomyces sp. S38 Xyl1 and thermophilic Thermomonospora fusca TfxA family 11 xylanases were built and compared with the three-dimensional (3D) structures of homologous enzymes. Some of the structural features identified as potential contributors to the higher thermostability of TfxA were introduced in Xyl1 by site-directed mutagenesis in an attempt to improve its thermostability and thermophilicity. A new Y11-Y16 aromatic interaction, similar to that present in TfxA and created in Xyl1 by the T11Y mutation, improved both the thermophilicity and thermostability. Indeed, the optimum activity temperature (70 vs. 60 degrees C) and the apparent Tm were increased by about 9 degrees C, and the mutant was sixfold more stable at 57 degrees C. The combined mutations A82R/F168H/N169D/delta170 potentially creating a R82-D169 salt bridge homologous to that present in TfxA improved the thermostability but not the thermophilicity. Mutations R82/D170 and S33P seemed to be slightly destabilizing and devoid of influence on the optimal activity temperature of Xyl1. Structural analysis revealed that residues Y11 and Y16 were located on beta-strands B1 and B2, respectively. This interaction should increase the stability of the N-terminal part of Xyl1. Moreover, Y11 and Y16 seem to form an aromatic continuum with five other residues forming putative subsites involved in the binding of xylan (+3, +2, +1, -1, -2). Y11 and Y16 might represent two additional binding subsites (-3, -4) and the T11Y mutation could thus improve substrate binding to the enzyme at higher temperature and thus the thermophilicity of Xyl1.     

Rational design of thermostability in bacterial 1,3-1,4-β-glucanases through spatial compartmentalization of mutational hotspots

Higher thermostability is required for 1,3-1,4-β-glucanase to maintain high activity under harsh conditions in the brewing and animal feed industries. In this study, a comprehensive and comparative analysis of thermostability in bacterial β-glucanases was conducted through a method named spatial compartmentalization of mutational hotspots (SCMH), which combined alignment of homologous protein sequences, spatial compartmentalization, and molecular dynamic (MD) simulation. The overall/local flexibility of six homologous β-glucanases was calculated by MD simulation and linearly fitted with enzyme optimal enzymatic temperatures. The calcium region was predicted to be the crucial region for thermostability of bacterial 1,3-1,4-β-glucanases, and optimization of four residue sites in this region by iterative saturation mutagenesis greatly increased the thermostability of a mesophilic β-glucanase (BglT) from Bacillus terquilensis. The E46P/S43E/H205P/S40E mutant showed a 20 °C increase in optimal enzymatic temperature and a 13.8 °C rise in protein melting temperature (T _m) compared to wild-type BglT. Its half-life values at 60 and 70 °C were 3.86-fold and 7.13-fold higher than those of wild-type BglT. The specific activity of E46P/S43E/H205P/S40E mutant was increased by 64.4 %, while its stability under acidic environment was improved. The rational design strategy used in this study might be applied to improve the thermostability of other industrial enzymes.

     

The structure of Rhodothermus marinus Cel12A,a highly thermostable family 12 endoglucanase,at 1.8 A resolution

Cellulose is one of the most abundant polysaccharides in nature and microorganisms have developed a comprehensive system for enzymatic breakdown of this ubiquitous carbon source, a subject of much interest in the biotechnology industry. Rhodothermus marinus produces a hyperthermostable cellulase, with a temperature optimum of more than 90 degrees C, the structure of which is presented here to 1.8 A resolution. The enzyme has been classified into glycoside hydrolase family 12; this is the first structure of a thermophilic member of this family to have been solved. The beta-jelly roll fold observed has identical topology to those of the two mesophilic members of the family whose structures have been elucidated previously. A Hepes buffer molecule bound in the active site may have triggered a conformational change to an active configuration as the two catalytic residues Glu124 and Glu207, together with dependent residues, are observed in a conformation similar to that seen in the structure of Streptomyces lividans CelB2 complexed with an inhibitor. The structural similarity between this cellulase and the mesophilic enzymes serves to highlight features that may be responsible for its thermostability, chiefly an increase in ion pair number and the considerable stabilisation of a mobile region seen in S. lividans CelB2. Additional aromatic residues in the active site region may also contribute to the difference in thermophilicity.     

Thermostable beta-galactosidase from the archaebacterium Sulfolobus solfataricus. Purification and properties

A thermophilic and thermostable beta-galactosidase activity was purified to homogeneity from crude extracts of the archaebacterium Sulfolobus solfataricus, by a procedure including ion-exchange and affinity chromatography. The homogeneous enzyme had a specific activity of 116.4 units/mg at 75 degrees C with o-nitrophenyl beta-galactopyranoside as substrate. Molecular mass studies demonstrated that the S. solfataricus beta-galactosidase was a tetramer of 240 +/- 8 kDa composed of similar or identical subunits. Comparison of the amino acid composition of beta-galactosidase from S. solfataricus with that from Escherichia coli revealed a lower cysteine content and a lower Arg/Lys ratio in the thermophilic enzyme. A rabbit serum, raised against the homogeneous enzyme did not cross-react with beta-galactosidase from E. coli. The enzyme, characterized for its reaction requirements and kinetic properties, showed a thermostability and thermophilicity notably greater than those reported for beta-galactosidases from other mesophilic and thermophilic sources.     

Three-dimensional structure of an alkaline xylanase Xyn11A-LC from alkalophilic Bacillus sp. SN5 and improvement of its thermal performance by introducing arginines substitutions

The alkaline xylanase Xyn11A-LC from the alkalophilic Bacillus sp. SN5 was expressed in E. coli, purified and crystallized. The crystal structure was determined at a resolution of 1.49 Å. Xyn11A-LC has the β-jelly roll structure typical of family 11 xylanases. To improve its thermostability and thermophilicity, a mutant SB3 was constructed by introducing three arginines on the different sides of the protein surface. SB3 increased the optimum temperature by 5 °C. The wild type and SB3 had the half-lives of 22 and 68 min at 65 °C at pH 8.0 (Tris/HCl buffer), respectively. CD spectroscopy revealed that the melting temperature (T _m) of the wild type and SB3 were 55.3 and 66.9 °C, respectively. These results showed that the introduction of arginines enhance the thermophilicity and thermostability of Xyn11A-LC.     

N-酰基高丝氨酸内酯酶的分子改造及酶学特性

革兰氏阴性细菌的群体感应系统利用N-酰基高丝氨酸内酯（N-acyl-homoserine lactone, AHL）作为主要信号分子诱导致病因子表达,造成细菌性病害. N-酰基高丝氨酸内酯酶（N-acyl-homoserine lactonase, AHLase）能水解AHL分子的内酯键,减弱致病菌的危害.本研究利用从苏云金芽孢杆菌克隆的N-酰基高丝氨酸内酯酶基因（auto inducer inactivation A, aiiA）,根据Swiss-model模拟aiiA所编码的AiiA蛋白三维结构,预测可能形成的分子内盐桥、活性中心位点等,利用环状诱变方法对AiiA进行定点突变,以期提高其酶活力和热稳定性等酶学性能.对AiiA及其突变蛋白酶学特性分析结果发现,突变体AiiA-N65K-A206E酶活力要比野生型AiiA-wild提高87.4%,并表现出良好的热稳定性和储存稳定性;37 ℃温浴30 min后酶活力剩余73;9%,比AiiA-wild有了大幅提高;4 ℃储存120 h后酶活力剩余12.9%,而AiiA-wild丧失酶活力.酶动力学分析表明,AiiA-N65K-A206E酶促反应的米氏常数Km为1.23 mmol/L,与野生型相当;最大反应速率Vmax为32.36 μmol/L/min,比野生型有较大提高.本研究表明,利用定点突变技术改造AiiA的分子结构,可有效提升AiiA酶活力、热稳定性和储存稳定性.本研究结果为进一步阐明AiiA结构与功能的关系,促进AiiA在植物病害生物防治上的应用,提供了有益的参考和新的思路.     

Functional analysis of active site residues of Bacillus thuringiensis WB7 chitinase by site-directed mutagenesis

To investigate the roles of the active site residues in the catalysis of Bacillus thuringiensis WB7 chitinase, twelve mutants, F201L, F201Y, G203A, G203D, D205E, D205N, D207E, D207N, W208C, W208R, E209D and E209Q were constructed by site-directed mutagenesis. The results showed that the mutants F201L, G203D, D205N, D207E, D207N, W208C and E209D were devoid of activity, and the loss of the enzymatic activities for F201Y, G203A, D205E, W208R and E209Q were 72, 70, 48, 31 and 29%, respectively. The pH-activity profiles indicated that the optimum pH for the mutants as well as for the wildtype enzyme was 8.0. E209Q exhibited a broader active pH range while D205E, G203A and F201Y resulted in a narrower active pH range. The pH range of activity reduced 1 unit for D205E, and 2 units for G203A and F201Y. The temperature-activity profiles showed that the optimum temperature for other mutants as well as wildtype enzyme was 60°C, but 50°C for G203A, which suggested that G203A resulted in a reduction of thermostability. The study indicated that the six active site residues involving in mutagenesis played an important part in WB7 chitinase. In addition, the catalytic mechanisms of the six active site residues in WB7 chitinase were discussed.     

Crystal Structure of the Hyperthermophilic Inorganic Pyrophosphatase from the Archaeon Pyrococcus horikoshii

A homolog to the eubacteria inorganic pyrophosphatase (PPase, EC 3.6.1.1) was found in the genome of the hyperthermophilic archaeon Pyrococcus horikoshii. This inorganic pyrophosphatase (Pho-PPase) grows optimally at 88°C. To understand the structural basis for the thermostability of Pho-PPase, we have determined the crystal structure to 2.66 Å resolution. The crystallographic asymmetric unit contains three monomers related by approximate threefold symmetry, and a hexamer is built up by twofold crystallographic symmetry. The main-chain fold of Pho-PPase is almost identical to that of the known crystal structure of the model from Sulfolobus acidocaldarius. A detailed comparison of the crystal structure of Pho-PPase with related structures from S. acidocaldarius, Thermus thermophilus, and Escherichia coli shows significant differences that may account for the difference in their thermostabilities. A reduction in thermolabile residues, additional aromatic residues, and more intimate association between subunits all contribute to the larger thermophilicity of Pho-PPase. In particular, deletions in two loops surrounding the active site help to stabilize its conformation, while ion-pair networks unique to Pho-PPase are located in the active site and near the C-terminus. The identification of structural features that make PPases more adaptable to extreme temperature should prove helpful for future biotechnology applications.