【作者】 付大伟；

【导师】 姚斌；

【作者基本信息】 中国农业科学院 ， 生物化学与分子生物学， 2010， 博士

【摘要】 植酸是豆类、谷类等作物种子中磷的主要储存形式。植酸酶(肌醇六磷酸磷酸水解酶)能够水解植酸的磷酸单酯键生成无机磷和磷酸肌醇衍生物。单胃动物饲料中添加微生物来源植酸酶能够有效地提高植酸磷的利用效率、降低动物排泄物中的磷污染，并能通过去除植酸的抗营养作用来提高饲料的营养价值。对具有高比活、良好热稳定性和酸稳定性植酸酶的需求，促进了新酶资源的开发以及酶的催化机制研究和蛋白质工程研究。HAP植酸酶(EC 3.1.3.8)由于其优良的催化性质而被广泛应用于动物饲料添加剂。Yersinia spp.是优良HAP植酸酶的主要来源，本实验室先后从Yersinia属11个种的基因组中克隆得到了10个HAP植酸酶基因，其中Yersinia intermedia、Y. rohdei来源的植酸酶具有良好的综合性质和潜在的应用价值。为了深入研究Yersinia spp.来源的植酸酶，本研究分别对Y. kristeensenii, Y.frederiksenii和Y. enterocolitica来源的三个植酸酶基因Ykappa, Yfappa和Yeappa进行了异源表达和性质研究，并通过定点突变对其相应的催化机制进行了研究。来源于Y. kristeensenii的植酸酶基因Ykappa在毕赤酵母中得到了活性表达。纯化的重组植酸酶YkAppa比活为2,656 U/mg (pH 4.5和37℃)，在pH 4.5和55℃条件下酶活最高，并在pH 2.0–6.5均有植酸酶活性。YkAppa具有优良的pH稳定性，在pH 1.5–11.0，37℃条件下处理3 h后仍保留90%以上的酶活。YkAppa也具有良好的热稳定性，在80℃处理10 min后还剩余初始酶活的46%。在体外水解豆粕实验中，YkAppa表现出高效的植酸降解能力。与其他的植酸酶相比，YkAppa具有优良的综合性状，如比活高、pH稳定性和热稳定性好、豆粕中植酸降解率高等，从而具有一定的商业化潜力。在大肠杆菌中表达的重组植酸酶YfAppa最适pH为2.5—显著低于其他微生物来源植酸酶(最适pH 4.5–6.0)。YfAppa的氨基酸序列与Y. intermedia来源的HAP植酸酶(最适pH为4.5)一致性高达84%。通过对YfAppa与相关植酸酶进行氨基酸序列比对和结构建模，发现在YfAppa催化中心附近只有一个差异氨基酸。为了研究YfAppa的适酸性，本文通过定点突变构建了五个突变体(S51A，S51T，S51D，S51K和S51I)，并对其在大肠杆菌中进行了表达、纯化和性质分析。Ser、Thr和Ile均为不带电荷的氨基酸，而相应的野生型和突变型植酸酶在最适pH方面表现出非常大的差异。突变体S51T和S51I的最适pH从2.5分别移到了4.5和5.0，确定了Ser51在YfAppapH特性中起关键作用。本研究发现了一个除静电荷之外的新的影响植酸酶pH特性的因子—活性中心附近氨基酸残基的侧链结构—对YfAppa的最适pH具有重要的影响。与野生型YfAppa相比，突变体S51T具有高比活、酸性范围内(pH 2.0–5.5)的高活性以及提高的热稳定性和酸稳定性等性质，从而具有潜在的商业价值。YeAppa在pH 5.0和45℃条件下酶活最高，其在生理温度条件下(pH 5.0和37℃)的比活为3.28 U/mg—为它同源植酸酶YkAppa (88%氨基酸序列一致性)比活的1/800。序列和结构比对分析显示YeAppa中的第79位氨基酸Arg79为非保守氨基酸，在YkAppa和其他HAP植酸酶中该氨基酸为Gly。YeAppa的R79G突变使其比活增加到2,615 U/mg，证明了Arg79为YeAppa低比活的原因。为了研究不同氨基酸残基在该位点对HAP植酸酶的影响，本文选择了晶体结构研究、性质研究很透彻的大肠杆菌植酸酶EcAppa为材料，将其相应位点的Gly73分别突变为Arg，Asp，Glu，Ser，Thr，Leu或Tyr。所有突变体的比活均低于野生型，并且随着氨基酸残基侧链体积的增大比活呈下降趋势,从而确定了一个新的影响HAP植酸酶比活的因素，即氨基酸残基侧链的体积。综上所述，Yersinia spp.来源的植酸酶在氨基酸序列上具有高度的一致性而在性质方面存在显著的差异，为植酸酶的结构与功能研究提供了优良材料。本文获得了一个具有优良综合性状并具有潜在商业价值的植酸酶YkAppa，确定了两个影响HAP植酸酶pH特性和催化效率的因子。因此，本研究具有重要的理论意义和实际应用价值。更多还原

【Abstract】 Phytate is the primary storage form of phosphorus in cereals and legumes. Phytases (myo-inositolhexakisphosphate phosphohydrolases) hydrolyze the phosphoester bonds of phytate to yield inorganicphosphate and less-substituted inositol. Monogastric animal feed supplemented with microbial phytaseseffectively improves phytate phosphorus utilization, reduces the excretion of phosphorus in animalmanure and improves the nutrient value by removing the anti-nutrient factor of phytate in intensivelivestock production. The industrial demand for phytase with high specific activity, excellentthermostability and acid stability continues to stimulate the search for new enzyme sources and thestudies on the catalytic mechanisms and protein-engineering of phytases.HAP phytases (EC 3.1.3.8) are a class of phytases with excellent catalysis capacity and have beenwidely used in animal feed. Yersinia spp. are excellent microbial sources of HAP phytases，and ten HAPphytase genes have been cloned from 11 species genomes of the genus Yersinia. Among them, HAPphytases from Yersinia intermedia and Y. rohdei had excellent comprehensive properties and applicationpotential. In this study, we heterologously expressed and characterized the genes Ykappa, Yfappa andYeappa from Yersinia kristeensenii, Y. frederiksenii and Y. enterocolitica, respectively. Furthermore, thestudies on catalytic mechanisms through site-directed mutagenesis were conducted.The gene Ykappa from Y. kristeensenii was expressed in Pichia pastoris. The purified recombinantphytase, YkAppa, had optimal activity at 55℃and pH 4.5, exhibited enzymatic activity between pH 2.0and 6.5, with a specific activity of 2,656 U/mg at pH 4.5 and 37℃. YkAppa was highly pH stable,retaining more than 90% of its initial activity after pre-incubation under varying pH conditions (pH1.5–11.0) at 37℃for 3 h. YkAppa was thermostable, and retained 46% of its initial activity afterincubation at 80℃for 10 min. YkAppa also showed efficiency in hydrolysis of phytate phosphorusfrom soybean meal in vitro. Compared with other well-known phytases, YkAppa has excellentcomprehensive properties, such as high specific activity, good pH stability and thermostability, highdegradation efficacy of soybean meal phytate and so on, and has significant potential in feed industryuse.The purified YfAppa, heterologously expressed in Escherichia coli, had optimal activity at pH2.5—substantially lower than that of most of microbial phytases (pH optima 4.5–6.0). The amino acidsequence of YfAppa has the highest identity (84%) to that of Y. intermedia HAP phytase (optimal pH4.5). Based on sequence alignment and molecular modeling of YfAppa and related phytases, only onedivergent residue in YfAppa, Ser51, was identified to be in close proximity to the catalytic site. Tounderstand the acidic adaptation of YfAppa, five mutants (S51A, S51T, S51D, S51K and S51I) wereconstructed by site-directed mutagenesis, expressed in E. coli, purified, and characterized. Ser, Thr andIle are all uncharged amino acids, but replacing Ser with Thr and Ile changed the pH optima a lot.Mutants S51T and S51I exhibited a shift in the optimal pH from 2.5 to 4.5 and 5.0, respectively,confirming the role of Ser51 in defining the optimal pH. Thus, a previously unrecognized factor other than electrostatics—presumably the side-chain structure near the active site—contributes to the optimalpH for YfAppa activity. Compared with wild-type YfAppa, mutant S51T showed higher specificactivity, greater activity over pH 2.0–5.5, and increased thermal and acid stability. These propertiesmake mutant S51T a better candidate than the wild-type YfAppa for use in animal feed.The maximum activity of YeAppa occurs at pH 5.0 and 45℃, and notably its specific activityunder physiological conditions (3.28 U/mg, pH 5.0 and 37℃) is 800-fold less than that of its Y.kristeensenii homolog (YkAppa; 88% amino acid sequence identity). Sequence alignment andmolecular modeling showed that the arginine at position 79 (Arg79) in YeAppa corresponding to Gly inYkAppa as well as other HAP phytases is the only non-conserved residue near the catalytic site.Site-directed replacement of Arg79 with Gly increased the specific activity of YeAppa to 2,615 U/mg,indicating that Arg79 is primarily responsible for the decreased activity. To characterize the effects ofother residues at this position on the specific activities of phytases, E. coli EcAppa, a well-characterizedphytase with a known crystal structure, was selected for mutagenesis—its Gly73 was replaced with Arg,Asp, Glu, Ser, Thr, Leu, or Tyr. The specific activities of all of the corresponding EcAppa mutants wereless than that of the wild-type phytase, and the activity levels were approximately proportional to themolecular volumes of the substituted residues’side chains. Thus, a new determinant that influences thecatalytic efficiency of HAP phytases—the molecular volumes of the substituted residues’side chainshas been identified.In summary, phytases from Yersinia spp. are highly identical in amino acid sequences but varied inproperties, and are good materials for structure-function studies. This study obtained a phytase YkAppawith excellent comprehensive properties and important application potential, identified two factorscontributing to pH optima and catalytic efficiency of HAP phytases, and thus had great significance intheory and application.更多还原