【作者】 张蕊；

【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， 2011， 硕士

【摘要】 植酸酶（Phytase,EC 3.1.3.8或EC 3.1.3.26）是一类能够降解植酸并释放无机磷和肌醇的磷酯酶。近些年,植酸酶作为一种新型的饲料添加剂和无机磷的替代物,在猪、鸡等单胃动物饲养业的应用价值已得到充分体现,而在水产养殖方面,具有应用价值的中性植酸酶亟需开发。昆虫肠道作为一个微生物数量众多且复杂多样的特殊环境,目前几乎没有相关植酸酶的报道。本论文的研究目的旨在从中性的昆虫肠道环境中获得具有新颖性和应用潜力的植酸酶。本研究从云斑天牛肠道环境来源细菌中挑选具有种属差异的20株细菌,利用简并PCR和TAIL-PCR技术,从其中3株细菌中克隆到4个植酸酶编码基因,分别为Pseudomonas sp. TN06来源的phyA06、Serratia sp. TN49来源的phyH49和phyB49、Janthinobacterium sp. TN115来源的phyA115。其中,phyH49为组氨酸酸性磷酸酶（HAP）基因,phyA06、phyB49和phyA115为β-折叠桶状植酸酶（BPP）基因。通过氨基酸序列比对分析,四个基因与已发表的植酸酶序列最高序列一致性为47–64%,三个BPP基因相互之间的一致性为39–51%,说明这四个昆虫肠道来源的植酸酶基因均具有较高的序列新颖性。通过同源建模分析,三个BPP植酸酶均具有双结构域,与大多数完成酶学性质分析的单结构域BPP植酸酶不同。Serratia sp. TN49是典型的肠杆菌科细菌,本研究首次从肠杆菌科中克隆得到BPP基因,并首次从单一菌株中克隆到多个植酸酶基因。进一步的系统进化树分析表明phyB49和与植物共生的Pseudomonas spp.来源的双结构域BPP植酸酶的亲缘关系最相近。四个植酸酶编码基因分别重组到pET-22b（+）载体,在大肠杆菌BL21（DE3）中表达。重组蛋白经Ni-NTA柱纯化达电泳纯后,进行了酶学性质分析。来源于Pseudomonas sp. TN06的PhyA06最适Ca²⁺浓度是2 mM,最适pH值为7.0,最适温度是55°C,并在30°C保持50%左右的活性。PhyA06具有较好的热稳定性,在65°C处理1 h后,还保留70%以上的相对酶活。PhyA06对胰蛋白酶的降解有抗性,胰蛋白酶处理1 h,酶活保持不变。该酶的性质特点符合水产养殖业对植酸酶的需求,具有潜在的应用价值。来源于Serratia sp. TN49的重组植酸酶PhyH49和PhyB49最适pH值分别为5.0和7.5,最适温度分别为60°C和45°C,在30°C时相对酶活力为30%和60%左右。PhyB49最适Ca²⁺浓度是1 mM。实验结果表明,同时具有两类植酸酶的Serratia sp. TN49在pH 2.0–9.0范围内都可以有效的利用植酸,增强了对不同生活环境的适应性。另外,从中性肠道微生物中分离得到中性植酸酶,也符合微生物对环境适应性和定向进化的趋势。来源于Janthinobacterium sp. TN115的PhyA115最适Ca²⁺浓度是1 mM,最适pH值为8.5,最适温度是45°C。PhyA115活性与Ca²⁺浓度、底物植酸浓度之间有密切的关系。在Ca²⁺浓度和植酸浓度一致时,PhyA115具有最高的酶活。在植酸浓度极低（0.1 mM）且无Ca²⁺时,PhyA115有较高酶活性,该现象至今尚未见报道。经结构和功能比较分析发现,不完整的N端结构域虽然没有降解植酸的能力,但可以改变PhyA115最适pH和pH作用范围。PhyA115是中性植酸酶基础理论研究及水产应用的良好材料。本研究首次对天牛肠道这个特殊生态环境中细菌来源的植酸酶进行了初步研究。结果表明该环境主要以双结构域BPP植酸酶为主。这些BPP植酸酶的序列和酶学性质具有较高的新颖性,为今后的基础研究和工业应用提供了良好的实验材料。更多还原

【Abstract】 Phytase （EC 3.1.3.8 or EC 3.1.3.26） is a group of enzymes that initiate the stepwise hydrolysis of phytic acid to generate inorganic orthophosphate, lower myo-inositol phosphoric esters, and free myo-inositol. Recently, dietary phytase, as a novel animal feed additive and as a replacement for inorganic phosphorus, has been proved to be efficient in the improvement of phytate-phosphorus utilization and bioavailability of minerals to swine and poultry. In aquaculture, neutral phytases with appropriate properties are being developed. Insect digestive tract harbors abundant and diverse microorganisms. To our knowledge, no phytase has been identified in these symbiotes. This study aims to obtain novel phytases with application potentials from the microbes of neutral insect digestive tract.Twenty bacterial stains of various taxa isolated from the gut of Batocera horsfieldi were selected as microbial sources. By using degenerate PCR and TAIL-PCR techniques, four phytase-encoding genes were cloned from three strains, including phyA06 from Pseudomonas sp. TN06, phyH49 and phyB49 from Serratia sp. TN49 and phyA115 from Janthinobacterium sp. TN115. Of them, phyH49 encodes a histidine acid phosphatase （HAP）, and phyA06, phyB49 and phyA115 encode threeβ-propeller phytases （BPPs）. Sequence homology analysis revealed that the deduced amino acid sequences of these four phytases had highest identities of 47–64% with known proteins in the public databases and 39–51% identities among three BPPs, suggesting that these phytase genes from insect gut have high sequence novelty. Homology modeling indicated that all three BPPs have dual-domain structures and are distinct from most single-domain well-characterized BPPs. Serratia is a typical genus of Enterobacteriaceae. It is the first report of a BPP gene in Enterobacteriaceae and of more than one type of phytase in a single strain. Further phylogenetic analysis revealed that deduced PhyB49 is closely related with the dual-domain phytases of Pseudomonas spp. accreted with plant. Four phytase-encoding genes were recombined with pET-22b（+） and transformed into Escherichia coli BL21 （DE3） for expression, respectively. The recombinant proteins were purified to electrophoretic homogeneity by Ni2+-NTA metal chelating affinity chromatography and further characterized.PhyA06 from Pseudomonas sp. TN06 exhibited maximal activity at pH 7.0 and 55°C in the presence of 2 mM Ca²⁺ and retained > 50% activity at 30°C. PhyA06 was thermostable, and retained 70% of the initial activity after incubation at 65°C for 1 h. PhyA06 was highly resistant to trypsin, retaining almost all of the activity after 1-h treatment. These properties make PhyA06 favorable for potential application in aquaculture.PhyH49 from Serratia sp. TN49 had pH and temperature optima of 5.0 and 60oC, respectively. PhyB49 showed optimal activity at pH 7.5 and 45oC in the presence of 1 mM Ca²⁺. PhyH49 and PhyB49 retained about 30 and 60% of the maximal activity at 30°C, respectively. The presence of two types of phytases makes Serratia sp. TN49 more efficient in phytate-phosphorus utilization over a board pH range （2.0–9.0） and more adaptive to various environments. Furthermore, symbiotes harboring neutralβ-propeller phytase correspond to their adaptation to environment changes and evolution trend. The optimal activity of PhyA115 from Janthinobacterium sp. TN115 was found to occur at pH 8.5 and 45°C in the presence of 1 mM Ca²⁺. PhyA115 activity was found to be related with concentrations of Ca²⁺ and substrate phytate. When the concentrations of Ca²⁺ and phytate were similar, PhyA115 exhibited the maximal phytase activity. When the phytate concentration was low （0.1 mM） and Ca²⁺ was absent, PhyA115 had high phytase activity. Structure-function analysis revealed that the N-terminal incomplete domain has no phytase activity but can influence the pH optimum of PhyA115. This is the first time to report such a phenomenon. PhyA115 is not only an important material for basic research ofβ-propeller phytase but also a potential feed additive in aquaculture.It is the first study to explore the phytases from bacteria harbored in the gut of B. horsfieldi larvae. In this special environment, dual-domain BPP phytases are predominant phytate-degraders. These BPP phytases share low identities with known proteins and have some novel enzyme properties, and may represent good materials for basic research and industrial applications in future.更多还原