青藏高原海晏湿地微生物几丁质低温降解酶资源的发掘和功能研究

Cold-adapted chitinases have a wide range of potential applications in biocontrol, medicine and environmental protection. A common way to obtain cold-adapted chitinases is to isolate chitinolytic bacteria from a cold environment and then obtain their chitinase or gene. Currently, 99% of environmental microorganisms are considered to nonculturable in the laboratory, which greatly limits the discovery of cold-adapted chitinases. Sophisticated metagenomic technologies have made it possible to obtain chitinase genes directly from the environments. Our previous work showed that microbes in the Qinghai-Tibetan Plateau wetlands can provide novel chitinases. This project intends to use deep metagenomic sequencing analysis to directly explore the chitinase gene resources in the microbial community from Haiyan wetland on the Qinghai-Tibetan Plateau, and to discover novel chitinase genes by amino acid sequence alignment and domain analysis; enzyme proteins will be expressed and purified by genetic engineering method; cold-adapted enzymes will be confirmed by low-temperature degradation experiments, their enzymatic properties will be clarified, and their abilities in antagonizing plant pathogenic fungi will be also assessed. In addition, the key chitinases will undertake X-ray crystallography study to clarify their structures, catalytic and low-temperature adaptation mechanisms from the atomic point of view. This project will not only enrich the scientific understanding on cold-adapted enzymes, but also guarantee the cold-adapted enzyme resource for the agricultural and industrial fields.

低温几丁质酶在生防、医药、环保等领域具有广阔的应用潜力。获得低温几丁质酶的常规方式是从低温环境中分离几丁质降解菌，再获得其几丁质酶或基因。目前99%环境微生物被认为是无法在实验室中培养的，因而极大的限制了低温几丁质酶资源的发现。日趋成熟的宏基因组学技术使得直接从环境中获得几丁质酶基因成为可能。我们前期工作显示青藏高原湿地微生物可以提供新型几丁质酶。本项目拟利用深度宏基因组学测序分析方法直接发掘青藏高原海晏湿地微生物群落中的几丁质酶基因资源，通过氨基酸序列比对和结构域分析，发现新型几丁酶基因；以基因工程手段表达、纯化酶蛋白，以低温降解实验确立低温几丁质酶，阐明其酶学性质，并初步评估其在拮抗植物病原真菌方面的应用潜能；同时对重点研究的几丁质酶进行X射线晶体学研究，从原子角度阐明其结构、催化及低温适应机制。该项目不仅可从理论上丰富人们对低温几丁质酶的科学认识，亦为低温几丁质酶的应用提供了资源保障

低温几丁质酶在生防、医药、环保等领域具有广阔的应用潜力。本项目利用深度宏基因组学测序分析方法直接发掘青藏高原海晏湿地微生物群落中的几丁质酶基因资源，通过氨基酸序列比对和结构域分析，共发现200多个潜在的新型几丁酶基因。通常一个几丁质酶具有一个催化域，而发现的几丁质酶P1724具有两个GH18催化结构域，结果表明P1724也具有新型的催化机制；基因P1724的全长序列，其N端GH18结构域[P1724(∆cGH18)]及其C端GH18结构域[P1724(∆nGH18)]，分别被克隆并在大肠杆菌BL21中表达。以胶体几丁质为底物，这些纯化的重组几丁质酶均在40°C，pH5.0-6.0和浓度为0-0.5M NaCl时显示出最大的水解活性，同样由于它们在4°C时仍具有活性，因此可以定义它们为冷适应酶；与P1724(∆nGH18)相比，P1724和P1724(∆cGH18)在温度和pH稳定性，NaCl耐受性和底物亲和力上具有更多相似性，这表明N端GH18结构域在P1724的生化特性方面贡献大于C端GH18结构域。P1724的kcat/Km值（催化效率）显著高于其N端和C端的GH18结构域的总和，这表明P1724的两个GH18域在降解几丁质上协同工作。项目还对新型的几丁质酶基因PcChi362、PcChi507和PcChi1295进行了进一步的研究；重组蛋白PcChi362表现出较高的几丁质酶活性；其最适反应温度为35℃，在4-40℃的范围内酶活比较稳定；最适反应pH为5，pH在4-10范围内酶活性保持稳定。由于通过宏基组获得的几丁质酶目前还没有表现出较好的病原真菌拮抗作用，这可能与酶蛋白活性易受环境影响有关；因此，我们增加了对可降解几丁质的微生物菌株的工作。经过分离、纯化、筛选，发现菌株CM134L-2T、CY25R-8T和ZN1L-CT均有较好的植物病原真菌的活性；其中菌株CM134L-2T的发酵液对赤霉病病原菌和西瓜枯萎病原菌有抑制作用，菌株CY25R-8T的对番茄早疫病病原菌的抑制作用，而菌株ZN1L-CT对黄瓜枯萎病病原菌的抑制作用。菌株CM134L-2T、CY25R-8T和ZN1L-CT不仅在植物生防方面有较好的应用前景，而经系统发育和比较基因组分析，它们分别代表了Pedobacter属、Variovorax属和Alcalige属的新种。