【作者】 李坤；

【导师】 李江华；

【作者基本信息】 江南大学 ， 发酵工程， 2014， 硕士

【摘要】 聚乙烯醇(Poly (vinyl) alcohol，简称PVA)，是目前已发现的唯一可溶于水且无毒的高聚物，具有很多优良的物理性质，如高粘度、成膜性、乳化性、拉伸性、对水、油脂和有机溶剂均有抵抗性等。基于以上特性，PVA广泛应用于粘合剂、涂料、乳化剂、造纸和纺织等行业。目前许多工业废水中含有大量的PVA，由于PVA的可降解性很差，会造成极大的环境污染。因此，研究PVA降解菌及PVA降解酶对于减轻环境污染将具有重大的意义。本论文从筛选PVA降解菌出发，详细研究了一个混菌体系SH-TD的PVA降解性质并通过响应面优化大大提高了其发酵产PVA降解酶的能力。设计了一个功能筛选基因文库的策略，尝试从中筛选出PVA氧化酶(Secondary alcohol oxidase, SAO)阳性克隆子进而获得SAO酶核酸序列，主要研究结果及内容呈现如下：1、利用以PVA为唯一碳源培养基从纺织印染厂土样及活性污泥中富集筛选到一个能在4天内完全降解PVA唯一碳源培养基中1g·L-1PVA的混菌体系SH-TD，从中纯化出3株纯培养的PVA降解菌，经鉴定分别为SH1(Aeromonas sp.)、TD5(Pseudomonassp.)以及TD33(Acinetobacter sp.)。其中TD5和TD33分别可以单独在220h和160h内完全降解发酵培养基中1g·L-1的PVA1799。然而SH1据推测只能利用小分子量的PVA寡聚物。通过测定酶活力，只在SH-TD的发酵液上清中检测到了SAO酶酶活。2、通过单因素实验对可能影响SH-TD对PVA降解效率的生长因子、氮源以及金属离子进行探索，确定了一个更适合SH-TD降解产酶的发酵优化起始培养基，其组成为：PVA、酵母粉、(NH4)2HPO4、KH2PO4、K2HPO4、MgSO4·7H2O、FeSO4·7H2O、CaCl2·2H2O、NaCl、Phe、Trp、Thr。选择了基础培养基中的8个主要因素进行PB实验，根据显著性顺序，得到三个影响显著的因素分别为酵母粉、(NH4)2HPO4、CaCl2·2H2O。通过爬坡实验和BB实验分析确定了当酵母粉、(NH4)2HPO4和CaCl2·2H2O的浓度分别为0.04、48.42、0.06g·L-1时，24h内SH-TD对PVA的降解效率由优化前的30%提高到78.76%。3、运用宏基因组技术，构建了一个包含SH-TD中全部基因组的Fosmid文库。通过功能筛选，从大约150000个Fosmid文库克隆中筛选得到FTD1-FTD42等42个可在筛选平板上形成透明圈的阳性克隆。其中FTD2SAO酶活力最大为57mU·mL-1。以三个阳性Fosmid克隆质粒为基因来源构建亚克隆文库。更多还原

【Abstract】 Poly (vinyl alcohol)(PVA), the only water soluble and non-toxic composite material, haslots of excellent physical properties such as high viscosity, film property, emulsifying propertyand resistance to water, grease and organic solvent, etc. Based on these characteristics, PVA iswidely applied to many industries like adhesive, coating, emulsifier, paper and textile etc.Nowadays, PVA pollution problem was more and more serious because of its deficiency inbiodegradation. Therefore, it is of great significance to carry out deep research on PVAdegrading bacteria and PVA degrading enzymes for reducing environmental pollution.In this work, a mixed PVA degradation culture SH-TD was isolated and the biodegradationcharacteristics of PVA by SH-TD were investigated. The production of PVA degrading enzymehas been significantly improved through the Response Surface Methodology (RSM) ofoptimization of culture medium. A function-based screening strategy was designed to screenthe gene of SAO from Fosmid and sub-cloning libraries. The main results are listed as follows:1. A PVA-degrading mixed culture was obtained from the activated sludge sample oftextile finishing operations. When0.1%PVA was used as a sole carbon source in culturemedium, it could be completely degraded in only4days. Three strains with PVA-degradingability were isolated from the mixed culture: SH1(Aeromonas sp.), TD5(Pseudomonas sp.),and TD33(Acinetobacter sp.). Over99%of PVA, at an initial concentration of0.1%, wasdegraded within220and160hours cultivation with the resting cells of TD5and TD33alone,respectively. However, SH1could only utilize low molecular weight PVA. The enzyme activityof SAO was only detectedin the fermentation supernatant of SH-TD.2. The initial fermentation culture medium consisting of PVA, Yeast Extract,(NH4)2HPO4,KH2PO4, K2HPO4, MgSO4·7H2O, FeSO4·7H2O, CaCl2·2H2O, NaCl, Phe, Trp and Thr, wasdetermined based on the single factor experiments about growth factors, nitrogen source andmetal ions. On the basis of Plackett-Burman experiments, three constituents from the startingculture medium were found to have significant influence on PVA degrading rate which wereYeast Extract,(NH4)2HPO4, BaCl2. Through ascent and Box-Behnken experiments, a RSMmodel was constructed. Data was analyzed using Design-Expert software. The optimalconcentrations (g·L-1) of the variables were determined as: Yeast Extract0.04g·L-1,(NH4)2HPO448.42g·L-1, BaCl20.06g·L-1. Under the optimal conditions, the PVA degradationrate after24h increased from30%to78.76%.3. By using metagenomic technology, a Fosmid library containing the whole genes of SH-TD was constructed. Among about150,000clones,42clones expressing the SAO activity wasobtained which were named FTD1-FTD42. They can form clear halos on the PVA-OPHscreening plate. The SAO enzyme activity of FTD2reached57mU mL-1. Several sub-clonelibraries whose genetic source was from the positive FTD strains were constructed and screenedas described.更多还原