【作者】 黄文；

【导师】 石碧；

【作者基本信息】 四川大学 ， 皮革化学与工程， 2002， 博士

【摘要】 本论文以植物单宁资源的精细化利用为出发点，研究了单宁生物降解的方法及其降解机理，其目的是通过生物降解，获得化学方法较难得到的单宁降解产物，开拓单宁的利用途径，提高单宁利用的附加值。与此同时，本论文对菌株降解性能、营养条件控制机制、代谢过程及其降解酶进行了深入研究，建立了一套适用于研究单宁生物降解的有效方法，其结果不仅对丰富天然产物化学及利用方法具有重要的理论意义，而且为利用生物技术处理单宁物质及大分子酚类物质提供了一条新颖、有价值的途径。 本论文的主要研究内容：分离筛选出了能有效降解橡椀单宁的三株菌株，并对其进行了鉴定，证明其分别是黑曲霉(Aspergillus niger)、内胞霉(Endomyces)和假丝酵母(Candida utilis)。 以没食子酸丙酯(PG)为模型化合物，研究各菌株对单宁酯键的降解性能及影响降解酶作用的因素，建立了酶解催化反应动力学方程。发现黑曲霉具有较高的单宁酶活性，内胞霉和假丝酵母单宁酶活相对较低。碳源葡萄糖对黑曲霉的单宁酯键降解活性有抑制作用，而对内胞霉和假丝酵母的活性有激活作用。 以邻苯二酚为底物时，发现黑曲霉不表现出多酚氧化酶活性，而内胞霉和假丝酵母具有多酚氧化酶活性。通过PPO底物专一性的研究，证实了PPO对间苯三酚不表现出活力，对邻苯二酚及邻苯三酚具有氧化作用。 在此基础上，通过对微生物间协同作用和菌间关系的分析，建立了黑曲霉和假丝酵母组合的最佳微生物共培养降解体系，即首先接入黑曲霉降解分子中的酯键，三天后接入具有单宁酶活性和多酚氧化酶活性的假丝酵母，在促进降解的同时，消除橡惋单宁降解产物中葡萄糖对单宁降解的阻碍作用，有效提高了赣花酸产率。 通过混合菌降解橡榆单宁产生躁花酸最优条件的研究，葱花酸产率由单一菌株培养降解的门二3％，提高到 28尸％。由于橡杭单宁组分中踱花酚基与分子主体部分的连接除了酯键外，还有碳－碳键和碳－贰键的结合，实验结果证实了橡杭单宁的降解既存在能使单宁酯键水解的单宁酶的催化作用，也有能使单宁分子的碳．碳键和碳－贰键断裂的酶在起作用，多酚氧化酶可能是发挥后一种作用的主要因素。 所建立的方法不仅可用于由橡婉单宁制备具有重要应用价值的精细化学品中间体躁花酸，而且对于利用生物降解法从含躁花单宁的植物中制备躁花酸提供了有意义的借鉴作用。更多还原

【Abstract】 Three strains, which could effectively degrade valonia tannins, were isolated and selected, and identified as Aspergillus niger, Endomyces and Candida utilis. Then, degradation ability of the strains on valonia tannins and factors influencing the biodegradation were comprehensively investigated.It was found that degradation rate of valonia tannin and yield of ellagic acid was limited when single-strain cultivation was employed. Using model compound, Propyl gallate (PG), the reasons for the lower biodegradation and the factors influencing activity of the degrading enzymes were evaluated.Furthermore, it was found that Aspergillus niger have higher activity of tannase than Endomyces and Candida utilis when using PG as substrate. And it was determined that hydrolysis function of Aspergillus niger on ester bond of tannin could be remarkably inhibited by additional carbon sources, glucose in particular, whilst that of Endomyces and Candida utilis was activatedIn addition, it was verified that no polyphenol oxidase (PPO) from Aspergillus niger could be determined, but activity of PPO derived from Endomyces and Candida utilis was obviously observed by using catechol as substrates. Meantime, it was approved that PPO could oxide pyrogallol and catechol, but not phloroglucinol.Based on the researches, a biodegradation system of valonia tannin byco-cultivation was established. The optimum combination and condition of co-cultivation were also obtained by the orthogonal experiment. It was showed that yield of ellagic acid would be remarkably increased when co-cultivation of strains Aspergillus niger and Candida utilis were employed. High ellagic acid yield (28.7%) was achieved in inorganic salt culture medium (pH 4.5) containing 25 g/L valonia tannin, 20g/L sucrose and 2g/L sodium nitrate, incubated for 8d at 28C in a rotary shaker (140r/min). The results showed that at least two kinds of enzymes, tannase and PPO, were involved in degradation of tannins.The approach established in this thesis and the data obtained in experiments are of theoretical importance and practical significance in both finer and value-added utilization of vegetable tannins and removal of tannin pollution in industries.更多还原