摘要
聚丁二酸丁二醇酯(PBS)于20世纪90年代进入材料研究领域,并迅速成为生物降解塑料研究热点。其合成原料来源既可以是石油资源,也可以通过生物资源发酵得到,PBS生物全降解塑料用途广泛,可用于包装、餐具、化妆品瓶及药品瓶、一次性医疗用品、农用薄膜、农药及化肥缓释材料、生物医用高分子材料等领域。与其它生物降解塑料相比,PBS在热性能、加工性能和性价比方面有独特的优势,因此引起科技和产业界高度关注。
本项工作的目的是从自然界中筛选出可降解PBS的菌株,并对降解菌株进行驯化、诱变,使其高效降解PBS,并分离纯化出PBS解聚酶,研究其酶学性质,为PBS的应用奠定基础。
本论文的主要结果如下:
1.由抚顺石油三厂排出的污泥中筛选出可降解PBS的四株霉菌,分别为XH0501、XH0502、XH0503、XH0504。对其中一种降解速率较快的真菌XH0501进行初步鉴定:XH0501属于真菌门、子囊菌纲、真子囊菌亚纲、曲霉目、曲霉科、杂色曲霉。
2.以XH0501为出发菌株,通过紫外线复合氯化锂诱变及微波诱变,采用摇瓶培养测定发酵液酶活力的复筛方法,获得具有遗传稳定性的PBS降解菌株,编号为XH0501-a,测得该菌株的酶活高于原始出发菌株的38.89%。
3.对XH0501-a菌株产生的PBS解聚酶进行了分离、纯化,并对其解聚酶的特性进行了研究。纯化倍数约为12.26,酶活力回收率12.07%,酶蛋白的相对分子质量约为44.7kD。
4.酶最适反应温度和pH分别为40℃和8.6。在温度30℃以下和pH8.0～9.4范围内稳定。金属离子Ca~(2+)、Fe~(2+)对PBS解聚酶有激活作用,而Cu~(2+)、Hg~(2+)等金属离子对此酶有抑制作用。
5.质谱仪测得酶降解PBS的产物均为单体,即丁二酸丁二醇酯。
6.扫描电镜观察降解后的PBS膜,结果表明PBS膜在没有降解之前,膜表面是光滑平整的,降解后膜表面能明显看到蜂窝状孔洞。
Poly(butylene succinate) got into the materials research region in the 1990s, and quickly become one of hotspot stuff of biodegradable plastics research. The source of synthesis material can be petroleum resource, and it also can be got by fermentation of biotic resources. PBS biodegradable plastic is used broadly; it is useful in casing, dinnerware, cosmetic case, drug bottle, agriculture film, pesticide, fertilizer slow release stuff, and high molecular material region. Compare to other biodegradable plastics, PBS have peculiar advantages in thermal property and process property. Therefore, science and technology and industrial community pay high attention on it.
The aim of this work is to try to find out the microorganisms from nature which can be able to degrade PBS, and the domesticated and mutagenic degradation strain can degrade PBS quickly. PBS depolymerase has been purified and some primary analysis has been done on it for lying the theoretical and practical foundations of PBS application.
The main results obtained from this work are as follows:
1. Four kinds of strains able to degrade PBS were isolates from the mud which was discharged by No.3 Petrol-Company of Fushun, coded as XH0501、XH0502、XH0503、XH0504. One kind of fungi which have a fast degradation rate, coded as XH0501, were identified preliminarily: XH0501 belongs to Aspergillus versicolor.
2. The Aspergillus versicolor XH0501, a strain of degrading PBS, was mutated by UV mutagenesis compound LiCl mutagenesis and microwave mutagenesis. Through screening a lot of mutants with the method of culture filtrate, one mutant which has stable character of heredity has been obtained, coded as XH0501-a. The activity of the enzyme which is abstracted from XH0501-a is 38.89% more than that is from XH0501.
3. Some research has been done on the PBS depolymerase which is obtained from XH0501-a. The final purified enzyme represented the 12.07% recovery and the 12.26-fold purified protein with a molecular weight of 44.7kD.
4. The most suitable reaction temperature of this PBS depolymerase is 40℃and pH is 8.6. Below temperature 30℃and between pH 8.4～9.0, the enzyme is stable. Ca~(2+) and Fe~(2+) can active the enzyme, but Cu~(2+) and Hg~(2+) will make it lose activation.
5. The degraded product with PBS depolymerase was monomer succinic acid butanediol ester analyzed by using mass spectrometer.
6. The degradation of PBS films was monitored by using SEM. Before degradation, the film was slide. But there are obvious holes on the film after degradation.
引文
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