降解PCL菌株的选育及其解聚酶的研究
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摘要
聚己内酯(poly caprolactone, PCL)是以石油为原料合成的一种可生物降解的脂肪族聚酯,在自然界中易被微生物或酶分解,最终产物为水和二氧化碳。目前,PCL作为一种新型的可生物降解高分子材料,在医学、药物、工业、农业等领域具有广泛的应用前景。
本项工作的目的是从自然界中筛选出可降解PCL的菌株,研究PCL降解菌株降解PCL的特性和降解规律,探讨PCL解聚酶的生化特性及其降解过程,为PCL的应用奠定理论和实践基础。
本论文的主要结果如下:
1.对不同地区和生态环境下采集的数个样品进行筛选分离,共获得四株可降解PCL的菌株,分别为YD0401、YD0402、YD0403、YD0404。对其中一种真菌YD0401进行初步鉴定:YD0401菌株属于半知菌纲,从梗孢目、从梗孢科、曲霉族、青霉属。
2.以青霉(Penicillium sp.)YD0401为出发菌株,通过紫外诱变分生孢子,采用透明圈初筛和摇瓶培养复筛的方法获得2株能稳定遗传的分泌PCL解聚酶的高产菌株YD0401-Ae和YD0401-Bc。
3.原始菌株对PCL膜的降解过程是缓慢降解—等速降解—快速降解;而YD0401-Ae及YD0401-Bc突变菌株对PCL膜的降解过程是缓慢降解—快速降解—缓慢降解。
4.测定原始菌株及突变株每日PCL解聚酶酶活时发现YD0401-Ae、YD0401-Bc突变株的酶活力比原始菌株要高,且有两个产酶高峰,分别在48h和120h,其PCL解聚酶酶活力分别比原始菌株提高32.1%、41.86 %和35.8 %、31.4%。
5.对原始菌株及YD0401-Ae突变株的PCL解聚酶粗酶液的基本性质进行了对照研究。原始菌株及YD0401-Ae突变株的PCL解聚酶粗酶液最适反应温度均为20℃。原始菌株及YD0401-Ae突变株的最适反应pH值为8.6。
6.质谱仪测得酶降解PCL的产物均为单体,即ε-羟基己酸。
7.研究了PCL膜的生物降解过程,PCL膜的生物降解首先发生在PCL表面的非晶部分,相继结晶部分开始降解,但结晶部分降解速度缓慢。
Poly caprolactone(PCL) produced by petroleum synthesis is an aliphatic polyester, and is easily decomposed by microbiology or enzyme in nature with the final productof CO2 and H2O. At present, PCL, as a novel biomaterial, has many potential uses in a wide range of medical, industrial and agricultureal applications.
The aim of this work is to try to find out the microorganisms which can be able to degrade PCL ,and to lay the theoretical and practical foundations of PCL application by studying their fundamental properties relative to PCL degradation, investigating the biochemistry characteristics of PCL depolymerase and degradation process.
The main results obtained from this work are as follows:
1. Four kinds of strains able to degrade PCL were isolates from different areas and ecological environments, coded as YD0401、YD0402、YD0403、YD0404. One kind of fungi, coded as YD0401, were identified preliminarily: YD0401 belongs to Penicillium.
2. The penicillium sp. YD0401, a strain of degrading PCL, was mutagenized by UV treatment. Through screening a lot of mutants with the method of transparent zones and culture filtrate, best two ones were obtained with high–yield of stable PCL depolymerase, coded as YD0401-Ae and YD0401-Bc.
3. The degradation rate of PCL film by the original one can be classified into the type for whole degradation process: slow-constant-fast,while the two YD0401-Ae and YD0401-Bc muntants was slow-fast-slow.
4. When the enzyme activity of the original one and the two muntants were surveyed with their daily crude enzyme, two highest enzyme peak production were found in 48h and 120h.The enzyme activity of the YD0401-Ae and YD0401-Bc muntants were 32.1%、41.86 % and 35.8 %、31.4% higher than the original one at two highest enzyme peak production respectively.
5. Comparative study on the basic enzyme property of the PCL depolymerase between the YD0401-Ae mutants and the original one. The optimum temperature and pH of the YD0401-Ae mutants and the original one was 20℃and 8.6.
6. The degraded product with PCL depolymerase was monomerε-hydroxy caproic acid analyzed by using mass spectrometer.
7. The degradation process of PCL films was monitored by using SEM.It was shown that PCL degradation occurred firstly in the amorphous part of PCL and then in the crystalline part. But the degradation rate of the crystalline part was slow.
本项工作的目的是从自然界中筛选出可降解PCL的菌株,研究PCL降解菌株降解PCL的特性和降解规律,探讨PCL解聚酶的生化特性及其降解过程,为PCL的应用奠定理论和实践基础。
本论文的主要结果如下:
1.对不同地区和生态环境下采集的数个样品进行筛选分离,共获得四株可降解PCL的菌株,分别为YD0401、YD0402、YD0403、YD0404。对其中一种真菌YD0401进行初步鉴定:YD0401菌株属于半知菌纲,从梗孢目、从梗孢科、曲霉族、青霉属。
2.以青霉(Penicillium sp.)YD0401为出发菌株,通过紫外诱变分生孢子,采用透明圈初筛和摇瓶培养复筛的方法获得2株能稳定遗传的分泌PCL解聚酶的高产菌株YD0401-Ae和YD0401-Bc。
3.原始菌株对PCL膜的降解过程是缓慢降解—等速降解—快速降解;而YD0401-Ae及YD0401-Bc突变菌株对PCL膜的降解过程是缓慢降解—快速降解—缓慢降解。
4.测定原始菌株及突变株每日PCL解聚酶酶活时发现YD0401-Ae、YD0401-Bc突变株的酶活力比原始菌株要高,且有两个产酶高峰,分别在48h和120h,其PCL解聚酶酶活力分别比原始菌株提高32.1%、41.86 %和35.8 %、31.4%。
5.对原始菌株及YD0401-Ae突变株的PCL解聚酶粗酶液的基本性质进行了对照研究。原始菌株及YD0401-Ae突变株的PCL解聚酶粗酶液最适反应温度均为20℃。原始菌株及YD0401-Ae突变株的最适反应pH值为8.6。
6.质谱仪测得酶降解PCL的产物均为单体,即ε-羟基己酸。
7.研究了PCL膜的生物降解过程,PCL膜的生物降解首先发生在PCL表面的非晶部分,相继结晶部分开始降解,但结晶部分降解速度缓慢。
Poly caprolactone(PCL) produced by petroleum synthesis is an aliphatic polyester, and is easily decomposed by microbiology or enzyme in nature with the final productof CO2 and H2O. At present, PCL, as a novel biomaterial, has many potential uses in a wide range of medical, industrial and agricultureal applications.
The aim of this work is to try to find out the microorganisms which can be able to degrade PCL ,and to lay the theoretical and practical foundations of PCL application by studying their fundamental properties relative to PCL degradation, investigating the biochemistry characteristics of PCL depolymerase and degradation process.
The main results obtained from this work are as follows:
1. Four kinds of strains able to degrade PCL were isolates from different areas and ecological environments, coded as YD0401、YD0402、YD0403、YD0404. One kind of fungi, coded as YD0401, were identified preliminarily: YD0401 belongs to Penicillium.
2. The penicillium sp. YD0401, a strain of degrading PCL, was mutagenized by UV treatment. Through screening a lot of mutants with the method of transparent zones and culture filtrate, best two ones were obtained with high–yield of stable PCL depolymerase, coded as YD0401-Ae and YD0401-Bc.
3. The degradation rate of PCL film by the original one can be classified into the type for whole degradation process: slow-constant-fast,while the two YD0401-Ae and YD0401-Bc muntants was slow-fast-slow.
4. When the enzyme activity of the original one and the two muntants were surveyed with their daily crude enzyme, two highest enzyme peak production were found in 48h and 120h.The enzyme activity of the YD0401-Ae and YD0401-Bc muntants were 32.1%、41.86 % and 35.8 %、31.4% higher than the original one at two highest enzyme peak production respectively.
5. Comparative study on the basic enzyme property of the PCL depolymerase between the YD0401-Ae mutants and the original one. The optimum temperature and pH of the YD0401-Ae mutants and the original one was 20℃and 8.6.
6. The degraded product with PCL depolymerase was monomerε-hydroxy caproic acid analyzed by using mass spectrometer.
7. The degradation process of PCL films was monitored by using SEM.It was shown that PCL degradation occurred firstly in the amorphous part of PCL and then in the crystalline part. But the degradation rate of the crystalline part was slow.
引文
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