新型中长链聚羟基脂肪酸酯的生物合成及其性能检测
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摘要
聚羟基脂肪酸酯(PHA)是细菌胞内发现的一种多聚物，具有生物可降解性、生物相容性等优良性能，有广泛的应用前景。由于PHA为R构型，其手性单元羟基脂肪酸(HA)是一些药物和农药的合成中间体，其经济价值也引起了有机合成工业的兴趣。
本论文围绕着新型中长链聚羟基脂肪酸酯(mcl-PHA)的生物合成及其性能进行研究。
首先，利用了Pseudomonas putida KT2442β-氧化最后两步反应所需的3-羟基酯酰辅酶A脱氢酶基因(3-hydroxyacyl-CoA dehydrogenase，fadB)及3-酮酰辅酶A硫解酶基因(3-ketoacyl-CoA thiolase，fadA)缺失突变株P．putida KTOY06为生产菌，以长链脂肪酸十四酸为碳源在不同浓度梯度和时间梯度下生产出具有高含量3-羟基十四酸单体(3HTD)的新型中长链PHA，3HTD的摩尔百分比从31mol％到49mol％不等。
其次，从细菌的干细胞内3HTD含量高于提取出来的mcl-PHA中3HTD的含量的情况，推断出非mcl-PHA 3HTD的存在。实验中提取并用核磁共振(NMR)分析了非mcl-PHA 3HTD在细胞中的存在方式为寡聚体，并通过凝胶渗透色谱GPC检测了分子量的分布。
最后，分析了所得几种mcl-PHA的分子量分布，热学性能，机械性能，热稳定性并与其他典型的mcl-PHA性能比较，新型mcl-PHA具有更好的结晶性和机械强度。
Polyhydroxyalkanoates, abbreviated as PHA, is a family of intracellularpolyesters synthesized by many bacteria. The piezoelectricity, biocompatibility andbiodegradability of PHA have offered these polymers many promising applications invarious areas. In addition, chiral monomers of PHA are highly interesting forapplication as intermediates for synthesis of pharmaceutics and pesticides.
This study mainly focused on biosynthesis and characterization of new type ofmedium chain length PHA.
Knockout mutant Pseudomonas putida KT2442 termed Pseudomonas putidaKTOY06 (P. putida KTOY06) which 3-ketoacyl-CoA thiolase (fadA) and3-hydroxyacyl-CoA dehydrogenase (fadB) in P. putida KT2442 was knocked out,respectively, was used to biosynthesized new mcl-PHA by tetradecanoate, fadA andfadB were catalyzed the last two steps inβ-oxidation pathway in P. putida KT2442. Inthis study, mcl-PHA which HTD monomer ranged from 31mo1% to 49mo1% wereobtained, the monomer composition is dependent on different cultivation conditions.
Non-mcl-PHA HTD was observed because of higher HTD content in lyophilizedcells than HTD content in the extracted mcl-PHA, indicating the possible existence ofnon-mcl-PHA HTD as intracellular substances. NMR was studied after thenon-mcl-PHA HTD being extracted showed that the free and/or oligo HTD werealmost oligo HTD, and average molecular weights were estimated by Gel PermeationChromatography (GPC).
The average molecular weight, thermal properties, mechanism properties,thermal stability and other properties of these high HTD content mcl-PHA wereanalyzed in this study. The mcl-PHA containing high HTD monomer contents wasfound to have both higher crystallinity and improved tensile strength compared withthat of typical mcl-PHA
本论文围绕着新型中长链聚羟基脂肪酸酯(mcl-PHA)的生物合成及其性能进行研究。
首先，利用了Pseudomonas putida KT2442β-氧化最后两步反应所需的3-羟基酯酰辅酶A脱氢酶基因(3-hydroxyacyl-CoA dehydrogenase，fadB)及3-酮酰辅酶A硫解酶基因(3-ketoacyl-CoA thiolase，fadA)缺失突变株P．putida KTOY06为生产菌，以长链脂肪酸十四酸为碳源在不同浓度梯度和时间梯度下生产出具有高含量3-羟基十四酸单体(3HTD)的新型中长链PHA，3HTD的摩尔百分比从31mol％到49mol％不等。
其次，从细菌的干细胞内3HTD含量高于提取出来的mcl-PHA中3HTD的含量的情况，推断出非mcl-PHA 3HTD的存在。实验中提取并用核磁共振(NMR)分析了非mcl-PHA 3HTD在细胞中的存在方式为寡聚体，并通过凝胶渗透色谱GPC检测了分子量的分布。
最后，分析了所得几种mcl-PHA的分子量分布，热学性能，机械性能，热稳定性并与其他典型的mcl-PHA性能比较，新型mcl-PHA具有更好的结晶性和机械强度。
Polyhydroxyalkanoates, abbreviated as PHA, is a family of intracellularpolyesters synthesized by many bacteria. The piezoelectricity, biocompatibility andbiodegradability of PHA have offered these polymers many promising applications invarious areas. In addition, chiral monomers of PHA are highly interesting forapplication as intermediates for synthesis of pharmaceutics and pesticides.
This study mainly focused on biosynthesis and characterization of new type ofmedium chain length PHA.
Knockout mutant Pseudomonas putida KT2442 termed Pseudomonas putidaKTOY06 (P. putida KTOY06) which 3-ketoacyl-CoA thiolase (fadA) and3-hydroxyacyl-CoA dehydrogenase (fadB) in P. putida KT2442 was knocked out,respectively, was used to biosynthesized new mcl-PHA by tetradecanoate, fadA andfadB were catalyzed the last two steps inβ-oxidation pathway in P. putida KT2442. Inthis study, mcl-PHA which HTD monomer ranged from 31mo1% to 49mo1% wereobtained, the monomer composition is dependent on different cultivation conditions.
Non-mcl-PHA HTD was observed because of higher HTD content in lyophilizedcells than HTD content in the extracted mcl-PHA, indicating the possible existence ofnon-mcl-PHA HTD as intracellular substances. NMR was studied after thenon-mcl-PHA HTD being extracted showed that the free and/or oligo HTD werealmost oligo HTD, and average molecular weights were estimated by Gel PermeationChromatography (GPC).
The average molecular weight, thermal properties, mechanism properties,thermal stability and other properties of these high HTD content mcl-PHA wereanalyzed in this study. The mcl-PHA containing high HTD monomer contents wasfound to have both higher crystallinity and improved tensile strength compared withthat of typical mcl-PHA
引文
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