【作者】 王岩；

【导师】 陈珊；

【作者基本信息】 东北师范大学 ， 微生物学， 2017， 硕士

【摘要】 在前期研究中,本实验室分离筛选出了一株能够降解聚-3-羟基丁酸酯(PHB)的细菌菌株DSWY01,除了能够高效降解PHB以外,该菌株对其他多种聚合物如聚乳酸(PLA)、聚丁二酸丁二醇酯(PBS)及聚ε-己内酯(PCL)等也具有降解作用,是研究及评估高分子聚合物生物降解机制与特性的极佳材料。前期的研究工作显示该菌株属于假单胞菌属,但其理化特性与属内其它种的标准菌株存在显著不同。因此本文采用多相分类鉴定的方法对菌株DSWY01进行了系统的菌种鉴定,确定该菌株为假单胞菌的一个新种,并将其命名为Pseudomonas changchunensis。同时,本论文还研究了菌株DSWY01的PHB解聚酶的结构域,确定了各结构域的基本功能以及其在PHB材料降解中的重要作用。具体研究结果如下:1.从表型、化学组分及遗传特征三个方面对菌株进行了系统鉴定,结果显示菌株DSWY01为革兰氏阴性菌,短杆状,无芽孢和荚膜,有单一的端生鞭毛。单菌落为圆形,黄白色,表面光滑且湿润,中间微凸且颜色比周围深。菌株在4°C时不生长,41°C时能够生长,最适生长温度是37°C;最适pH是7.0;大于4%的NaCl会抑制其生长。菌株在碳源氮源利用、糖醇发酵及酶反应方面与参比的标准菌株存在显著差异。DSWY01主要的细胞壁脂肪酸有11种,C18:1ω7c/C18:1ω6c与C16:0为其中含量最多的两种,分别约占总量的36.91%和19.55%。基于16S rDNA的系统发育分析结果显示该菌株与P.mendocina,P.alcaliphila,P.oleovorans在亲缘关系上最为接近,但其它遗传特征方面具有明显差异,菌株DSWY01全基因组DNA(G+C)含量约为63.65%(Tm),与P.mendocina,P.alcaliphila,P.oleovorans标准菌株的DNA杂交率分别为44.24%,21.62%和41.92%。最终根据多相分类鉴定所得结果,确定菌株DSWY01为Pseudomonas属的一个新种,命名为Pseudomonas changchunensis。2.对菌株DSWY01的PHB解聚酶基因序列进行了分析,预测了其可能的功能结构域,包括一个催化结构域(CD),一个连接区(LD),以及两个底物结合结构域(SBDI和SBDII)。本研究通过对菌株DSWY01 PHB解聚酶基因进行截短突变获得了含有不同结构域的重组突变体;通过优化诱导表达最终获得了五种重组酶;并进一步对重组酶进行了降解及吸附性能的检测,以探究PHB解聚酶各个结构域的功能,结果显示:(1)只含有催化域CD的重组酶con具有对p-NP6水溶性底物的降解能力,但对PHB乳化底物的降解大大减弱,仅为野生型重组酶的1/5;且重组酶丧失了PHB膜片降解能力,对PHB粉末的吸附率约为22%。(2)含有催化域CD和连接区LD的重组酶link对p-NP6水溶性底物具有降解能力;对PHB乳化底物的降解活性约为重组酶S1+2的1/3;重组酶link不能够降解PHB膜片,,对PHB粉末的吸附率约为35%。(3)含有CD、LD和一个底物结合域SBDI的重组酶S1及含有CD、LD和另一个底物结合域SBDII的重组酶S2对p-NP6水溶性底物具有降解能力;对PHB乳化底物的降解活性约为重组酶S1+2的1/2;低浓度下重组酶S1和S2均不能降解PHB膜片;重组酶S1与S2的吸附能力相差不大,吸附率约为80%。(4)同时含有CD、LD和两个底物结合域的野生型重组酶S1+2对p-NP6水溶性底物具有降解能力;重组酶S1+2对PHB乳化底物的降解能力最强,对PHB粉末的吸附能力也最强,几乎达到100%;此外在浓度较低的情况下,只有含有两个底物结合域的重组酶S1+2能够降解PHB膜片。上述结果表明,解聚酶中催化结构域CD为独立完整的酯酶催化结构域,具有水解酯类底物的功能,但解聚酶对PHB固相材料的降解需要底物结合结构域的参与。另一方面,单一的底物结合结构域SBDI和SBDII具有对固相材料的吸附能力,但对PHB薄膜的降解作用依然较弱,而同时具有两个底物结合结构域的重组酶则显示了更高的吸附性以及高效的PHB降解能力,该结果证实了底物结合结构域对于酶吸附固相底物是必需的,是固相聚酯酶解的前提条件;但同时也显示出,底物结合结构域除了完成对底物的吸附功能以外,可能还在维持酶的稳定性及构象方面具有重要作用。更多还原

【Abstract】 Our laboratory isolated a strain which was able to degrade the poly-3-hydroxybutyrate(PHB)in previous study,called DSWY01.In addition to degrade the PHB efficiently,strain DSWY01 can also degrade other polymers,such as the polycaprolactone(PCL),polybutylene succinate(PBS)and polylactic acid(PLA)and so on,so it is an excellent material to evaluate the biodegradation characteristics of polyester and the mechanism of biodegradation.Previous studies showed that the strain belonged to Pseudomonas sp.,but its physical and chemical properties were significantly different from other standard strains of the genus.In this paper,the strain DSWY01 was identified by the method of Polyphasic Taxonomy and finally confirmed that it was a new strain of Pseudomonas genus,named as Pseudomonas changchunensis.At the same time,we studied the structure of the PHB depolymerase produced by it as well,determined the basic function of each domain and their important roles in the degradation of PHB materials.The detail results are as follows:1.The strains were identified in three aspects: phenotype,chemical composition and genetic characteristics.And the results show that the strain is gram-negative,short rod,no spore and capsule,with a single terminal flagellum.Single colony is round,yellow-white,the middle is slightly convex and the color is deep,the surface is moist and smooth.The strain didn’t grow at 4°C but could grow at the temperature of 41°C,its optimum growth temperature is about °C,and its optimum pH is 7.The NaCl of more than 4% can inhibit the growth of the strain.furthermore,There are significant differences in the utilization of carbon source nitrogen source,sugar and alcohol fermentation and enzyme reaction between the strain DSWY01 and reference strains.There are mainly 11 kinds of fatty acids in the cell wall of DSWY01,the two most abundant species inside are C18:1ω7c/C18:1ω6c and C16:0,accounting for about 36.91% and 19.55% of the total,respectively.The phylogenetic analysis results based on 16 S rDNA show that this strain is closest to P.mendocina,P.alcaliphila and P.oleovorans on the genetic relationship,but they have obvious differences in other genetic characteristics,the genome DNA(G+C)of strain DSWY01 was about 63.65%(Tm).The hybridization rates between DSWY01 and P.mendocina,P.alcaliphila and P.oleovorans are about 44.24%,21.62% and 41.92%,respectively.According to the results of polyphasic taxonomy,we finally confirm that strain DSWY01 is a new species of the genus Pseudomonas,named as Pseudomonas changchunensis.2.PHB depolymerase gene sequence of strain DSWY01 was analyzed and the possible functional domains was predicted,including a catalytic domain(CD),a connecting region(LD),and two substrate binding domain(SBDI and SBDII).In this study,the PHB depolymerase gene of strain DSWY01 was truncated and five recombinant mutant containing different domains were obtained.Fanally,five kinds of recombinant enzymes were obtained by optimizing the expression,and the degradation and adsorption properties of the recombinant enzyme were examined to explore the function of each domain of the PHB depolymerase,the detail results are as follows:(1)The recombinant enzyme con which contains only catalytic domain of CD has the ability to degrade p-NP6 water soluble substrates,but the degradation ability of PHB emulsion substrate is greatly weakened,which is only 1/5 of the wild type recombinant enzyme.Moreover,the recombinant enzyme con lost the degradation ability of PHB film.And the adsorption rate of PHB powder is about 22%.(2)The recombinant enzyme link which contains the catalytic domain CD and the connection LD has the ability to degrade p-NP6 water soluble substrate,but the degradation ability of PHB emulsion substrate is about 1/3 of wild type recombinant enzyme.Moreover,recombinant enzyme link could not degrade PHB film,the adsorption rate of PHB powder is about 35%.(3)The recombinant enzyme S1 which contains CD,LD and a substrate binding domain SBDI and the recombinant enzyme S2 which contains CD,LD and another substrate binding domain SBDII have the ability to degrade p-NP6 water soluble substrate,the degradation ability of PHB emulsion substrate is about 1/2 of wild type recombinant enzyme.in the case of low concentration,neither S1 nor S2 can degrade the PHB film.Moreover,the adsorption capacity of S1 and S2 is not significant,their adsorption rate of PHB powder was about 80%.(4)The wild type recombinant enzyme S1+2 which contains the catalytic domain CD,the connection LD and two substrate binding domains meanwhile has the ability to degrade p-NP6 water soluble substrate,its degradation ability of PHB emulsion substrate is the strongest,its adsorption capacity of PHB powder is also the strongest,Almost 100%.In addition,in the case of low concentration,only the wild type recombinant enzyme S1+2 can degrade PHB film.The results above show that the catalytic structure domain CD of the depolymerase is an independent and complete esterase catalytic structure domain,which has the function of hydrolysis of ester substrate.However,the degradation of PHB solid phase material by the depolymerase requires the participation of the binding substrate domain.On the other hand,single substrate binding domain of SBDI or SBDII has adsorption capacity of solid materials,but their degradation of PHB film is still weak,while the recombinant enzyme with two substrate binding domain shows higher adsorption capacity and efficient PHB degradation ability.This result confirms that the substrate binding domain is necessary for enzyme to adsorb to solid substrate,it is also a prerequisite for the enzymolysis of solid-phase polyesters.At the same time,this result also shows that the substrate binding domain plays an important role in maintaining the stability and conformation of the enzyme besides the adsorption function to the substrate.更多还原