Polyphasic Taxonomy Of Strain DSWY01 And Study About The Function Of Each Domain Of Its PHB Depolymerase

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 C18:1?7c/C18:1?6c 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.