| Polyvinyl alcohol?PVA?,a biodegradable and water-soluble high molecular polymer.PVA has recently gained wide attention due to its biodegradable potential.China could consume a great number of PVA every year,due to its various useful properties,such as thermostability,film-forming ability,emulsibility and high-viscosity.Under such circumstances,a large amount of waste water containing PVA is poured into rivers,leading to serious environmental pollution problems.Although PVA could be biodegraded to some extent,the biodegradation rate and efficiency is very low.Therefore,in this study,we investigated the biodegradation of PVA from PVA degrading bacteria and PVA degrading enzymes this two aspects to increase the biodegradation efficiency of PVA.The major results were summarized as follows:?1?PVA-degrading mixed cultures were obtained from a textile factory.After cultivated in selective medium for several generations,BQ-2,one of the mixed cultures,was obtained as the best one.When 1 g·L-1 PVA was used as a sole carbon source in culture medium,it could be completely degraded within only 48 h.F2,which can degrade PVA with high-efficiency,were isolated from BQ-2.After 16 S rDNA identification,F2 belongs to Sphingopyxis sp.When 1g·L-1 yeast extract were added into culture medium,F2 could grow well and 1 g·L-1 PVA could be degraded over 90% within 120 h.Compared with the degradation efficiency of 50% in the culture medium without yeast extract,the degrading ability of F2 was increased by over 80%.The enzyme activity of polyvinyl alcohol dehydrogenase?PVADH?and oxidized polyvinyl alcohol hydrolase?OPH?were detected in the supernatant after cells were breaked.?2?ARTP mutagenesis was conducted to F2 as the original strain,to improve the PVA degradation efficiency.Based on lethal rate,the mutagenesis time was defined as 12 s.Using high-throughput method to screen the positive mutants and after 72 h fermentation,a mutant named SM2 with an increase of 53% in PVA degradation efficiency was finally obtained.Cultivating SM2 for 10 generations,the PVA degradation efficiency of each generation changed within 10%,which showed that SM2 possessed good genetic stability.?3?The genes of pvadh and oph were from the genome of F2,and the high-level expression of them were studied.The pvadh gene,after codon optimizing based on the bias of P.pastoris,was inserted into pPIC9 K.The recombinant plasmid was linearized by Sal I and was transformed into P.pastoris GS115.A positive transformant of P.pastoris GS115/pPIC9K/ pvadh selected from YPD plates containing 3 mg·m L-1 G418 was chosen for expression.After 120 h induction,its maximum activity reached 523 U·m L-1 in shake flasks;The oph gene was inserted into pET-20b?+?and the recombinant plasmid was transformed into E.coli BL21?DE3?.A positive transformant of E.coli BL21?DE3?/p ET-20b?+?/oph was selected for expression.After 120 h induction,its maximum activity reached 20.6 U·m L-1 in shake flasks.?4?The reaction conditions of PVA biodegradation by using PVADH and OPH were optimized.The effects of 6 single factors including PVADH concentration,OPH concentration,enzymatic temperature,pH,Ca2+ concentration and PVA concentration on PVA degradation efficiency were studied.Three significant factors,that is PVADH concentration,pH and enzymatic temperature,were determined through single factor experiments.Based on the ascent experiments,Box-Behnken design and response surface strategy,the optimal conditions were set as follows: PVADH 123 U·mL-1,pH 7.7,enzymatic temperature at 41?,OPH 12 U·mL-1,PVA concentration 1 g·L-1,Ca2+ concentration 1 mmol·L-1 and PQQ concentration 6 ?mol·L-1.Under these optimal conditions,the degradation efficiency can reach 34.07% after 1 h enzymatic reaction and reach over 95% after 4 h enzymatic reaction. |
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