An extracellular polyhydroxybutyrate（PHB） depolymerase was purified to homogeneity from the culture supernatant of a PHB-degrading bacterium, Pseudomonas mendocina DSWY0601, which was isolated from brewery sewage fo...An extracellular polyhydroxybutyrate（PHB） depolymerase was purified to homogeneity from the culture supernatant of a PHB-degrading bacterium, Pseudomonas mendocina DSWY0601, which was isolated from brewery sewage for the ability to form clear zones on the PHB mineral agar plates. The molecular weight of the purified PHB depolymerase as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis（SDS-PAGE） was ap- proximately 59800 at the optimal temperature and pH value being 50 ℃ and 8.5, respectively. PHB depolymerase was stable in a temperature range of 20--50 ℃ and sensitive to pH value within a pH range of 8.0-9.5. PHB depo- lymerase degraded poly-3-hydroxybutyrate-co-4-hydroxybutyrate（P3/4HB） and poly-3-hydroxybutyrate-co-3- hydroxyvalerate（PHBV） but did not degrade poly（lactic acid）（PLA）, poly（butylene succinate）（PBS） or poly- （caprolactone）（PCL）. PHB depolymerase was sensitive to phenylmethylsulfonyl fluoride（PMSF）, H202 and SDS. The main product after enzymatic degradation of PHB was indentified as 3-hydroxbutyrate monomer（3HB） by mass spectrometric analysis, suggesting that PHB depolymerase acted as an exo-type hydrolase. Analysis ofphaZpm gene reveals that PHB depolymerase is a typical denatured short-chain-length PHA（dPHAscL, PHA=polyhydroxyalkanoate） depolymerase containing catalytic domain, linker and substrate-binding domain.展开更多
The bacteriophage P13 that infects Klebsiella serotype K13 contains a heat-stable depolymerase capable of effective degradation of exopolysaccharide(EPS) produced by this microorganism. In this study, the titer of pha...The bacteriophage P13 that infects Klebsiella serotype K13 contains a heat-stable depolymerase capable of effective degradation of exopolysaccharide(EPS) produced by this microorganism. In this study, the titer of phage P13, initially 2.0 × 107 pfu mL-1, was found increasing 20 min after infection and reached 5.0 × 109 pfu mL-1 in 60 min. Accordingly, the enzyme activity of depolymerase approached the maximum 60 min after infection. Treatment at 70℃ for 30 min inactivated all the phage, but retained over 90% of the depolymerase activity. Addition of acetone into the crude phage lysate led to precipitation of the protein, with a marked increase in bacterial EPS degradation activity and a rapid drop in the titer of phage. After partial purification by acetone precipitation and ultrafiltration centrifugation, the enzyme was separated from the phage particles, showing two components with enzyme activity on Q-Sepharose Fast Flow. The soluble enzyme had an optimum degradation activity at 60℃ and pH 6.5. Transmission electron microscopy demonstrated that the phage P13 particles were spherical with a diameter of 50 nm and a short stumpy tail. It was a double-strand DNA virus consisting of a nucleic acid molecule of 45976 bp. This work provides an efficient purification operation including thermal treatment and ultrafiltration centrifugation, to dissociate depolymerase from phage particles. The characterization of phage P13 and associated EPS depolymerase is beneficial for further application of this enzyme.展开更多
Klebsiella pneumoniae is one of the major pathogens causing global multidrug-resistant infections.Therefore,strategies for preventing and controlling the infections are urgently needed.Phage depolymerase,often found i...Klebsiella pneumoniae is one of the major pathogens causing global multidrug-resistant infections.Therefore,strategies for preventing and controlling the infections are urgently needed.Phage depolymerase,often found in the tail fiber protein or the tail spike protein,is reported to have antibiofilm activity.In this study,phage P560isolated from sewage showed specific for capsule locus type KL47 K.pneumoniae,and the enlarged haloes around plaques indicated that P560 encoded a depolymerase.The capsule depolymerase,ORF43,named P560dep,derived from phage P560 was expressed,purified,characterized and evaluated for enzymatic activity as well as specificity.We reported that the capsule depolymerase P560dep,can digest the capsule polysaccharides on the surface of KL47 type K.pneumoniae,and the depolymerization spectrum of P560dep matched to the host range of phage P560,KL47 K.pneumoniae.Crystal violet staining assay showed that P560dep was able to significantly inhibit biofilm formation.Further,a single dose(50μg/mouse)of depolymerase intraperitoneal injection protected 90%–100%of mice from lethal challenge before or after infection by KL47 carbapenem-resistant K.pneumoniae.And pathological changes were alleviated in lung and liver of mice infected by KL47 type K.pneumoniae.It is demonstrated that depolymerase P560dep as an attractive antivirulence agent represents a promising tool for antimicrobial therapy.展开更多
Bacteriophages infected different serotypes of Klebsiella were isolated from sewage. Among them, a heatstable polysaccharide depolymerase enzyme which could degrade bacterial exopolysaccharide effectively was prepared...Bacteriophages infected different serotypes of Klebsiella were isolated from sewage. Among them, a heatstable polysaccharide depolymerase enzyme which could degrade bacterial exopolysaccharide effectively was prepared from the phage infecting Klebsiella K13. Treatment at 60℃ for 30 min could inactivate most of the K13 phage, with the titration decreasing from 6.4×10^8 PFU/mL to 1.6×10^6 PFU/mL. However, no obvious loss of phage enzyme activity was found after this treatment. The optimum hydrolytic temperature of phage enzyme was 60℃, with an activity 57 % higher than that at 30℃. The addition of phage enzyme could result in a rapid decrease of viscosity of exopolysaccharide （EPS） solution within minutes, indicating that K13 phage polysaccharide depolymerase acts as a kind of endo-glycanohydrolase. HPLC and reducing sugar analysis showed that the hydrolysis of EPS approached approximately the maxi-mum at 4h when the final concentration of phage was 6.0 x los PFU/mL. The results showed that K/eb-siella K13 phage depolymerase enzyme could be used as a good tool for the preparation of EPS oligosac- charide.展开更多
基金Supported by the National Natural Science Foundation of China (Nos.31100046,31100099)
文摘An extracellular polyhydroxybutyrate（PHB） depolymerase was purified to homogeneity from the culture supernatant of a PHB-degrading bacterium, Pseudomonas mendocina DSWY0601, which was isolated from brewery sewage for the ability to form clear zones on the PHB mineral agar plates. The molecular weight of the purified PHB depolymerase as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis（SDS-PAGE） was ap- proximately 59800 at the optimal temperature and pH value being 50 ℃ and 8.5, respectively. PHB depolymerase was stable in a temperature range of 20--50 ℃ and sensitive to pH value within a pH range of 8.0-9.5. PHB depo- lymerase degraded poly-3-hydroxybutyrate-co-4-hydroxybutyrate（P3/4HB） and poly-3-hydroxybutyrate-co-3- hydroxyvalerate（PHBV） but did not degrade poly（lactic acid）（PLA）, poly（butylene succinate）（PBS） or poly- （caprolactone）（PCL）. PHB depolymerase was sensitive to phenylmethylsulfonyl fluoride（PMSF）, H202 and SDS. The main product after enzymatic degradation of PHB was indentified as 3-hydroxbutyrate monomer（3HB） by mass spectrometric analysis, suggesting that PHB depolymerase acted as an exo-type hydrolase. Analysis ofphaZpm gene reveals that PHB depolymerase is a typical denatured short-chain-length PHA（dPHAscL, PHA=polyhydroxyalkanoate） depolymerase containing catalytic domain, linker and substrate-binding domain.
基金supported by the National Natural Science Foundation of China(No.41076087)
文摘The bacteriophage P13 that infects Klebsiella serotype K13 contains a heat-stable depolymerase capable of effective degradation of exopolysaccharide(EPS) produced by this microorganism. In this study, the titer of phage P13, initially 2.0 × 107 pfu mL-1, was found increasing 20 min after infection and reached 5.0 × 109 pfu mL-1 in 60 min. Accordingly, the enzyme activity of depolymerase approached the maximum 60 min after infection. Treatment at 70℃ for 30 min inactivated all the phage, but retained over 90% of the depolymerase activity. Addition of acetone into the crude phage lysate led to precipitation of the protein, with a marked increase in bacterial EPS degradation activity and a rapid drop in the titer of phage. After partial purification by acetone precipitation and ultrafiltration centrifugation, the enzyme was separated from the phage particles, showing two components with enzyme activity on Q-Sepharose Fast Flow. The soluble enzyme had an optimum degradation activity at 60℃ and pH 6.5. Transmission electron microscopy demonstrated that the phage P13 particles were spherical with a diameter of 50 nm and a short stumpy tail. It was a double-strand DNA virus consisting of a nucleic acid molecule of 45976 bp. This work provides an efficient purification operation including thermal treatment and ultrafiltration centrifugation, to dissociate depolymerase from phage particles. The characterization of phage P13 and associated EPS depolymerase is beneficial for further application of this enzyme.
基金the National Natural Science Foundation of China(U1803109)the National Key Research and Development Program of China(2018YFC1602500)+6 种基金the National Natural Science Foundation of China(81572032)Major scientific and technological innovation projects in Shandong Province(2019JZZY010719)National Key Research and Development Program(2019YFA0904003)Six Talent Peaks Project in Jiangsu Province(2016-WSN-112)Key research and development project of Jiangsu provincial science and Technology Department(BE2017654)Gusu key health talent of Suzhou,Jiangsu youth medical talents program(QN-867)the Science and Technology Program of Suzhou(SZS201715)。
文摘Klebsiella pneumoniae is one of the major pathogens causing global multidrug-resistant infections.Therefore,strategies for preventing and controlling the infections are urgently needed.Phage depolymerase,often found in the tail fiber protein or the tail spike protein,is reported to have antibiofilm activity.In this study,phage P560isolated from sewage showed specific for capsule locus type KL47 K.pneumoniae,and the enlarged haloes around plaques indicated that P560 encoded a depolymerase.The capsule depolymerase,ORF43,named P560dep,derived from phage P560 was expressed,purified,characterized and evaluated for enzymatic activity as well as specificity.We reported that the capsule depolymerase P560dep,can digest the capsule polysaccharides on the surface of KL47 type K.pneumoniae,and the depolymerization spectrum of P560dep matched to the host range of phage P560,KL47 K.pneumoniae.Crystal violet staining assay showed that P560dep was able to significantly inhibit biofilm formation.Further,a single dose(50μg/mouse)of depolymerase intraperitoneal injection protected 90%–100%of mice from lethal challenge before or after infection by KL47 carbapenem-resistant K.pneumoniae.And pathological changes were alleviated in lung and liver of mice infected by KL47 type K.pneumoniae.It is demonstrated that depolymerase P560dep as an attractive antivirulence agent represents a promising tool for antimicrobial therapy.
基金the National Natural Science Foundation of China(No40506027 and No30771646)the Doctoral Foundation of Shandong Province(No2005BS02015)
文摘Bacteriophages infected different serotypes of Klebsiella were isolated from sewage. Among them, a heatstable polysaccharide depolymerase enzyme which could degrade bacterial exopolysaccharide effectively was prepared from the phage infecting Klebsiella K13. Treatment at 60℃ for 30 min could inactivate most of the K13 phage, with the titration decreasing from 6.4×10^8 PFU/mL to 1.6×10^6 PFU/mL. However, no obvious loss of phage enzyme activity was found after this treatment. The optimum hydrolytic temperature of phage enzyme was 60℃, with an activity 57 % higher than that at 30℃. The addition of phage enzyme could result in a rapid decrease of viscosity of exopolysaccharide （EPS） solution within minutes, indicating that K13 phage polysaccharide depolymerase acts as a kind of endo-glycanohydrolase. HPLC and reducing sugar analysis showed that the hydrolysis of EPS approached approximately the maxi-mum at 4h when the final concentration of phage was 6.0 x los PFU/mL. The results showed that K/eb-siella K13 phage depolymerase enzyme could be used as a good tool for the preparation of EPS oligosac- charide.
