摘要
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.
The bacteriophage P13 that infects Klebsiella serotype K13 contains a heat-stable depolymerase capable of effectivedegradation of exopolysaccharide （EPS） produced by this microorganism. In this study, the titer of phage P13, initially 2.0 x 107 pillmL-1, was found increasing 20rain after infection and reached 5.0 x 109 pfumL-1 in 60min. Accordingly, the enzyme activity of de-polymerase approached the maximum 60 min after infection. Treatment at 70°C for 30 rain inactivated all the phage, but retained over90% of the depolymerase activity. Addition of acetone into the crude phage lysate led to precipitation of the protein, with a markedincrease in bacterial EPS degradation activity and a rapid drop in the titer of phage. After partial purification by acetone precipitationand ultrafiltration centrifugation, the enzyme was separated from the phage particles, showing two components with enzyme activityon Q-Sepharose Fast Flow. The soluble enzyme had an optimum degradation activity at 600C and pH 6.5. Transmission electron mi-croscopy demonstrated that the phage P13 particles were spherical with a diameter of 50nm and a short stumpy tail. It was a dou-ble-strand DNA virus consisting of a nucleic acid molecule of 45976bp. This work provides an efficient purification operation in-eluding thermal treatment and ultrafiltration centrifugation, to dissociate depolymerase from phage particles. The characterization ofphage P13 and associated EPS depolymerase is beneficial for further application of this enzyme.
基金
supported by the National Natural Science Foundation of China(No.41076087)
