The Seperation And Identification Of The Parasitic Bacteria From Lampreys And Its Characteristics And Activities Of The Extracellular Fibrino(Gen) Lytic Enzyme

Lampreys(Lampetra japonica)are one of the most primitive vertebrates,which usually feed on the host fish flesh and blood.And its buccal gland secretion may contain active peptides or proteins to prevent the formation of blood clots.Previous studies have shown that the buccal gland secretion was degraded during the incubation at room temperature or 37 ?for several days.Compared with the secretion without degradation,the degraded buccal gland secretion showed higher fibrinogenolytic activity,with the degradation of fibrinogen A? chain,B? chain,and ? chain in a short time.This suggests that novel active components might appeared in the degraded secretion which might result from lampreys or bacteria present in the buccal gland.In order to illuminate the mechanisms of higher fibrinogenolytic activity in the degraded secretion,a bacteria strain with higher fibrinogenolytic activity was isolated in the degraded buccal gland secretion for the first time.This suggests that the higher fibrinogenolytic activity in the degraded secretion might result from the bacteria present in the buccal gland.Through the morphological observation and the sequencing of 16 S rDNA,the separated bacteria strain was identified as Stenotrophomonas maltophilia strain LJ1.Subsequently,the culture conditions of the LJ1 strain were optimized,including cultivation temperature(28?),cultivation time(48 h),incubated volume(50 mL LB in 500 mL bottle).A single protein with the molecular weight of 44 kDa and fibrinogeolytic activity was purified from the supernatant of LJ1 strain by using ammonium sulfate precipitation,dialysis,lyophilization and the DEAE anion exchange chromatography.And it was named as Stenotrophomonas maltophilia strain LJ1 fibrinogenase(SMLJ1F).N-terminal sequencing results showed that the N-terminal amino acid sequence of SMLJ1F was NH2-LAPNDPYYQQ.And the amino acid sequence of random peptides of SMLJ1F was ETARPFPVAIPAATPIGTGILDAK,YRPASCDGVVTVGATR and ITGGITYYSNYGTR by using mass spectrometry identification.Function analysis showed that SMLJ1F could completely degrade several proteins related to the coagulation cascade processes,including fibrinogen,fibrin,plasminogen,and thrombin which suggests that SMLJ1F might inhibit the blood coagulation by degradation of fibrinogen and thrombin,and might show the thrombolysis properties by degradation of fibrin or activation of plasminogen.According to the results of N-terminal sequencing and mass spectrometry identification,the nucleotide sequence and amino acid sequence of SMLJ1F was obtained through the gene cloning method.All of the above not only provide the basis for further studies of the structure and function of SMLJ1F,but also provide the possibilities to develop novel anticoagulant and thrombolytic drugs in the near future.