The Gene Cloning And Biological Activities Of An Extracellular Fibrino(Gen)lytic Enzyme SMLJ1F From Stenotrophomonas Maltophilian

Stenotrophomonas maltophilia(S.maltophilia)are extensively existed in the environment,and are also known as gram-negative bacteria which need oxygen strickly.Previous studies have shown that a bacterium with relatively higher fibrinogenolytic activity was isolated from the degraded buccal gland secretion of lampreys and identified as S.maltophilia strain LJ1.Furthermore,a S.maltophilia strain LJ1 fibrinogenase(SMLJ1F)was proved to be secreted from LJ1 strain.In order to further study the biological functions of SMLJ1F,LJ1 strain was firstly identified through physiological and biochemical methods in the present study,which further confirmed the relationship between LJ1 strain and with S.maltophilia.Meanwhile,ammonium sulfate precipitation,dialysis,lyophilization and ion exchange chromatography were further optimized to purify SMLJ1F.In the present study,the purified SMLJ1F was electrophoresed as a single band on SDS-PAGE.According to the degradation manner of fibrinogen,SMLJ1F was classified as an alpha fibrinogenase.In addition,enzyme activity analysis showed that SMLJ1F has higher fibrinogenolytic activity at 40°C and at p H value of 7.Furthermore,KCl and Ca Cl₂was found to increase the activity of SMLJ1F;Na Cl and EDTA has no effect on the activity of SMLJ1F;While PMSF was found to inhibit its activity.This suggested that SMLJ1F is a member of the serine protease family.Chromogenic substrate assays showed that SMLJ1F have thrombin-like and FXa-like activity,which could hydrolyze the peptide bonds containing arginine in the C terminal of its sequences.Thus,SMLJ1F was also identified as a tryptase.Combining with results from previous studies,SMLJ1F has effects on a variety of substrates.In order to further confirm whether SMLJ1F has anticoagulant thrombolytic effects,coagulation time,hemorheology and bleeding time in mice were analyzed in the presence of SMLJ1F.The results showed that SMLJ1F was able to prolong the blood coagulation time,such as APTT and PT,but not TT.Furthermore,SMLJ1F could also decrease the whole blood viscosity and prolong the tail bleeding time(BT)in mice,suggested that SMLJ1F might have the potential to be used as an anticoagulant.As S.maltophilia are also known as the pathogenic bacteria,they may cause diseases.In order to weaken its impact on the environment and obtain more SMLJ1F,the bioinformatics analysis and expression of SMLJ1F was also performed in the present study.After IPTG induction,recombinant SMLJ1F(r SMLJ1F)was expressed as the soluble protein partly.In conclusion,the deep study on SMLJ1F from S.maltophilia would provide the clues for the pathogenic mechanism and novel drug development,which has important medical value and practical significance.