A comparative study on fibrinolytic enzymes extracted from six Bacillus spp. isolated from fruit-vegetable waste biomass

Abstract

Fibrinolytic enzymes are considered as a significant therapeutic agent for thrombosis. Keeping the quest for identification of effective fibrinolytic enzyme(s) from microbial source, a comparative study was done on six Bacillus spp. isolated from fruit and vegetable waste. The fibrinolytic activities of the isolates were confirmed by a fibrin plate assay. Six potential isolates were identified by 16s rRNA sequencing as Bacillus altitudinus HEM05, Bacillus halotolerance ARKH1, Bacillus safensis CO05, Bacillus subtilis subsp. inaquorum BE1, Bacillus tequilensis SAS23 and Bacillus siamensis BHO27. Extracellular fibrinolytic enzymes were purified to homogeneity from all six isolates by acetone precipitation, anion exchange chromatography and gel filtration chromatography. Purified fibrinolytic enzymes showed fibrinolytic specific activity ranging from 60.92 U mg-1 to 220.19 U mg-1. Molecular weight (SDS PAGE) of purified enzymes ranged from 30,200 kDa to 69,500 kDa. Enzymes were effective over wide range of pH from 5 to 10, with optimum pH between 7 and 10. The enzymes were active between 37 °C to 60 °C. Effect of inhibitory activity of metal ions, protease inhibitors and amidolytic activity on purified enzymes indicated fibrinolytic enzymes from Bacillus altitudinus HEM05, Bacillus halotolerance ARKH1, Bacillus safensis CO05 as plasmin serine protease, from Bacillus subtilis subsp. inaquorum BE1, Bacillus tequilensis SAS23 as subtilin chymotrypsin like metalloprotease and from Bacillus siamensis BHO27 as subtilin chymotrypsin. In vitro assays for the purified enzyme preparations from all six isolates confirmed enzymes could activate plasminogen and significantly degraded the fibrin net of blood clot.