Isolation And Identification Of Intraceller Substances Of Brevibacillus Brevis FJAT-0809-GLX And Its SOD Gene Analysis

Brevibacillus brevis was distributed widely in nature. In different growth stages, the colony morphology would present diversity. At present, the whole genomic sequences of two strains B. brevis were sequenced, which a number of functional genes had been studied. B. brevis FJAT-0809-GLX used in this study was isolated from the soil in Fuzhou City, the bacterial displayed a broad spectrumant agonistic activity to pathogenic fungi and bacteria, including Verticillium dahliae, V.dahliae, V. alboatrum, V.fungicola, Colletotrichum gloeosporioides, Monilialaxa and Ralstonia solanaceaum. The fermentation broth displayed a broad bacteriostatic activity, and its extracellular substances have strong stability.The extracellular substances of B. brevis FJAT-0809-GLX had been reported, but their intracellular substances remains unknown. Extracellular substances synthesis, secretion and intracellular substances are closely related, so ultrasonic extraction separation to prepare intracellular substances for identification was conducted. The response surface method was used in the test to screen optimal cell wall broken condition, ultrasonic power, ultrasonic time and feed liquid ratio as the main influence factors, the substances yield as an index to screen optimum conditions. The results showed that the cell wall broken was affected by the ultrasonic power, ultrasonic broken time, feed liquid ratio, e.g. ranging with feed liquid ratio> ultrasonic broken time> ultrasonic power. The best technological conditions are ultrasonic power 318.68 W, ultrasonic time 10 min and feed liquid ratio 1:25, theoretical predictions value 13.3586 mg/g, the experimental value 12.9878 mg/g. The simulation showed good results.Intracellular substances compositions was analyzed using GC-MS on the basis of the cell ball broken. The results showed that ultrasonic power, ultrasonic time and feed liquid ratio influenced type and content of intracellular substances. When ultrasonic time was 40 min, antioxidant 1076 was obtained that is a high molecular weight phenolic ester. It was spontaneous synthetic in the process of intracellular substances extraction and provides a new way for antioxidant synthesis.In spite of structural genomics of B. brevis FJAT-0809-GLX was sequenced, but the functional genemics of this strain is just beginning. The SOD gene was amplified from the genomic DNA. Amino acids’sequences deduced by this gene showed the highest homology with B. brevis NBR10059. The cloned SOD gene was inserted into the expression vector pET-28a to construct recombinant plasmid pET-28a/SOD.The SOD protein with molecular weight of about 22 kD was expressed in E.cok after induction with IPTG. The response surface method was used to screen optimal conditions of protein expression. The best technological conditions are the cell concentration 0.68; the Mn ion concentration 1.65; the IPTG concentration 0.84; and cultivate 12 h in 30℃ at constant temperature incubator shaker. Theoretical predictions value is 3761.68 U/mg, and the experimental value is 3435.23 U/mg, both values demonstrated the good simulation effects. All results might be helpful to further studies on the structure, function research and a convenient and efficient way to produce MnSOD, and also to development and application of SOD.