Identification And Molecular Biological Characteristics On The Novel Multi-Functional Fibrinolytic Enzymes

The fibrinolytic enzymes in the Chongguk- Jiang of Korea, Dou-Shi of China, Natto of Japan have some advantages such as the high safety, mass production, oral administration, high specify to fibrin and direct dissolving fibrin, so many researches on this enzyme had been done. Till now, all researchers have investigated by using the enzymes purified from commodity food, but no one has researched the characteristics of this fibrinolytic enzyme such as anti-oxidation, cell protection and targeting by using purified from the culture of the strain which have screened from these foods by measuring fibrinolytic activity.In this treatise, it was subscribed the multi-functional properties of the fibrinolytic enzymes purified from the culture of the strain with the high fibrinolytic activity which was screened from natural source by fibrin plate method.We have isolated the strain with high fibrinolytic activity from the soybean food fermented by packing with rice leaves and identified it as Bacillus subtilis QK02 to be deserved in China Center for Type Culture Collection (CCTCC) with accession numberNo. M203078. The fibrinolytic activity of B. subtilis QK02 was higher 5-60 times than those of the other strains isolated from the fermented soybean food. Two fibrinolytic enzymes (called as Subtilisin, QK-1 and QK-2) purified from the culture medium of B. subtilis QK02 were exhibited 42,000 and 28,000 kD of molecular weight, 1930 IU/mg and 41,000 IU/mg of specific activity, respectively. They all directly dissolved the Act-, B|3-,y- fragment of fibrin and have activity on the fibrin plate after being digested by trypsin. The fibrinolytic activity of QK-2 which is smaller molecular weight had not inhibited by any substances in blood such as plasminogen activator inhibitor-1. The N-terminal amino acid sequence of QK-2 wasA-Q-S-V-P-Y-G-I-S-Q-I-K-A-P-A-L-H-S-Q-G, which is identical with those ofSubtilisin NAT Subtilisin J Subtilisin E Subtilisin Amylosacchariticus Mensentricopeptidase and from the biochemical characteristics it was named as subtilisin QK.In reference of the N-terminal amino acid sequence of subtilisin QK (QK) and the C-terminal amino acid sequence of subtilisin NAT (NK), the primers was designed and synthesized and the DNA fragment, qk gene, encoded subtilisin QK was amplified from B. subtilis QK02 genome DNA by PCR. The qk gene was composed with 828bpnucleotides encoded a protein consisted by 275 amino acids which had Asp32, His64Ser221 as catalytic center, Ser126 Leu127 Gly128 as the substrate binding site that are aliketo NK. A comparison of the deduced amino acid sequence of QK and restriction enzyme sites of qk gene with those of above five enzymes showed that QK was different from those and the high homolog enzyme with it was subtilisin NAT (96.8%). Through the 3D structure modeling of QK protein by using those of subtilisin BPN', it was indicated that there were six regions of a-helical and nine P-strand structure and one cleft structure formed with catalytic center and substrate binding site in order to have fibrin easily contact and combine. Through the general molecular cloning method, qk gene was inserted into the expression vector to be expressed in E. coli and the expressionproduct, r-QK, had the fibrinolytic activity like to natural protein.The anti-nitrotyrosine formation activity of QK in BSA was analyzed by using spectrophotometer, ELISA, Westhern blot and fluorescence emission spectra assay, in vitro. The protein protection action of QK against protein nitration, in vivo, was investigated with the homogenates of brain, heart, lung, liver, kidney, spleen, and skeletal muscle between the ribs from the mice injected with oxidants. As results, QK had significantly exhibited the anti-protein nitration activity in vitro and in vivo. And QK had be able to inhibit the oxidation of oxy-hemoglobin by nitrite, which was not concerned with its fibrinolytic activity, and also protect the human...