好食脉孢菌纤溶酶的分离纯化

Purification of the fibrinolytic enzyme from Neurospora sitophila

以豆渣和麸皮为固体发酵主要原料培养好食脉孢霉,用生理盐水浸提取发酵后培养基、硫酸铵40%,80%两次盐析后,用Octyl-Sepharose FF疏水色谱分离出两个组分,均为纤溶酶。再顺次采用Sephadex G-25凝胶色谱、SP-Sepharose HP强阳离子色谱和Superdex 75凝胶色谱对好食脉孢霉发酵产生的纤溶酶组分Ⅰ进一步分离纯化,获得纯度较高的纤溶酶,其比活力为97.52 U/mg,其相对于发酵液的活力回收率为0.74%,总纯化倍数为87.07。经SDS-PAGE、酶谱法活性电泳和等电聚焦电泳,切割活性条带进行Q-TOF质谱测序,获得纤溶酶的部分氨基酸序列,分别为:N-P-S-G-S-L-S-N-G-A-G-L-E-P-K;P-V-Q-P-G-S-G-Q-S-D-G-T-A-D-V-E-K;S-S-V-M-D-S-E-A-N-M-A-W-N-D-E-R。通过NCBI-BLAST数据库与已报道的纤溶酶氨基酸序列比对,发现此脉孢霉纤溶酶为新型蛋白质。

The purification protocol of a fibrinolytic enzyme from bean dregs and wheat bran fermented by Neurospora sitophila was explored and established.The culture medium was composed of bean dregs and wheat bran, and dry weight ratio of them was 4： l.The fibrinolytic enzyme was separated using fractional salting-out with （ NH4）2SO4 firstly, and then three fibrinolytic active components were found by Octyl-Sepharose Fast Flow hydrophobic interaction chromatography. The active component I was further purified using Sephadex G-25 gel filtration chromatography, SP-Sepharose HP ion exchange chromatography and Superdex 75 gel filtration chromatography. A single active peak was received in Superdex 75 gel filtration chromatography. After the enzyme was purified, the specific activity was 97.52U/ mg. The purification protocol resulted in a 87.07-fold purification of the enzyme, with a final yield of 0.74%. SDS polyacrylamide gel electrophoresis, active spectrum electrophoresis and isoelectric focusing electrophoresis of the active component obtained from Superdex 75 gel filtration chromatography were conducted, the active gel stripes were cut for sequences determination by Q-TOF. The amino acids sequence of 62 peptide fragments of the fibrinolytic enzyme were respectively. Through the NCBI BLAST database and has reported the amino acid sequence alignment of fibrinolytic enzyme, to find three peptide fragments of the fibrinolytic enzyme, wereN-P-S-G-S-L-S-N-G-A-G-L-E-P-K; P-V-Q-P-G-S-G-Q-S-D-G-T-A-D-V-E-K; S-S-V-M -D-S-E-A-N-M-A-W-N-D-E-R.