一株高产纳豆激酶菌株的鉴定与分析

Identification and Analysis of a Nattokinase-producing Strain

鉴定高产纳豆激酶菌株Td,分析纳豆激酶的分子特征。利用菌体形态、生理生化特征、以及分子生物学方法对菌株Td进行鉴定;并采用MALDI-TOF质谱测定与分析、SDS-PAGE和纤溶活性测定等方法检测纳豆激酶特性,利用PCR方法扩增纳豆激酶的基因全长。结合菌体形态、生理生化特征和16S r DNA、gyr A基因序列、DNA-DNA杂交率等实验结果,鉴定菌株Td为枯草芽孢杆菌枯草亚种(Bacillus subtilis subsp.subtilis);菌株Td发酵产生的纳豆激酶产量可达300 mg/L以上,占发酵液总蛋白的40%以上;纤溶活性达230 U/m L以上;氨基酸序列与subtilisin E的序列相似性最高;基因全长序列为1 143 bp。枯草芽孢杆菌枯草亚种Td是一株高产、高活性纳豆激酶的产生菌,具有优良的工业化开发价值。

The objective of this work is to identify an isolate Td of yielding high nattokinase, and to analyze the molecular characteristics of its nattokinase. The morphologic, biochemical and physiological characterizations, and molecular biological methods were used to identify the taxonomic position of isolate Td;mass spectrometric determination and analysis of MALDI-TOF, SDS-PAGE and fibrinolytic activity were employed to measure the characteristics of nattokinase, and PCR method was adopted to clone the full length of nattokinase gene. The bacterial strain Td was identified as Bacillus subtilis subsp. subtilis according to the results of morphologic, biochemical and physiological characterizations, as well as 16 S r DNA, the sequences of 16 S r DNA gyr A, hybrid rate of DNA-DNA. The yield of nattokinase produced by strain Td reached 300 mg/L, accounted for over 40% of the total protein. Fibrinolytic activity was over 230 U/m L. Amino acid sequence of nattokinase produced by Td showed the highest similarity to the subtilisin E. and the full length of the gene was 1143 bp. Conclusively, as a high-yield and high-activity strain for nattokinase, B. subtilis subsp. subtilis Td is a promising candidate for industrial development and utilization.