Details

Autor(en) / Beteiligte

• Yao, Zhuang
• Jeon, Hye Sung
• Yoo, Ji Yeon
• Kang, Yun Ji
• Kim, Min Jae
• Kim, Tae Jin
• Kim, Jeong Hwan

Titel

DNA Shuffling of aprE Genes to Increase Fibrinolytic Activity and Thermostability

Ist Teil von

• Journal of microbiology and biotechnology, 2022-06, Vol.32 (6), p.800-807

Ort / Verlag

한국미생물생명공학회

Erscheinungsjahr

2022

Quelle

EZB Electronic Journals Library

Beschreibungen/Notizen

• Four aprE genes encoding alkaline serine proteases from B. subtilis strains were used as template genes for family gene shuffling. Shuffled genes obtained by DNase I digestion followed by consecutive primerless and regular PCR reactions were ligated with pHY300PLK, an E. coli-Bacillus shuttle vector. The ligation mixture was introduced into B. subtilis WB600 and one transformant (FSM4) showed higher fibrinolytic activity. DNA sequencing confirmed that the shuffled gene (aprEFSM4) consisted of DNA mostly originated from either aprEJS2 or aprE176 in addition to some DNA from either aprE3-5 or aprESJ4. Mature AprEFSM4 (275 amino acids) was different from mature AprEJS2 in 4 amino acids and mature AprE176 in 2 amino acids. aprEFSM4 was overexpressed in E. coli BL21 (DE3) by using pET26b(+) and recombinant AprEFSM4 was purified. The optimal temperature and pH of AprEFSM4 were similar to those of parental enzymes. However, AprEFM4 showed better thermostability and fibrinogen hydrolytic activity than the parental enzymes. The results indicated that DNA shuffling could be used to improve fibrinolytic enzymes from Bacillus sp. for industrial applications.