Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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DNA Shuffling of aprE Genes to Increase Fibrinolytic Activity and Thermostability

  • Yao, Zhuang (Division of Applied Life Science (BK21 4), Graduate School, Gyeongsang National University) ;
  • Jeon, Hye Sung (Division of Applied Life Science (BK21 4), Graduate School, Gyeongsang National University) ;
  • Yoo, Ji Yeon (Division of Applied Life Science (BK21 4), Graduate School, Gyeongsang National University) ;
  • Kang, Yun Ji (Division of Applied Life Science (BK21 4), Graduate School, Gyeongsang National University) ;
  • Kim, Min Jae (Division of Applied Life Science (BK21 4), Graduate School, Gyeongsang National University) ;
  • Kim, Tae Jin (Division of Applied Life Science (BK21 4), Graduate School, Gyeongsang National University) ;
  • Kim, Jeong Hwan (Division of Applied Life Science (BK21 4), Graduate School, Gyeongsang National University)
  • Received : 2022.02.12
  • Accepted : 2022.04.20
  • Published : 2022.06.28
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Abstract

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.

Keywords

Acknowledgement

This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2017R1D1A1B03030037). Yao Z, Jeon HS, Yoo JY, Kang YJ, Kim MJ, and Kim TJ were supported by the BK21 4 Program (MOE), Republic of Korea.

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