• 한국미생물·생명공학회지
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• 제46권3호
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• pp.234-243
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• 2018

남극해에는 산업적으로 유용한 신규 효소촉매를 생산하는 미생물들이 들어 있다. 우리는 로스해(Ross Sea)로부터 분리한 여러 저온성 박테리아를 조사하였으며, 그 중에서 지방분해 능력이 뛰어난 Croceibacter atlanticus (No. 40-F12)를 찾았다. Shotgun 클로닝 방법으로 리파아제 유전자(lipCA)를 찾았으며 Escherichia coli 균에서 LipCA 효소를 발현하였다. Spain Arreo metagenome alpha/beta hydrolase를 기준으로 LipCA 상동구조모델을 만들어서 분석한 결과, ${\alpha}/{\beta}$ hydrolase fold, Gly-X-Ser-X-Gly motif, 그리고 lid 구조를 갖고 있기 때문에 전형적인 리파아제 효소임이 밝혀졌다. Ammonium sulfate 침전법과 겔여과 크로마토그래피를 통해서 세포추출액으로부터 LipCA 효소를 순수하게 분리한 후, 최적 온도, pH, 안정성, 기질특이성, 유기용매 안정성 등의 효소특성을 규명하였다. LipCA를 cross-linked enzyme aggregate (CLEA) 방법으로 고정화하고 효소특성을 조사, 비교하였다. 고정화를 통해 온도, pH, 유기용매에 대한 안정성이 증가하였고 기질특이성의 변화는 관찰되지 않았다. $LipCA^{CLEA}$는 원심분리 방법으로 쉽게 회수되었고 4번의 재사용 후에 40% 이상의 활성이 잔재하였다. 이 논문은 C. atlanticus 리파아제의 발현, 특성규명, Cross-linked Enzyme Aggregated 고정화를 바탕으로 안정성을 높여 산업적 활용 가능성을 제시한 최초의 보고이다.

• Journal of Microbiology and Biotechnology
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• 제31권6호
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• pp.882-889
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• 2021

In order to use an enzyme industrially, it is necessary to increase the activity of the enzyme and optimize the reaction characteristics through molecular evolution techniques. We used the error-prone PCR method to improve the reaction characteristics of LipCA lipase discovered in Antarctic Croceibacter atlanticus. Recombinant Escherichia coli colonies showing large halo zones were selected in tributyrin-containing medium. The lipase activity of one mutant strain (M3-1) was significantly increased, compared to the wild-type (WT) strain. M3-1 strain produced about three times more lipase enzyme than did WT strain. After confirming the nucleotide sequence of the M3-1 gene to be different from that of the WT gene by four bases (73, 381, 756, and 822), the secondary structures of WT and M3-1 mRNA were predicted and compared by RNAfold web program. Compared to the mean free energy (MFE) of WT mRNA, that of M3-1 mRNA was lowered by 4.4 kcal/mol, and the MFE value was significantly lowered by mutations of bases 73 and 756. Site-directed mutagenesis was performed to find out which of the four base mutations actually affected the enzyme expression level. Among them, one mutant enzyme production decreased as WT enzyme production when the base 73 was changed (T→ C). These results show that one base change at position 73 can significantly affect protein expression level, and demonstrate that changing the mRNA sequence can increase the stability of mRNA, and can increase the production of foreign protein in E. coli.