남극 로스해에서 분리한 Croceibacter atlanticus 균 유래 리파아제의 생산, 고정화, 효소특성 연구
Production, Immobilization, and Characterization of Croceibacter atlanticus Lipase Isolated from the Antarctic Ross Sea
2
조회
0
다운로드
1
인용
0
참고문헌
키워드
초록
남극해에는 산업적으로 유용한 신규 효소촉매를 생산하는미생물들이 들어 있다. 우리는 로스해(Ross Sea)로부터 분리한 여러 저온성 박테리아를 조사하였으며, 그 중에서 지방분해 능력이 뛰어난 Croceibacter atlanticus (No. 40-F12)를찾았다. Shotgun 클로닝 방법으로 리파아제 유전자(lipCA) 를 찾았으며 Escherichia coli 균에서 LipCA 효소를 발현하였다. Spain Arreo metagenome alpha/beta hydrolase를기준으로 LipCA 상동구조모델을 만들어서 분석한 결과, α/β hydrolase fold, Gly-X-Ser-X-Gly motif, 그리고 lid 구조를 갖고 있기 때문에 전형적인 리파아제 효소임이 밝혀졌다. Ammonium sulfate 침전법과 겔여과 크로마토그래피를통해서 세포추출액으로부터 LipCA 효소를 순수하게 분리한후, 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. LipCACLEA는 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 활용가능성을 제시한 최초의 보고이다.
Abstract
The Antarctic Ocean contains numerous microorganisms that produce novel biocatalysts that can have applications in various industries. We screened various psychrophilic bacterial strains isolated from the Ross Sea and found that a Croceibacter atlanticus strain (Stock No. 40-F12) showed high lipolytic activity on a tributyrin plate. We isolated the corresponding lipase gene (lipCA) by shotgun cloning and expressed the LipCA enzyme in Escherichia coli cells. Homology modeling of LipCA was carried out using the Spain Arreo lake metagenome alpha/beta hydrolase as a template. According to the model, LipCA has an α/β hydrolase fold, Gly-X-Ser-X-Glymotif, and lid sequence, indicating that LipCA is a typical lipase enzyme. Active LipCA enzyme was purified fromthe cell-free extract by ammonium sulfate precipitation and gel filtration chromatography. We determined its enzymatic properties including optimum temperature and pH, stability, substrate specificity, and organic solvent stability. LipCA was immobilized by the cross-linked enzyme aggregate (CLEA) method and its enzymatic properties were compared to those of free LipCA. After cross-linking, temperature, pH, and organic solvent stability increased considerably, whereas substrate specificities did not changed. The LipCA CLEA was recovered by centrifugation and showed approximately 40% activity after 4th recovery. This is the first report of the expression, characterization, and immobilization of a C. atlanticus lipase, and this lipase could have potential industrial application.
참고문헌 (23)
- Sharma R, "Production, purification, characterization, and applications of lipase", Biotechnol. Adv., 19, pp.627~662 (2001)[DOI]
- Datta S, "Enzyme immobiliza- tion: an overview on techniques and support materials", 3 Biotech., 3, pp.1~9 (2013)
- Sheldon RA, "Cross-linked enzyme aggregates as industrial biocatalysts", Org. Process Res. Dev., 15, pp.213~223 (2011)[DOI]
- Lenfant N, "ESTHER, the database of the <TEX>${\alpha}", {\beta}$<, Nucl. Acids Res., pp.2013, 41[DOI]
- Arpigny JL, "Bacterial lipolytic enzymes: classification and properties", Biochem. J., 343, pp.177~183 (1999)[DOI]
- Martinez-Martinez M, "Biochemical diversity of carboxyl esterases and lipases from lake Arreo (Spain): a metagenomic approach", Appl. Environ. Microbiol., 79, pp.3553~3562 (2013)[DOI]
- Kartal F, "Improved esterification activity of Candida rugosa lipase in organic solvent by immobilization as cross-linked enzyme aggregates (CLEAs)", J. Mol. Catal. B: Enzym., 71, pp.85~89 (2011)[DOI]
- Rehman S, "Cross-linked enzyme aggregates (CLEAs) of Pencilluim notatum lipase enzyme with improved activity, stability and reusability characteristics", Int. J. Biol. Macromol., 91, pp.1161~1169 (2016)[DOI]
- Iftikhar T, "Purification and characterization of extracellular lipase", Pak. J. Bot., 43, pp.1541~1545 (2011)
- Perez D, "A novel halophilic lipase, LipBL, showing high efficiency in the production of eicosapentaenoic acid (EPA)", PLoS One (2011)[DOI]
- Kim HK, "Gene cloning and charac- terization of thermal stable lipase from Bacillus stearothermophilus L1", Biosci. Biotechnol. Biochem., 62, pp.66~71 (1998)[DOI]
- Cho JC, "Croceibacter atlanticus gen.nov., sp. Nov., A Novel Marine Bacterium in the Family Flavobacteriaceae", Syst. Appl. Microbiol., 26, pp.76~83 (2003)[DOI]
- Lai Q, "Alcanivorax marinus sp. Nov., isolated from deep-sea water", Int. J. Syst. Evol. Microbiol., 63, pp.4428~4432 (2013)[DOI]
- Saxena RK, "Purification strategies for microbial lipase", J. Microbiol. Methods., 52, pp.1~18 (2003)
- Li M, "Screening, purification and characterization of a novel cold-active and organic solvent-tolerant lipase from Stenotrophomonas maltophilia CGMCC 4254", Bioresour. Technol., 148, pp.114~120 (2013)[DOI]
- Wang Q, "Purification and biochemical characterization of a cold-active lipase from Antarctic sea ice bacteria Pseudoalteromonas sp. NJ 70", Mol. Biol. Rep., 39, pp.9233~9238 (2012)[DOI]
- Gauthier MA, "Arginine-specific protein modification using <TEX>${\alpha}$<", TEX>-oxo-aldehyde functional polymers prepared by atom transfer radical polymerization, 2011, pp.2, 1490~1498
- Farris S, "Alternative reaction mechanism for the cross-linking of gelatin with glutaraldehyde", J. Agric. Food Chem., 58, pp.998~1003 (2010)[DOI]
- Migneault I, "Glutaraldehyde: behavior in aqueous solution, reaction with proteins, and application to enzyme crosslinking", Biotechniques., 37, pp.790~802 (2004)[DOI]
- Yedavalli P, "Engineering the loops in a lipase for stability in DMSO", Protein Eng. Des. Sel., 26, pp.317~324 (2013)[DOI]
- Dachuri V, "Organic solvent-tolerant, cold-adapted lipase PML and LipS exhibit increased conformational flexibility in polar organic solvents", J. Mol. Catal. B: Enzym., 131, pp.73~78 (2016)[DOI]
- Lopez-Serrano P, "Cross-linked enzyme aggregates with enhanced activity: application to lipases", Biotechnol. Lett., 24, pp.1379~1383 (2002)[DOI]
- Valdes EC, "Influence of the pH of glutaraldehyde and the use of dextran aldehyde on the preparaton of cross-linked enzme aggregates (CLEAs) of lipase from Burkholderia cepacia", Electronic J. Biotechnol., 14 (2011)[DOI]