Purification and properties of the extracellular lipase, Lip A, from Acinetobacter sp. RAG-1.

摘要

The major objectives of this research were: (a) understand the temporal and spatial distribution of lipase production by Acinetobacter sp. RAG-1 during growth on hexadecane and triglycerides; (b) purify the extracellular lipase; and (c) to examine its role in modifying the fatty acid component of the bioemulsifier, emulsan, produced by this bacterium. To achieve these objectives, the lipase was produced and purified from RAG-1 cells grown on hexadecane and the properties of the protein investigated. The majority of the enzyme was released into the growth medium during transition to stationary phase. The lipase showed high stability in hexadecane medium where it remained active for longer than 48 hr. at 30°C. Therefore, minimal medium supplemented with 10 mM hexadecane was selected for purification of the lipase. An 8% yield and greater than 10-fold purification were achieved. The protein demonstrated little affinity for anion exchange resins. However, contaminating proteins were removed by passing crude supernatants over a Mono Q column. The lipase was further purified by hydrophobic interaction chromatography, employing a butyl sepharose matrix, and eluted with an increasing Triton X-100 gradient. The protein has an apparent molecular weight of 33 kDa, determined from SDS PAGE. LipA was found to be stable at pH values of 5.8--9.0 and showed optimal activity at approximately pH 9.0. The lipase remained highly active at temperatures up to 70°C and showed a 3-fold increase in activity over the standard assay temperature (30°C) at its temperature optimum (55°C). LipA was found to be active against a wide range of fatty acid esters of p-nitrophenyl but demonstrated higher activity toward medium length acyl chains (C6, C8). The enzyme demonstrated hydrolytic activity toward emulsions of both medium and long chain triglycerides, determined by zymogram. RAG-1 lipase was stabilized by Ca+2. Loss in activity was not observed in preparations containing the cation, whereas 70% loss was observed over a 30-hr period in its absence. The lipase was strongly inhibited by EDTA, Hg+2, and Cu +2, but did not lose activity after incubation with Ca+2, Mg+2, Zn+2, Fe+3, Co+2 , and Rb+. The protein retained more than 75% of the initial activity after exposure to most of the organic solvents employed in this study. It was, however, rapidly inactivated by pyridine. LipA was found to be highly active in organic media, as demonstrated by transesterification of vinyl butyrate with 1-octanol. Similar conditions were employed to examine transesterification activity of LipA toward emulsan fatty acids.; However, it could not be conclusively demonstrated that LipA-catalyzed trans-acylation of the polymer occurred using an exogenous n-alkanoic acid. RAG-1 lipase was found to increase emulsifying activity of emulsan. Emulsan incubated in the presence of LipA showed a greater than 2-fold increase in emulsifying activity over control experiments containing emulsan and selected proteins. A model was proposed to show LipA as an emulsan-associated protein.