Purification and Properties of the Extracellular Lipase, Lip A, from Acinetobacter SP.RAG-1
Abstract
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 deg 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 deg C and showed a 3-fold increase in activity over the standard assay temperature (30 deg C) at its temperature optimum (55 deg 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, C5).
Document Details
- Document Type
- Technical Report
- Publication Date
- Feb 19, 2002
- Accession Number
- ADA399380
Entities
People
- Erick A. Snellman
Organizations
- Air Force Institute of Technology