High level expression and characterization of a novel thermostable, organic solvent tolerant, 1,3-regioselective lipase from Geobacillus sp. strain ARM.
ABSTRACT The mature ARM lipase gene was cloned into the pTrcHis expression vector and over-expressed in Escherichia coli TOP10 host. The optimum lipase expression was obtained after 18 h post induction incubation with 1.0mM IPTG, where the lipase activity was approximately 1623-fold higher than wild type. A rapid, high efficient, one-step purification of the His-tagged recombinant lipase was achieved using immobilized metal affinity chromatography with 63.2% recovery and purification factor of 14.6. The purified lipase was characterized as a high active (7092 U mg(-1)), serine-hydrolase, thermostable, organic solvent tolerant, 1,3-specific lipase with a molecular weight of about 44 kDa. The enzyme was a monomer with disulfide bond(s) in its structure, but was not a metalloenzyme. ARM lipase was active in a broad range of temperature and pH with optimum lipolytic activity at pH 8.0 and 65°C. The enzyme retained 50% residual activity at pH 6.0-7.0, 50°C for more than 150 min.
Full-textDOI: · Available from: Afshin Ebrahimpour, May 16, 2014
- SourceAvailable from: Donald A. Cowan[Show abstract] [Hide abstract]
ABSTRACT: In this work we report the whole genome sequences of six new Geobacillus xylanolytic strains along with the genomic analysis of their capability to degrade carbohydrates. The six sequenced Geobacillus strains described here have a range of GC contents from 43.9% to 52.5% and clade with named Geobacillus species throughout the entire genus. We have identified a ~200 kb unique super-cluster in all six strains, containing five to eight distinct carbohydrate degradation clusters in a single genomic region, a feature not seen in other genera. The Geobacillus strains rely on a small number of secreted enzymes located within distinct clusters for carbohydrate utilization, in contrast to most biomass-degrading organisms which contain numerous secreted enzymes located randomly throughout the genomes. All six strains are able to utilize fructose, arabinose, xylose, mannitol, gluconate, xylan, and α-1,6-glucosides. The gene clusters for utilization of these seven substrates have identical organization and the individual proteins have a high percent identity to their homologs. The strains show significant differences in their ability to utilize inositol, sucrose, lactose, α-mannosides, α-1,4-glucosides and arabinan.Frontiers in Microbiology 05/2015; 6. DOI:10.3389/fmicb.2015.00430 · 3.94 Impact Factor
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ABSTRACT: A gene (1,254 bp) encoding a lipase was identified from a deep-sea hydrothermal field thermophile Geobacillus sp. EPT9. The open reading frame of this gene encoded 417 amino acid residues. The gene was cloned, overexpressed in Escherichia coli, and the target protein was purified to homogeneity. The purified recombinant enzyme presented a molecular mass of 44.8 kDa. When p-nitrophenyl palmitate was used as a substrate, the recombinant lipase was optimally active at 55 °C and pH 8.5. The recombinant enzyme retained 44 % residual activity after incubation at 80 °C for 1 h, which indicated that Geobacillus sp. EPT9 lipase was thermostable. Homology modeling of strain EPT9 lipase was developed with the lipase from Bacillus sp. L2 as a template. The core structure exhibits an α/β-hydrolase fold and the typical catalytic triad might consist of Ser142, Asp346, and His387. The enzymatic activity of EPT9 lipase was inhibited by addition of phenylmethylsulfonyl fluoride, indicating that it contains serine residue, which plays an important role in the catalytic mechanism.World Journal of Microbiology and Biotechnology 11/2014; 31(2). DOI:10.1007/s11274-014-1775-0 · 1.35 Impact Factor
Article: Solvent tolerant lipases: A review[Show abstract] [Hide abstract]
ABSTRACT: Lipases have proven to be useful in different hydrolytic and synthetic reactions of industrial importance. Microbial strains from natural and extreme environments produce lipases with unique characteristics. The ability of lipase to withstand different environmental conditions during reactions, including temperature and pH, is essential. Solvent systems tend to affect lipase-catalyzed reactions, and thus the careful selection of both the medium and the lipase source is necessary. This review considers different solvent systems used in lipase-catalyzed reactions and some of the enzymatic properties required for function. Other properties of interest besides enzyme activity include tolerance, stability and compatibility to different reaction media, such as acid, alkaline, salt, organic solvents and other compatible solvents (ionic liquids and detergents). For lipase to be used in a detergent, its thermostability and alkaline tolerance must be well pronounced. In addition, organic solvent stability plays an essential role in employing lipases for biodiesel production. Thus, the selection of the lipase for each application is based on specificity and stability in different solvent systems, which gives lipases many potential applications in aqueous and non-aqueous biocatalysis.PROCESS BIOCHEMISTRY 11/2014; 50(1). DOI:10.1016/j.procbio.2014.10.019 · 2.52 Impact Factor