High level expression and characterization of a novel thermostable, organic solvent tolerant, 1,3-regioselective lipase from Geobacillus sp. strain ARM

Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia.
Bioresource Technology (Impact Factor: 4.49). 07/2011; 102(13):6972-81. DOI: 10.1016/j.biortech.2011.03.083
Source: PubMed


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.

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Available from: Afshin Ebrahimpour, May 16, 2014
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    • "Many genes of thermophilic enzymes have been isolated in recent years from the genomes of Geobacillus for their further heterological expression in mesophylic bacteria. Among these enzymes, lipases are found (AbdellFatt tah, Gaballa, 2008; QuintanaaCastro et al., 2009; Cheong et al., 2011; Ebrahimpour et al., 2011; Balan et al., 2012). They take part in the decomposition of lignocellulosic biomass: βglucosidase (Shallom et al., RUSSIAN JOURNAL OF GENETICS: APPLIED RESEARCH Vol. 4 No. 3 2014 2005; BenDavid et al., 2007; Wagschal et al., 2009; Ratnadewi et al., 2013), endoglucanase (Ng et al., 2009), xylanase (Wu et al., 2006; Canakci et al., 2007, 2012; Gerasimova, Kuisiene, 2012; Liu et al., 2012; Verma et al., 2013), and many other proteins. "
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    ABSTRACT: Thermophilic bacteria are being extensively used in biotechnology. Species of the genus Geobacillus rank among the most prospective thermophiles. The current methods of genetic and metabolic engineering of these microorganisms are considered in the article. Examples of their use in various branches of biotechnology are presented.
    Full-text · Article · May 2014 · Russian Journal of Genetics: Applied Research
    • "The specific activity of purified T1 (Leow et al. 2007), L2 (Shariff et al. 2011) and ARM (Ebrahimpour et al. 2011) lipases is 958.2, 75.8 and 7092 U/mg, respectively, using olive oil as a substrate. Specific activity of purified JC lipase (Jiang et al. 2010) is 515.6 U/mg when p-NP palmitate is used as a substrate. "
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    ABSTRACT: GD-95 lipase from Geobacillus sp. strain 95 and its modified variants lacking N-terminal signal peptide and/or 10 or 20 C-terminal amino acids were successfully cloned, expressed and purified. To our knowledge, GD-95 lipase precursor (Pre-GD-95) is the first Geobacillus lipase possessing more than 80 % lipolytic activity at 5 °C. It has maximum activity at 55 °C and displays a broad pH activity range. GD-95 lipase was shown to hydrolyze p-NP dodecanoate, tricaprylin and canola oil better than other analyzed substrates. Structural and sequence alignments of bacterial lipases and GD-95 lipase revealed that the C-terminus forms an α helix, which is a conserved structure in lipases from Pseudomonas, Clostridium or Staphylococcus bacteria. This work demonstrates that 10 and 20 C-terminal amino acids of GD-95 lipase significantly affect stability and other physicochemical properties of this enzyme, which has never been reported before and can help create lipases with more specific properties for industrial application. GD-95 lipase and its modified variants GD-95-10 can be successfully applied to biofuel production, in leather and pulp industries, for the production of cosmetics or perfumes. These lipases are potential biocatalysts in processes, which require extreme conditions: low or high temperature, strongly acidic or alkaline environment and various organic solvents.
    No preview · Article · Nov 2013 · Extremophiles
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    • "SBS-4S lipase when exposed for 100 min at 60°C [69], and the lipase from G. stearothermophilus JC after 30 min at 70°C [13], while the Geobacillus sp. strain ARM lipase had half lives of only few minutes at 60 and 70°C [68]. "
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    ABSTRACT: In a bioprospecting effort towards novel thermostable lipases, we assessed the lipolytic profile of 101 bacterial strains isolated from the volcanic area of Santorini, Aegean Sea, Greece. Screening of lipase activity was performed both in agar plates and liquid cultures using olive oil as carbon source. Significant differences were observed between the two screening methods with no clear correlation between them. While the percentage of lipase producing strains identified in agar plates was only 17%, lipolytic activity in liquid culture supernatants was detected for 74% of them. Nine strains exhibiting elevated extracellular lipase activities were selected for lipase production and biochemical characterization. The majority of lipase producers revealed high phylogenetic similarity with Geobacillus species and related genera, whilst one of them was identified as Aneurinibacillus sp. Lipase biosynthesis strongly depended on the carbon source that supplemented the culture medium. Olive oil induced lipase production in all strains, but maximum enzyme yields for some of the strains were also obtained with Tween-80, mineral oil, and glycerol. Partially purified lipases revealed optimal activity at 70-80°C and pH 8-9. Extensive thermal stability studies revealed marked thermostability for the majority of the lipases as well as a two-step thermal deactivation pattern.
    Full-text · Article · May 2013
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