Cloning, expression and characterization of a novel lipase gene lipB from Aspergillus niger F044

Abstract:

Abstract: [Objective] We cloned, expressed and characterized a novel lipase gene lipB from Aspergillus niger F044, to facilitate the large scale production and application of that enzyme. [Method] We cloned lipB gene and the cDNA sequence by PCR and RT-PCR, and then cloned the open reading frame of lipB into pET28a vector and expressed by isopropyl β-D-1-thiogalactopyranoside (IPTG) induction. After Ni-agarose purification, the characteristics were determined and the conformation change was checked by circular dichroism methods. [Results] The novel lipase genes cDNA of lipB were cloned from Aspergillus niger F044 (GenBank: FJ536287, FJ536288) and expressed in Escherichia coli. The molecular weight of LipB was about 43 kDa. The optimal substrate of this enzyme is 4-nitrophenyl octanoate (pNPC-C8) with Km=5.98 mmol/L. The optimal temperature and pH was 50oC and pH 6.0. The enzyme was stable below 40 oC. After incubated at 60 oC for 1 h, only 18.8% activity remained. After treated by 2 mmol/L Ca2+ for 1 h, the activity improved 2.6-fold. [Conclusion] Enzymatic characteristics of LipB determined showed this enzyme might have potential in industrial applications.