Biochemical Characterization, Gene Cloning And Expression And Structure Prediction Of Lipase From Aspergillus Niger

Biodiesel, a novel alternative energy, can be attained as by-product in the structured lipids synthesis when the transesterification reaction is catalyzed by lipases with sn-1, 3-regionselectivity. It's important for deeply processing of agriculture products and enhancing value of the oils and fats. However, there are very few reports on the lipases with sn-1, 3-regionselectivity and structured lipids synthesis in China. In this study, a lipase with sn-1, 3-regionselectivity from Aspergillus niger 404 was purified and biochemically characterized, and the lipase gene was cloned and expressed in E. coli and P. pastoris, respectively, for the first time in China. Furthermore, based on the homology of its amino acid sequence with T. lanuginosus lipase, the 3D-model of the A. niger lipase was predicted. The main results were as follows:A novel filamentous fungi strain producing lipase with acid-tolerant, thermostable and high-specific activity characters was screened. On the selective media with rhodamine B as the indicator, a high-level lipase-producing strain was isolated from oil-polluted soil samples. The strain was identified and named as A. niger F044 based on the colony morphology characters and the ITS sequence analysis. The lipase from A. niger F044 was purified to homogeneity using ammonium sulfate precipitation, dialysis, DEAE-Sepharose Fast Flow anion exchange chromatography, and Sephadex G-75 gel filtration chromatography. This protocol resulted in a 73.71-fold purification with 33.99 % final yield, and the relative molecular weight of the lipase was determined to be approximately 35-40 kDa using SDS-PAGE. The optimal pH and temperature for lipolytic activity of the lipase were 7.0 and 45℃, respectively. It was stable at temperature below 60℃and retained 98.70 % of its original activity for 30 min, while its activity declined rapidly as soon as the temperature rose over 65℃. The lipase was highly stable in the pH range from 2.0 to 9.0 for 4 h. Ca2+ ions stimulated its lipolytic activity, whereas Mn2+, Fe2+, and Zn2+ ions caused inhibition. The values of Km and Vmax calculated from the Lineweaver–Burk plot using p-nitrophenyl palmitate as hydrolysis substrate were 7.37 mmol/l and 25.91μmol·min-1·mg-1, respectively. The N-terminal amino acid sequence of the lipase was Ser/Glu/His-Val-Ser-Thr-Ser-Thr-Leu-Asp-Glu-Leu-Gln-Leu-Phe-Ala -Gln.It is the first time that the lipase gene from A. niger was cloned and reported. Based on the N-terminal amino acid sequence of the lipase from A. niger F044 and the same conserved sequences with filamentous fungi lipases, a potential homologous gene A84689 to the anl (the gene encoding the lipase from A. niger F044) was identified in NCBI nucleotide database by means of bioinformatics. A pair of primers was designed according to the nucleotide sequence of A84689, and the anl was directly cloned by PCR. Nucleotide sequencing revealed that the anl has an ORF of 1, 044 bp, containing three introns of 54 bp, 45 bp and 51 bp. The deduced amino acid sequence corresponds to 297 amino acid residues including a signal sequence of 27 amino acid residues. Nucleotide sequence alignment through BLAST reveal that the anl had no sequence identity to any known lipase genes except for another A. niger lipase gene (GenBank access No. DQ680030). The overall amino-acid sequence identity of the ANL (the mature A. niger lipase) to those of A. flavus lipase, T. lanuginosus lipase, P. allii lipase was 40%, 50% and 40%, respectively.It is also for the first time that the lipase gene from A. niger was expressed successfully in heterogeneous host. The cDNA coded for the ANL was cloned into pET28a vector and the recombinant plasmid was transformed into E. coli BL21 (De3). The anl was overexpressed in E. coli BL21 (De3) after induction by IPTG, and the rANL accumulated in the cells in an insoluble form as inclusion bodies. The production level of the anl in the cells was 50 mg rANL/g wet cells. The recombinant ANL with 6-His-tag was purified by Ni-NTA Agrose chromatography. The denatured recombinant ANL by 8mol/l urea was refolded in vitro by dilution and DEAE Sepharose Fast Flow chromatography. The specific activity of the renatured rANL was 221.49 U/mg.The cDNA fragment coding the ANL with 6-His encoding sequence at N-terminus was cloned into the pPIC9K vector and the reconstructed expression plasmid was then transformed into P. pastoris GS115. The anl was actively expressed in P. pastoris GS115 after induction by mehanol and the rANL was secreted into the fermentation broth. The measured lipolytic activity of the fermentation broth was 15.50 U/ml. The rANL with 6-His-tag at N-terminus was purified by Ni-NTA Agrose chromatography. Two kinds of rANL, ANLⅠand ANLⅡ, were detected in the fermentation broth by SDS-PAGE analysis. After isolation by Sephadex G-75 gel filtration chromatography, the specific activity of the ANLⅡwas 812 U/mg.We have tried for the first time to annotate the primary structure, the secondary structure, and the three-dimensional structure of the A. niger lipase through the homologous lipase with known structure in BioEdit software, PSIPRED server and SwissModel sever, respectively. The predicted structures of the A. niger lipase were consistent with those of the serine hydrolase. The molecular structure models of the A. niger lipase and the A. niger feruloyl esterase were superimposed on each other to study the structure difference, and the result suggested that the main structure difference lied in the lid structure covering the active site.