ABSTRACT
A lipase from Aspergillus 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 purification protocol resulted in a 73.71-fold purification of lipase with 33.99% final yield, and the relative molecular weight of the enzyme was determined to be approximately 35-40kD using SDS-PAGE. The optimum pH and temperature for lipolytic activity of the lipase was 7.0 and 45 degrees C , respectively. It was extremely stable at 60 degrees C and retained 98.70% of its original activity for 30min. The stability declined rapidly as soon as the temperature rose over 65 degrees C . The lipase was highly stable in the pH range from 2.0 to 9.0 for 4h. Ca2+ and Mg2+ ions stimulated lipolytic activity, whereas Mn2+ , Fe2+ and Zn2+ ions caused inhibition. The values of Km and Vmax calculated from the Lineweaver-Burk plot using pNPP as hydrolysis substrate were 7.37mmol/L and 25.91 micromol/(min x mg), respectively. The N-terminal sequence of the lipase was Ser/Glu/His-Val-Ser-Thr-Ser-Thr-Leu-Asp-Glu-Leu-Gln-Leu-Phe-Ala-Gln, which is highly homogeneity with that of lipase, as reported by Torossian.
Subject(s)
Amino Acid Sequence , Aspergillus niger , Chromatography, Gel , Chromatography, Ion Exchange , Electrophoresis, Polyacrylamide Gel , Enzyme Stability , Fungal Proteins , Chemistry , Metabolism , Hydrogen-Ion Concentration , Lipase , Chemistry , Metabolism , Molecular Sequence Data , Molecular Weight , Sequence Analysis, Protein , TemperatureABSTRACT
The fermentation conditions of alkaline lipase producing by Pseudomonas cepacia PCL-3 were optimized.Based on the analysis of single factorial experiments,dextrin was the most suitable carbon source,peptone and urea were the suitable compound nitrogen sources among the examined materials.Three significant factors(urea,inoculum and initial pH) were selected from the eight factors related to lipase production by Plaekett-Burman method,and were further optimized with response surface analysis.And then,steepest ascent procedures were applied to define the optimal response region of the three factors.The obtained optimal conditions were urea 0.15%,inoculum 3.05% and initial pH 8.38,under which conditions,the enzyme activity was improved from 25.37 U/ml to 48.88 U/ml,enhanced 1.93 folds.Starting from the flask conditions,the highest lipase activity of 47.69U/ml was achieved by batch fermentation in a 10 L fermentor after 52 h of the cultivation.
ABSTRACT
Bioimprinting is a new developed technique to improve the characteristics of enzymes.Bioimprinting by lauric acid was conducted to improve the esterification activity of lipase PS in sol-gel immobilization process with methyltrimethoxysila(MTMS) and tetramethoxysila(TMOS) as the precursors.Results generated by checking the esterification activity and scanning electron microscope showed that bioimprinting can enhance the specific activity and thermal stability of lipase PS.The bioimprinting system was optimized by orthogonal experiment,and the optimal condition for lipase bioimprinting is water/silane molar ration(R) 12,polyethylene glycol(PEG) 120?l,and lauric acid 0.15 mmol.Compared with the free enzyme and the non-imprinted enzymes,the specific activity of imprinted enzymes has been improved 44.3 fold and 2.4 fold,respectively.Imprinted lipase show better thermal stability,and the relative activity is 58% after incubated in 80 ℃ for 0.5 h,while no activity was detected for the free enzyme.
ABSTRACT
The fermentation conditions of lipase production by Geotrichum candidum Y162 were optimized. Initially, the most suitable carbon olive oil, nitrogen source soybean flour and NH4Cl, salt BaCl2 and MgCl2 were selected according to single factorial experiments respectively. Based on the result, screening methodology Plackett-Burman design was used to evaluate the effects of twelve factors related to lipase production and three statistically significant factors olive oil, BaCl2 and NH4Cl were selected. The path of steepest ascent was used to approach the optimal region of lipase production subsequently. Then, the optimal combined concentration for maximum enzyme activity were further optimized by response surface methodology and determined as follows: olive oil 2.35%, BaCl2 0.36%,and NH4Cl 4.69%.The optimization of culture conditions of G.candidum Y162 led to a 2.25-fold increase in lipase production relative to initial result 14.16 U/ml, which indicate that single factor in combination with response surface methodology is an effective method for optimization of lipase production conditions by G.candidum Y162.
ABSTRACT
From the N-terminal amino acid sequence of the Aspergillus niger F044 lipase,a potential homologous gene A84689 to the anl(the gene encoding the Aspergillus niger lipase)was found by means of bioinformatics.Based on the nucleotide sequence of the A84689,primers were designed to amplify anl.Nucleotide sequencing of the genomic anl gene revealed an open reading frame of 1 044 nucleotides,containing three introns(54,45 and 51 nucleotides).The deduced amino acid sequence of the anl gene corresponds to 297 amino acid residues including a signal sequence of 27 amino acid residues.The cloned cDNA coding for mature Anl(the protein of the Aspergillus niger lipase)was overexpressed in Escherichia coli BL21(De3),and the recombinant Anl was purified.The denatured recombinant Anl by 8mol/L urea was refolded in vitro by dilution and DEAE Sepharose Fast Flow chromatography.