| Filamentous fungi has a strong ability of protein secretion. It not only has complete eukaryotic cells transcription and translation system, but also has a protein posttranslational processing modification system.In many categories of filamentous fungi, Aspergillus niger is the most representative. Surface display technology of microbial can take the gene of foreign proteins with the gene of a specific anchor protein to express together, and through the role of the anchor protein, enzyme immobilizes on the cell surface. The immobilization method, compared with the conventional immobilization methods, has many advantages.At present, there exists fermentation technology research of heterologous protein expression system of Aspergillus niger, and the research on mechanism of Aspergillus niger metabolism also increases gradually, but there is no research on the surface display CALB fermentation conditions of Aspergillus niger.AN-CALB-CWPA Aspergillus niger strain in this study was a specialized structures strain of cell walls also without spores. and the Aspergillus niger saccharifying enzyme promoter gla A as a strong induction type promoter, and we take the type of carbon source and carbon source concentration control as the main goal to study the fermentation conditions of the strains, and temperature, p H value, dissolved oxygen as important factors of fermentation content of this study. This experiment combines research methods of shake flask fermentation condition and the research method of 2L fermentation tank conditions, investigating the five factors above. The fermentation conditions of final 2 L fermentation tank were determined: quantity of 20%, seed liquid OD = 4 to 6, age 2 d, fermentation canned fluid volume 1 L, medium APY, carbon sources, Sucrose, 1% of initial concentration of carbon source, constant p H6.0(ammonia flow), constant temperature 30 ℃, constant speed 500 rpm(DO not control), 3.0 Lpm constant volume; During induction period, the concentration of residual glucose is 5mmol/L, maintaining DO 30%, other conditions remain the same. Under these conditions, the enzyme activity displayed on surface can reach 1001 U/g, when it reaches the highest enzyme activity, OD600 can reach 5.7, as dry weight 19 g/L.The highest enzyme activity increased to 242.8% of enzyme activity in shake flask level.As for the applications of Aspergillus niger CALB, we carry out the experiment of Aspergillus niger CALB catalytic lauric acid butyl ester synthesis. We investigated the reaction time, concentration of substrate, substrate molar ratio, enzyme amount, reaction temperature. We prove that Aspergillus niger surface displayed CALB has biosynthesis potential in long chain fatty acids of low carbon alcohol ester compounds. The optimum conditions: hexane solvent, reaction system 10 m L, lauric acid concentration tendency for 75 mmol/L, lauric acid butyl alcohol mole ratio of 1:1.2, aspergillus Niger surface CALB 0.1 g, reaction temperature 60℃, molecular sieve 0.2 g, stirring speed 200 r/min, reaction time 3 h. Under this condition, the product yield of lauric acid butyl ester reached 92.1%, and the conversion mass is 2.3mmol·g-1·h-1 in unit mass enzyme and unit time. Under the optimal technology, we carry out 10 batches of repetition of catalytic reaction, and the relative conversion rate fell by 4.27%, proved that the surface of the Aspergillus niger surface displayer CALB has strong stability under the catalysis of long chain fatty acids low carbon alcohol ester reaction, provided a relatively complete lauric acid butyl ester product preparation. |
PDF Full Text Request