Uv Mutation Of Pectinase-producing Aspergillus Niger And Proteomic Analysis Of Synthetic Regulatory Mechanisms

A pectinase-producing Aspergillus niger strain EIM-6 was mutagenized by using UV radiation and the mutant EIMU2 was obtained after screening. The pectinase activity of EIMU2 was increased to 32161 U/mL,1.212 times higher than that of the original A. niger EIM-6. At the same time, morphological characteristics of EIMU2 changed obviously. The color of spore became deeper and the spore balls grew bigger. Through Response Surface Methodology experiment, the optimal submerged fermentation condition was obtained to further improve the production of pectinase from A. niger mutant EIMU2. The optimum medium contained (w/v) 1.83% beet residue,1.69% peanut cake powder,0.5% (NH4)2SO4,0.3% K2HPO4,0.2% CaCO3,0.15% MgSO4,6% inoculum and 21.36 mL liquid volume. The optimum pectinase activity was increased by 207% compared to that of the previous.To understand the mechanism for increasing pectinase activity of A. niger EIM-6, the total amount of enzyme protein expression, gene mutation and proportion of the multi-enzyme components were studied. Inoculated the wild strain EIM-6 and mutant EIMU2 under the same conditions, and their extracellular total proteins were extracted separately. The results indicated that extracellular total protein of EIMU2 increased 14-17% than EIM-6 after weighing, suggested that the improment of the total enzyme protein expression was one of the reasons for high pectinase enzyme activity for A. niger mutant. Furthermore, the pectinase enzyme components of A. niger were identified by using two-dimensional electrophoresis and mass spectrometry analysis. There were 5 protein spots to relate the reported pectinase, including polygalacturonase-X2, three isomers of pectate lyase, and pectinesterase precursor. The tesult of MS analysis showed that the isoelectric point of pectinase components were between the pI4.0-6.0, which was consistent with the previous acidic pectinase conclusions. Then PelA and peg were cloned from A. niger wild strain EIM-6 and mutant EIMU2, respectively. The PelA and peg genes from both EIM-6 and EIMU2 were consistent, suggested that increased pectinase activity of mutant strains was not derived from gene mutation of a single pectinase component.The extracted extracellular total proteins from the wild strain EIM-6 and mutant EIMU2 were further compared through two-dimensional electrophoresis maps with same amount.4 differential-expressed proteins spots were identified. All of these 4 protein spots of EIMU2 were significantly higher than the EIM-6. The results of mass spectrometry showed that the differential-expressed proteins include:a pectinesterase precursor, a actibind protein, a hypothetical protein An14g01620 and a mitochondrial DNA-directed RNA polymerase. Pectinesterase precursor is an important component of the pectinase. The results indicated that the improvement of mutant EIMU2 pectinase activity was also related with the enzymes synthesis and regulation of A. niger.