Study On Highly Expression Of Prolyl Endopeptidase In Non-spore A.niger SH-2 Via The Strain Reformation

Prolyl endodeptidase(EC3.4.21.26,PEP),a new type of serine proteinase S28,has the function that PEP can have a reaction with the peptide bond between a proline residue specificly.Proline exists in biologically active peptide of many diseases,including melancholia,Parkinson's disease and celiac disease.Even though PEP is widely used in some areas such as biological medicine,polypeptide,food and brewing industry etc,the fewer microbiology sources,the low yield and the low enzymatic activity are still problems.What does this dissertation use is the Aspergillus niger SH-2 which does not produce reproductive spores,has morphological mutation and forms strong and branchy hydha.What's more,this kind of Aspergillus niger SH-2 has a strong ability of expression about protein secretion so that it is used as the host to express homologous or heterologous protein in industry.At the same time,Aspergillus niger SH-2 is also one kind of microbiology approved by GRAS because of its strong and impeccable ability on posttranslational modification of proteins.Besides,Asperillus niger SH-2 is safe,highly productive and efficient when it expresses the homologous or heterologous protein.Therefore,this dissertation revolves around expressing PEP highly and does the following studies.This paper adopted the method of homologous recombination and marker gene recycle to acheived multiple copies endogenous secretory protein genes of the host strain such as amylase.The host strain reduced protein endogenous exocytosis protein expression to improve the purpose protein that we researched in the dissertation.This dissertation completed a copy acid amylase and two copies of the neutral amylase knock-out in host strain,fermentation supernatant on SDS-PAGE analysis showed that endogenous three amylase protein bands disappear,endogenous secretory protein content declined dramatically.We further built a termination and recycled marker genes expression plasmid commonality of filamentous fungi,and based on the background of building endogenous secretory protein knock out the host of peptide enzyme in optimization of PEP expression,study two different strong promoter,including amylase promoter,preserved under the hybrid promoter of peptide enzyme in PEP expression;amylase promoter and hybrid promoter under the control of PEP expression frame and integrated into the host chromosome.Based on 5 L fermentation tank tested the first copy of the product of the enzyme activity reached 6.89 U/ml,compared with the laboratory prophase research express quantity increased.relatively very potential campared to DSM PEP concentration Brewer Clarex whose enzyme activity is 11.7nkat/ml.