Identification Of Xylanase-producing Thermophilic Fungi, Gene Cloning And Construction Of Aspergillus Oryzae Expression System

Xylanase is a kind of xylan degrading enzyme system,widely used in food and feed fields.However,the application of the enzyme is currently restricted by factors such as low enzyme activity,poor thermal stability,and insecurity.In this paper,we combined the food-grade Aspergillus oryzae system and the xylanase-producing thermophilic strain to obtain xylanase-Aspergillus oryzae strain,which provides a new idea for using the genetic engineering technique researching xylanase with high enzyme activity and low cost.The main experimental results are as follows:(1)Using morphology,18 S rDNA and enzyme properties to identify the xylanase producing fungi TL01 which is saved by laboratory,the results show that the fungi is Thermomyces lanuginosus,the crude enzyme activity is 0.250?0.03 U/mL;Cloning and analysising its two major xylanase genes(xyn11A and xyl43).The bioinformatics software predicts that the molecular weight of xyn11 A is 24.36 kDa and isoelectric point is 4.77,it has a signal peptide sequence which contain 19 amino acidsand.This protein is a hydrophilic protein with no transmembrane domain.Its secondary structure is mainly composed of random coil(44.89%);Otherwise,the molecular weight of protein xyl43 is 38.24 kDa and isoelectric point is 5.23,the protein is a hydrophilic protein with no transmembrane domain.Its secondary structure is mainly composed of random coil(59.76%).(2)A pyrG auxotroph strain RIB40?pyrG was obtained by UV mutagenesis conbined with phenotype and sequenceing.The expression vector pBC-Hygro.4 was constructed with pyrG selected marker,promoter and signal peptide amyB,His-tag and the reporter gene GFP.Using the PEG method to transfer the vector into Aspergillus oryzae RIB40?pyrG,the fluorescent mycelium was observed by fluorescence microscopy,which proved that the vector was successfully constructed.(3)The xylanase-Aspergillus oryzae expression vectors pBC-Hygro-xyn11 A and pBC-Hygro-xyl43 were constructed and transferred into Aspergillus oryzae RIB40?pyrG.Then,two expression host strains were successfully screened by colony PCR.In a conclusion,this experiment is the first time to construct a xylanase-Aspergillus oryzae expression system,which provides a foundation for further research on the efficient expression of xylanase.