Breeding Of Selenium Rich Strains Of Ganoderma Applanatum And Research Of Enzymes For Cell Wall Disruption To Extract Selenoproteins

Ganoderma applanatum is a large fungus with high medicinal value,which can effectively treat diseases such as tumors,hepatitis,pneumonia,hypertension,and hyperglycemia.In recent years,selenium rich foods have become a hot topic in the field of health food research due to their anticancer,antioxidant and other functions.Many varieties of Ganoderma lucidum have received widespread attention and research due to their high selenium conversion rate.However,there have been no reports on the selenium rich research of Ganoderma applanatum with high medicinal value.Selenoprotein are the main forms of organic selenium in Ganoderma lucidum and other large fungi.They can form a series of antioxidant enzymes in the human body,promote the metabolism and repair of their own cells,and also play a certain role in preventing immune system diseases,diabetes,thyroid diseases,cardiovascular diseases,hepatitis diseases,brain nerve diseases and reproductive system diseases,How to effectively use selenoprotein has attracted much attention in the field of food processing.Ultrasonic assisted alkaline extraction is a commonly used method to extract protein from plants.However,the quality of selenoprotein extraction will be affected due to the solid cell wall structure of large fungi and the long ultrasonic time.Enzymatic extraction of selenoprotein is relatively mild,but expensive.The ability to break the cell wall will affect the efficiency of protein extraction.Therefore,it is of great practical significance to develop an efficient extraction process for selenoprotein.In this study,a mutant YL108 of Ganoderma applanatum obtained by screening and mutagenesis breeding was used as a selenium rich carrier to obtain selenoprotein,screen wall breaking enzymes that can effectively degrade fungal cell wall,and found that theβ-glucanase AnEglA6 derived from Aspergillus niger and chitinase ECH42 derived from Trichoderma harzianum can degrade the cell wall of Ganoderma applanatum.Based on this,this study further modified these two enzymes to enhance their ability to degrade Ganoderma lucidum.After enzymolysis,the selenoprotein in Ganoderma applanatum could be effectively extracted by short-term ultrasound.The main research contents of this paper are as follows:（1）A selenium tolerant Ganoderma applanatum XQ20115 was screened from abandoned selenium mines in Lichuan County,Hubei Province.Adopting ⁶⁰Co-γRadiation induced breeding to obtain a highly selenium tolerant mutant strain YL108 of Ganoderma applanatum.The biomass of YL108 in a selenium containing medium of 90 mg·L^-1 is 11.9 g·L^-1,and the selenium content of bacterial protein is 937 mg·kg^-1.（2）The cellulose binding domain（CBD）of AnEglA6 was replaced by the CBMs family that can degrade insoluble cellulose.Among them,the recombinase An Eg-CBM9 and An Eg-CBM10 from CBM9 and CBM10 families showed high activity,and the range of substrate utilization was significantly expanded,and the ability to degrade the cell wall of tongue was also significantly improved.In order to improve the degradation ability of chitinase on Ganoderma applanatum cell walls,a CRISPR/Cas9 system of Pichia pastoris was constructed.The vacuolar sorting receptor protein genes Vps10p-1 and Vps10p-2 of Pichia pastoris were knocked out,which increased the extracellular secretion of chitinase ECH42 in Pichia pastoris.After knocking out Vps10p-2,the expression of chitinase ECH42 increased by 50%.（3）The method of BGL6906+An Eg-CBM10+ECH42 was used to hydrolyze the mycelium of tongue to greatly weaken the strength of cell wall.The ultrasonic time was 5 min,and the protein extraction rate of Ganoderma applanatum reached 90%.Further,the ultrasonic assisted enzymatic extraction process of selenoprotein from tongue was optimized through response surface experiments,and the protein extraction rate was finally increased to 94.8±0.5%.（4）Quantitative analysis of selenocysteine,selenomethylcystine,and selenomethionine in extracted selenium rich Ganoderma applanatum protein by HPLC-ICP-MS showed that they were 389 mg·kg^-1,2 mg·kg^-1 and 107 mg·kg^-1 mg·kg^-1,respectively.It can be seen that selenocysteine and selenocysteine mainly exist in the form of selenocysteine and selenocysteine in selenium rich tree tongue Ganoderma lucidum protein.