Heterologous Expression Of Hyaluronidase In Ganoderma Lucidum And Determination Of Key Residue Sites

Hyaluronidase（HAase）is a glycosidase that catalyzes the degradation of Hyaluronic acid（HA）byβ-1,3 orβ-1,4 glycosidic bonds,to some extent,it can also catalyze chondroitin and chondroitin to a certain extent.It is widely used in clinical medicine,leather making technology and so on.At the same time,it has attracted extensive attention as the main preparation of low molecular weight hyaluronic acid.At present,the research on hyaluronidase mainly focuses on animals and bacteria,and there are few reports on hyaluronidase in fungi,in this paper,the hyaluronidase from Ganoderma lucidum was heterologous expressed,the properties of hyaluronidase were studied,and site directed mutation was carried out.The main findings and findings are as follows:1.Construction and heterologous expression of hyaluronidase recombinant plasmidThe hyaluronidase gene from Ganoderma lucidum（Gen Bank:LR729866.1）was analyzed and primers were designed.The target gene was obtained by PCR,the size of which was 1683bp,and the recombinant plasmid was constructed by pET-15b vector,it was named pET-15b-HAase and transformed into E.coli BL21 for induced expression.The protein was purified by Ni-NTA column and analyzed by SDS-PAGE.The target protein was about 62 k Da.2.Characterization of wild-type enzymesThe enzymatic properties of wild-type were studied by reducing sugar detection（DNS）.The optimum reaction temperature was 40℃;The optimum p H value is 5.0;The results of substrate specificity showed that it had the highest activity to hyaluronic acid and had a certain degradation effect to chondroitin sulfate;The inhibitory effect of monovalent K⁺,divalent Mg²⁺,Cu²⁺and trivalent Al³⁺on the enzyme increases with the increase of concentration,among which Cu²⁺has the most obvious inhibitory effect,Na⁺has the opposite inhibitory effect with the increase of concentration,trivalent Fe³⁺has obvious activation at low concentration,and also shows an inhibitory trend with the increase of concentration;Methanol and glycerol in organic solvents showed a trend of low concentration promoting high concentration inhibition,and the inhibitory effect of ethanol and dimethyl sulfoxide was stronger with the increase of concentration;Using hyaluronic acid as substrate,the enzymatic reaction kinetic curve conforms to Michaelis equation.Under the optimum reaction conditions,Km=0.502 mg/ml,Vmax=1.419μmol/m L/min.3.Identification of critical residuesThe crystal structure of hyaluronidase was constructed by homologous modeling technology.Use the online evaluation website SAVES to evaluate the model,the highest score model was selected to dock with the substrate HA,and the important conserved sites were determined in combination with homologous sequence alignment.Finally,the 79th arginine residue was selected as the mutation site,and the whole plasmid PCR technology was used for site-specific mutation to obtain the mutant R79A.4.Characterization of enzyme properties of mutant R79AThe enzymatic properties of the mutant R79A were determined by reducing sugar method.The optimum reaction temperature was 37℃and p H was 5.0.Substrate specificity showed that the degradation effect of chondroitin sulfate was better than that of hyaluronic acid;All metal ions showed inhibition,and the degree of inhibition was divalent>trivalent>monovalent;The four organic solvents showed the trend of low concentration inhibition and high concentration promotion,among which dimethyl sulfoxide was the most effective;Using hyaluronic acid as substrate,the kinetic parameters measured by Michaelis equation were Km=0.591 mg/m L and Vmax=1.154μmol/m L/min.Compared with the wild-type mutant,it was found that some properties of the enzyme changed greatly,among which the optimum reaction p H did not change obviously,and the optimum reaction temperature changed slightly,from 40°C to 37°C.It becomes very sensitive to metal ions,and its ability to degrade chondroitin sulfate is greatly improved.Through the observation of enzyme kinetics,it is found that the K_m value of the mutant increases,indicating that the affinity for the substrate is weakened,which is not conducive to the nucleophilic attack between the enzyme and the substrate.According to the changes of enzyme activity,it is speculated that the arginine located at site 79 may have played an important role in the specific recognition of the substrate.