Heterologous Expression Of Maltase-glucoamylase In Ganoderma Lucidum And Identification Of Key Residue Sites

Maltase-glucoamylase（MGAM;EC3.2.1.20 and EC3.2.1.3）are glycoside hydrolases that mainly hydrolyzeα-1,4-glycosidic bonds.They are brush-edge membrane enzymes at the end of small intestine and provide theoretical help for the production of type 2 diabetes inhibitors.It is widely used in medical diagnosis,biological repair and other fields.Since MGAM in Ganoderma lucidum has the most abundant content and high activity,this study took MGAM as the research object and carried out heterologous expression test and spatial structure study,which has important theoretical significance and medical application value for the production and preparation of single MGAM.At present,there are many studies on MGAM from animals,but there are few reports on MGAM from large fungi.In this paper,MGAM from Ganoderma lucidum was cloned,expressed,isolated and purified,and its enzymatic properties were characterized.In addition,the spatial structure of MGAM was reconstructed,and the important amino acid residue sites were determined by combining bioinformatics techniques.Mutants were obtained by site-directed mutation and characterized by enzyme properties,so as to explore its influence on enzyme activity.Main research content:MGAM gene was amplified by PCR,recombinant plasmid p ET-15b-MGAM was constructed,and induced expression was conducted in Escherichia coli.The recombinant protein was obtained after separation and purification,and its properties were characterized.Bioinformatics methods were used to conduct homologous modeling and substrate docking analysis to determine the key residue sites for substrate binding.Combined with the results of homologous sequence alignment,the key residue sites for substrate binding were selected and site-directed mutations were performed.The mutants were screened by full-plasmid PCR,and the induced expression,isolation and purification of the mutants were analyzed,and the enzymatic properties of the wild-type and mutant were compared to explore their effects on enzyme activity.Research results:1.The maltase-glucoamylase gene of Ganoderma lucidum（Gen Bank:LR729362.1）,primers were designed and the target gene was amplified by PCR.After double enzyme digestion,the target gene was connected to plasmid p ET-15b,and the recombinant plasmid p ET-15b-MGAM was successfully constructed and transformed into EScherichia coli BL21,which was efficiently induced at 28℃and 120rpm.High purity protein was obtained by ni-nta affinity chromatography.Sds-page showed that the molecular weight was about 90k DA.2.Characterization of wild-type properties:MGAM is a water-soluble protein.The optimal p H and temperature of the wild-type are 6.0 and 65℃respectively.The monovalent metal ions Na⁺and K⁺and bivalent metal ions Cu²⁺and Mg²⁺show activation or inhibition at different concentrations,while the trivalent metal ions Al³⁺and Fe³⁺always show activation,especially the activation effect of Al³⁺is most obvious.Low concentration of organic solvents methanol,ethanol and DMSO all had activation effects on wild-type p ET-15b-MGAM in different degrees,and DMSO>propanol>methanol>ethanol.But all of them showed inhibition at high concentration.The Km and Vmax of wild-type p ET-15b-MGAM with p-Nitrophenyl-β-D-Galactopyranoside（pNPG）as substrate were 1.129m M and 6.471μmol/ml/min,respectively.3.Determination of key residue sites:The crystal structure of maltase-glucoamylase was constructed by homology modeling using bioinformatics methods.By docking with substrate pNPG,it was found that D246 site was bound to substrate through hydrogen bond.Homologous sequence alignment revealed that D246 site was an absolutely conserved site in the family,so the amino acid residue at site 246 was selected for site-directed mutation.The negative polar aspartic acid was mutated into non-polar alanine by full plasmid PCR,and the mutant D246A was obtained.4.Characterization of mutant properties:The optimal p H and temperature of mutant D246A were 7.5 and 60℃respectively.The monovalent and divalent metal ions inhibited the enzyme activity at low concentration,and activated it with the increase of concentration.Different concentrations of trivalent metal ions Al³⁺and Fe³⁺showed activation effect,and the activation effect of Al³⁺was the most obvious.Organic solvents with different concentrations of glycerol,ethanol,methanol and DMSO have different degrees of activation at low concentration,but show inhibition at high concentration.The kinetic curve of mutant D246A,which was based on pNPG,was in accordance with Miltonian equation,with Km and Vmax of 1.29 m M and 1.68μmol/ml/min,respectively.Compared with the wild type,Km became larger and Vmax decreased by 4 times,which may be because the number of hydrogen bonds between the mutant and the substrate was reduced and the combination with the substrate was not firm,and the new spatial conformation was not conducive to the nucleophilic attack of the enzyme on the substrate.