Study Of Glucose Oxidase From Penicillium Chrysogenum A4 And Its Heterologous Expression

Glucose oxidase?GOD;E.C.1.1.3.4?catalyzes the oxidation of P-D-glucose to gluconic acid and hydrogen peroxidase.The catalytic reaction is fast,safe and high-specific,so the enzyme has gained extensive commercial applications,used as food preservatives and stabilizers,bleaching agents in textile industry,the biosensor of glucose concentration in fermentation and medical diagnostics.Traditional commercial preparations of glucose oxidase are mostly derived from the genera Aspergillus and Penicillium.Glucose oxidase from Penicillium spp.have more advantages than those extracted from A.niger.The former are extracellular enzyme that are easier to be extracted as well as the latter are intracellular enzyme.Glucose oxidase from Penicillium spp.have enhanced kinetic parameters and stability.Its application can reduce the industrial cost from the separation and purification process,as well as improve the sensitivity and application effect when used as biosensor and glucose quantitative detection.In this study,a high glucose oxidase producing strain was sereened and isolated from soil and rotten fruit,which was identified as P.chrysogenum A4,and the medium components and fermentation condition were optimized for higher glucose oxidase production.The gene of glucose oxidase was cloned,analysed and heterologous expressed in Pichia pastoris.The purification,characterization of the native and the recombinant enzyme were also studied,aimed at providing theoretical and practical evidence for developing a new glucose oxidase.The main results are as follows:1.Screening and identification of extracellular glucose oxidase producer,and the characterization of the native enzyme were studied.34 strains of glucose oxidase producing fungi from soil and rotten fruit in Jiangsu province were preliminary screened and isolated by Fiedure K.J.colorimetric method.A strain named A4 was selected for its high level of glucose oxidase activity in the liquid fermentation condition using o-dianisidine-H2O2 colorimetric method.It was identified as Penicillium chrysogenum by multi-sequenses analysis of 18S,ITS,BenA,CO1,polygenetic dendrogram construction and morphological observation.The glucose oxidase of P.chrysogenum A4 was purified by 75%?NH4?2SO4 precipitate and 50 kDa ultrafiltration from the culture filtrate and partially characterised.It was identified as a homo-dimeric protein with a relative molecular mass of 149 kDa by gel filtration chromatography while denaturing SDS-PAGE indicated a single subunit band of 75 kDa.The enzyme was highly specific for ?-D-glucose and show only marginal activities with D-galactose.The enzyme displayed an optimal activity for the oxidation of glucose at 35? and pH 5,and exhibited a relatively broad temperature profile between 25-60?.Tween 20,SDS and Ca2+ activate glucose oxidase activity.Fe2+,Fe3+,Cu2+,Mn2+show various degrees of inhibition.The Km and Vmax values of the enzyme were 1.3 mM and 7.5 U/mg,respectively.2.Optimization of liquid fermentation conditions for P.chrysogenum A4 producing extracellular glucose oxidase was studied.Effects of different kinds of carbon source,organic nitrogen source and inorganic nitrogen source on extracellular glucose oxidase production by P.chrysogenum A4 were studied by single-factor tests,respectively.The addition amount of glucose,malt extract and NaNO3 were optimized by orthogonal test.Results showed that the optimal addition amounts were as follow:glucose 10%,malt extract 0.5%,NaNO3 1.7%.The culture condition was also studied with the optimal medium.Results showed that the optimal fermentation conditions were as follow:1 mL spore suspension was incubated in 50 mL of culture medium at 180 rpm and 30? for 6 d.The glucose oxidase activity was 17.2 times that of initial enzyme activity.3.The gene encoding glucose oxidase from P.chrysogenum A4 was cloned,characterized and heterologous expressed in Pichia pastoris,and the characterization of the recombinant enzyme was studied.The glucose oxidase gene from P.chrysogenum A4 consisting of 1815 bp.was acquired by PCR and confirmed by DNA sequencing.It encoded a putative peptide of 604 amino acid residues with a theoretical molecular mass of 66.3 kDa.Comparison of the glucose oxidase gene sequences with other strains in the GenBank database,P.chrysogenum?accession number JN809249.1?and Aspergillus terreus?accession number XM_<sub>001215424.1?showed identity degrees of 99%and 75%,respectively.The putative amino acid sequence of P.chrysogenum A4 GOD have 76-99%similarity with these two strains of filamentous fungi.The open reading frame of the glucose oxidase consisted of a secretion signal peptide of 18 amino acids,4 putative N-glycosylation sites?Asn111?Asn278?Asn374?Asn409?and 3 conserved Cys residues at conserved region?Cys 186,Cys 228,Cys 542?.The expression plasmid GODP-pPICZaA was constructed and transformed into P.pastoris SMD1168.A strain R2 producing 0.5 U/mL of heterologous glucose oxidase after 4 d of fermentation was selected.Effects of liquid fermentation conditions on recombinant glucose oxidase production by P.pastoris R2 were studied by single-factor tests,respectively.Results showed that the highest catalysis activity of 1.1 U/mL was obtained when the inoculum was cultivated in 40 mL of BMMY medium?pH 6.0?with 2.0%methanol addition every 24 h for 5 d.The recombinant glucose oxidase was purified by 80%?NH4?2SO4 precipitate and 30 kDa ultrafiltration.The subunit had a molecular weight of 75 kDa by SDS-PAGE,comparison of carbohydrate moieties showed a same glycosylation level of the recombinant glucose oxidase and the native enzyme.The recombinant enzyme was optimally active for the oxidation of glucose at 50? and pH 6.0,and a higher thermal stability than the native enzyme.The Km and Vmax values of the recombinant enzyme were 44.9 mM and 9.8 U/mg,respectively.