Gene Cloning, Expression And Catalytic Features Of Thioredoxin And Thioredoxin Reductase From Aspergillus Niger

Aspergillus niger is a filamentous fungus with importance in basic research and industrial application.Intracellular redox system includes glutathione system and thioredoxin system,which consists of thioredoxin(Trx),thioredoxin reductase(TrxR) and nicotinamide adenine dinucleotide phosphate(NADPH).The Trx system in Escherichia coli enables to reduce disulfide bonds of proteins and thus can be used as a food additive to reduce food allergy.However,little is known about the Trx system in A.niger.This study sought to identify thioredoxin and thioredoxin reductase from A.niger,i.e.,AnTrx and AnTrxR.The method of splicing-by-overlap extension was used to construct fusions of both proteins with designed peptide linkers alter the conserved active region of AnTrx was determined by site-directed mutation.Two selected fusions and AnTrx/AnTrxR expressed in E.coli were compared for their activities in the reduction of disulfide bonds in wheat gliadins,a source of food allergy.The results are summarized below.Gene cloning,expression and characterization of AnTrx.The gene encoding A. niger thioredoxin(AnTrx) was cloned from the fungal genome.Three site-directed mutations(C34S,C37S and C34S-C37S) of the AnTrx gene were conducted,forming the mutants AnTrx-C34S,AnTrx-C37S and AnTrx-C34S-C37S.The wild-type and mutated genes were separately transformed into E.coli for expression.As a result,the three mutated proteins purified from E.coli cultures showed no substantial activity in the reduction of insulin compared to the wild-type AnTrx.Interestingly,AnTrx-C34S significantly enhanced the activity of the purified AnTrx when an equal-volume mixture of both proteins was included in the reaction system but no difference in activity was detected when AnTrx and AnTrx-C37S were mixed.The results indicates that the Cys residue at site 34 is involved only in the first step of the reduction reaction as usual in microbial Trx whereas the Cys residue at site 37 may participate in the second step of the reaction to generate reduced protein and disulfide-vectoring Trx.Gene cloning,expression and characterization of AnTrxR.The gene encoding Aspergillus niger thioredoxin reductase(AnTrxR) was cloned from the genome of the fungus and well expressed in engineered E.coli.The recombinant AnTrxR purified from the cell culture was assayed for its activity in the reduction of 5,5-dithiobis(2-nitrobenzoic acid) (DTNB) at gradient concentrations(x) by monitoring OD₄₁₂ change(y) during the NADPH inclusive reaction.The observations were well fitted to the non-linear Michaelis-Menten equation y=V_maxx/(K_m+x).As a result,the kinetic parameters K_m and V_max(±SE) for the purified AnTrxR reducing DTNB were estimated as 1.8(±0.22) mM and 114.6(±8.4) U/mg. The catalytic efficiency(K_cat/K_m) for the reduction of DTNB to TNB was up to 42.4 mM^-1 s^-1.This efficiency is 6.7 fold of the A.nidulans TrxR catalyzing the same reduction reaction and 2.1 fold of the TrxR from mouse liver.AnTrx/AnTrxR fusion,expression,characterization and potential use for inactivating gliadin allergen.A peptide linker that naturally links Trx and Trx in Mycobacterium leprae(Linker A) and a flexible linker(GGGGS)₃(Linker B) were used to link AnTrx and AnTrxR by means of the method of splicing-by-overlap extension,forming four fusions AnTrxR-Linker A-AnTrx(RMT),AnTrxR-Linker B-AnTrx(RGT),AnTrx-Linker A-AnTrxR(TMR) and AnTrx-Linker B-AnTrxR(TGR).The fusions and their parental proteins were well expressed in transgenic E.coli.Purified fusion enzymes and parental moieties were compared for their activities and specificity to the substrates NADPH,DTNB, AnTrx,oxidative glutathione(GSSG) and bovine insulin.The two fusions RMT and RGT were capable of catalyzing disulfide bonds and acted as a complete Trx system in the presence of NADPH but TMR and TGR showed weak activities.The AnTrxR moiety fused to the N-terminus was more active.The catalytic efficiencies(K_cat/K_m) of the AnTrxR moiety of RMT and RGT for reducing DTNB and NADPH were greatly enhanced compared to the parental enzyme.The efficiencies of RMT and RGT were enhanced by 65.8 and 33.5 fold in the DTNB reduction and by 3.7 and 1.4 fold in the NADPH reduction,respectively.However,the catalytic efficiencies of the AnTrx moiety of of RMT and RGT were reduced to 31.8%and 7.5%in the dithiothreitol-induced insulin reduction and to 46%and 68%in the GSSG reduction, respectively,compared to those of parental AnTrx.Moreover,both RMT and RGT were proven to reduce disulfide bonds in wheat gliadins in the presence of NADPH and were similar to the two parental proteins in the extent of reduction after 3-h incubation based on SDS-PAGE analysis under fluorensence.This indicates that the fusions are of potential use for reducing food allergens.