Research And Application Of D-lactate-oxidizing Enzymes From Gluconobacter Oxydans

Gluconobacter oxydans,a Gram-negative,obligate aerobic,acetic acid bacterium,is famous for its unique capability to enable the rapid and incomplete oxidation of a wide range of sugars and alcohols.With this unique capability,G.oxydans has been widely used not only in numerous traditional production processes for the synthesis of compounds such as vitamin C,vinegar,gluconic acids,and dihydroxyacetone,but also in new processes for the synthesis of compounds such as D-tagatose,miglitol,and chiral aldehydes and acids.Due to the sequencing of the G.oxydans genome at 2005,more and more researchers are focus on the studies about the metabolic pathways of G.oxydans at molecular level and the biochemical and physiological characteristics of the enzymes from G.oxydans in recent years.The habitats of G.oxydans contain numerous organic compounds,such as sugars,alcohols,amino acids,and organic acids.Utilization of these organic compounds is vital for the survival of G.oxydans.The growth of G oxydans 621H on various sugars and alcohols has been investigated in details.In this study,the growth of G.oxydans on proteinaceous amino acids and common organic acids was examined.It was found that only several 2-hydroxy acids,particularly D-lactate,supported the growth of G.oxydans 621H.However,pyruvate,the product of D-lactate oxidation,did not support the growth of G.oxydans in our observations.Thus,the driving force of D-lactate on G.oxydans 621H growth should derive solely from D-lactate oxidation,which was distinct from that in other lactate utilization bacteria.Thus,this study mainly focused on the growth mechanism of G.oxydans 621H on D-lactate,the properties and applications of the D-lactate-oxidizing enzymes involved.Lactic acid,i.e.,2-hydroxy-propionic acid,is the most common 2-hydroxycarboxylic acid.It exists extensively in nature and human body.The molecule of lactic acid contains a symmetric carbon atom.Thus,it has two isomers,L-lactic acid and D-lactic acid,based on optical rotation.Lactic acid,containing two functional groups,hydroxy and carboxyl,involves in many microbial metabolism processes which play important roles in the survival,applications and pathogenicity of microorganism.Thus,the studies on microbial lactate metabolism are significant.The key enzymes involved in microbial lactate metabolism are NAD-dependent lactate dehydrogenase(nLDH)and NAD-independent lactate dehydrogenase(iLDH).nLDH catalyzes pyruvate to lactate and the reverse reaction is also permitted at specific conditions.Thus,nLDH plays important roles in lactate anabolism.In contrast,iLDH catalyzes lactate into pyruvate and no reverse reaction activity can be detected.Thus,iLDH plays important roles in lactate catabolism and usually acts as a key enzyme to support the growth of microorganism on lactate.In this study,to understand the utilization mechanism of D-lactate by G.oxydans 621H,its genome was analyzed and two putative D-iLDHs(GOX1253 and GOX2071)were identified.The two putative D-iLDHs were characterized and their physiological functions were investigated to clarify the unusual mechanism of G.oxydans growth on D-lactate,At first,the genes of these two putative D-iLDHs were knockout individually or in combination in G.oxydans 621H and their respective complement strains were also constructed.The results revealed that both GOX1253 and GOX2071 can catalyze the oxidation of D-lactate and are constitutive expression but they have different cellular locations,GOX1253 on the cytoplasmic membrane while GOX2071 in the cytoplasm.GOX1253 and GOX2071 were overexpressed inEscherichia coli and purified.The purified enzymes contained FAD as cofactor.Active GOX1253 exists as a tetramer while active GOX2071 exists as a dimer.Bioinformatics analysis of these two putative D-iLDHs revealed that the predicted FAD-binding domain of GOX1253 is located near the N-terminus of the protein,while the C-terminus contains a predicted membrane-bound lactate dehydrogenase domain.The predicted domains of GOX1253 are similar to that of respiratory D-iLDH from E.coli and this type of D-iLDH is only found in bacteria now.GOX2071 has an FAD-binding domain at the N-terminus,but an FAD-oxidase domain was found at the C-terminus.GOX2071 shared low homology with the well-studied D-iLDHs and its homologous proteins widely distribute in the three domains of life.The substrate spectra of the enzymes were determined using MTT as an electron acceptor.The result showed that although the specific activity of GOX1253 for D-lactate reached 22.31 U mg-1,GOX1253 had narrow substrate specificity and incomplete stereoselectivity for the D-isomer(i.e.,low but detectable activity for L-lactate),while GOX2071 showed a relatively low specific activity for D-lactate(0.35 U mg-1),but had broad substrate specificity and high stereoselectivity.Their preferences for different artificial electron acceptors and their ability to use molecular oxygen as electron acceptor were assessed.The results showed that GOX1253 is a typical D-iLDH,while GOX2071 appears to be a novel type of D-iLDH,a D-lactate oxidase.The further investigation of the mechanism of GOX2071 showed that GOX2071,as a D-lactate oxidase,catalyzed the oxidation of D-lactate to pyruvate and H2O2 and that the generated H2O2 oxidized pyravate to acetate.D-Lactate-dependent growth of the mutant and complement strains were determined and it showed that only GOX1253 is the key enzyme for its D-lactate-dependent growth.Combined with the results of this study and some speculations,a model for D-lactate oxidation in G.oxydans 621H was proposed.D-Lactate may enter into cell through lactic acid permease(GOX2098),GOX1253,located on the inner of membrane,oxidizes D-lactate to pyruvate and transfers the electrons to quinone then to quinone oxidases.The quinone oxidases use the electrons to reduce O2 into H2O with pump-out of hydrogen proton.The formed electrochemical proton gradient was used by F1F0 ATP synthase to generate ATPs that provide energy for the growth of the strain.Due to the incomplete TCA cycle and EMP of G.oxydans,pyruvate derived from D-lactate cannot enter into the TCA cycle for further metabolism or for production of C6-sugars via gluconeogenesis,and has to enter a non-energy generation pathway with acetate as the final product.Namely,pyruvate may be catalyzed to acetaldehyde by pyruvate decarboxylase(GOX1081)and the latter may be further oxidized to acetate by acetaldehyde dehydrogenase(GOX2018).In the cytoplasm,GOX2071 may also play minor roles in oxidation of D-lactate to acetate.This study not only clarified the mechanism of G.oxydans 621H growth on D-lactate,but also experimentally identified a bacteria containing two D-lactate-oxidizing enzymes for the first time.Since GOX2071 is the first identified D-lactate oxidase,it is necessary and meaningful to investigate the enzyme characteristics and catalytic mechanism of GOX2071 deeply.In this study,the enzyme characteristics of GOX2071 was assessed using MTT as an electron acceptor.The results showed that its optimal pH and temperature are pH 8 and 55?;respectively,and the enzyme is relatively stable in the buffer of pH 7-9 below 50?.Cd2+,Zn2+,Co2+,Cu2+,Mn2+,and Fe3+ showed obvious inhibition to GOX2071,especially Cu2+ and Fe3+ inhibiting more than 90%activity.The kinetic constants of D-lactate oxidase GOX2071 were evaluated by a Clark-type oxygen electrode(Oxytherm)using O2 as an electron acceptor.The estimated apparent Km and Vmax of GOX2071 towards D-lactate were 3.62±0.53 mM and 0.52± 0.05 U mg-1,respectively.The estimated apparent Km and Vmax for O2,the other substrate,were 0.16 ± 0.01 mM and 0.97 ± 0.03 U mg-1,respectively.The anaerobic reduction of GOX2071 by D-lactate was investigated and showed that FAD(yellow)bounded to GOX2071 was reduced to FADH2(colourless)in the presence of D-lactate under anaerobic condition,which indicated that the reaction of the GOX2071-catalyzed D-lactate oxidation starts with the combination of D-lactate and the enzyme followed by the oxidation of D-lactate and the electrons transferring to FAD of the enzyme.Steady-state kinetics of GOX2071 revealed that it follows Ping-Pong bisubstrate-biproduct(Bi Bi)mechanism.Further,the effects of two catalysates(pyruvate and H2O2)on activity of GOX2071 were evaluated.The results showed that the first product,pyruvate,exhi'bited competitive inhibition towards the second substrate,O2;the second product,H2O2,exhibited competitive inhibition towards the first substrate,D-lactate,which indicated that GOX2071 follows a single-site ping pong Bi Bi mechanism.Optically active 2-hydroxycarboxylic acids(2-HAs)are versatile building blocks for the asymmetric synthesis of various significant compounds and widely used in pharmaceutical,chemical,biological synthesis and other fields.Abundant racemic 2-HAs are relatively easy to obtain from biological sources and chemical processes.Hence,kinetic resolution,particularly enzymatic resolution with high stereoselectivity and mild conditions,is a promising alternative for preparing enantiopure 2-HAs.Although the production of(R)-2-HAs via oxidase-catalyzed resolution was achieved at the end of last century,currently,there is no report of(S)-2-HA production from racemates using oxidase due to lack of the related oxidase.Aiming at this problem,in this study,suitable enzymes were systematically screened by analyzing numerous putative D-lactate oxidase sequences and sequentially identifying several required properties.D-lactate oxidase from G.oxydans 621H,with several advantageous characteristics,such as good solubility,broad substrate spectrum,and high stereoselectivity,was selected for resolving 2-HAs into(S)-2-HAs.A variety of(S)-2-HAs were successfully produced using this D-lactate oxidase with excellent enantiomeric excess values(>99%)and high yields.By coupling a catalase to eliminate the by-product H2O2,another type of important organic synthesis and pharmaceutical intermediates,2-oxocarboxylic acids,were co-produced with high product concentration close to their theoretical values.This is the first study to produce(S)-2-HAs via enzymatic resolution using oxidase.The novel approach developed in this study is a promising alternative to existing methods of producing enantiopure(S)-2-HAs.(R)-2-Hydroxy-4-phenylbutyric acid[(R)-HPBA],an important 2-hydroxycarboxylic acid,is a key precursor for the production of angiotensin-converting enzyme inhibitors.However,the product yield and concentration of reported(R)-HPBA synthetic processes remain unsatisfactory.The Y52L/F299Y mutant of NAD-dependent D-lactate dehydrogenase(D-nLDH)in Lactobacillus bulgaricus ATCC 11842 was found to have high bio-reduction activity towards 2-oxo-4-phenylbutyric acid(OPBA).The mutant D-nLDHY52L/F299Y was then coexpressed with formate dehydrogenase in E.coli BL21(DE3)to construct a novel biocatalyst E.coli DF.Thus,a novel bio-reduction process utilizing whole cells of E.coli DF as the biocatalyst and formate as the co-substrate for cofactor regeneration was developed for the production of(R)-HPBA from OPBA.The optimum conditions were pH 6.5,OPBA 75 mM,and catalyst of 6 g DCW L-1.Under the optimum conditions,73.4 mM OPBA was reduced to 71.8 mM(R)-HPBA in 90 min and both its product enantiomeric excess(>99%)and productivity(47.9 mM h-1,)reached relatively high levels currently.