| Glucose dehydrogenase (GDH) is a kind of oxidoreductase, which catalyzes theoxidation of glucose to generate gluconic acid-lactone. GDH is now widely used in bloodsugar detecting, biofuel cell, implanted pacemaker and industrial glucose monitoring. GDH isdivided into three parts according to their bound cofactors, which includes nicotinamideadenine dinucleotide-dependent glucose dehydrogenase (NAD-GDH), pyrrolequinine-dependent glucose dehydrogenase (PQQ-GDH) and flavin adeninedinucleotide-dependent glucose dehydrogenase (FAD-GDH). At present, glucose oxidase(GOD) is widely usedin blood sugar detectionbut GOD utilizes oxygen as electron acceptor inglucose oxidation, it resultsin the fluctuatation of testing result when the partial pressure ofoxygen in the sample is changed. GDH can use many artificial electronacceptors exceptoxygen to finish the catalytic reactionandavoid this drawback of GOD made and was thoughtto be the optimal candidate in glucose detecting. It was found that NAD did not bind withGDH tightly and will be lost easily, which would lead to poor stability for the testing of bloodglucose when NAD-GDH was employed. Unlike NAD-GDH, the cofactor PQQ can bindtightly to GDH to make the holoenzyme. However, the substrate specificity of PQQ-GDH islow and the testing result is inaccurate when several common monosaccharide ordisaccharidewas presented in the samples. Compared with NAD-GDH and PQQ-GDH，FAD-GDH is superior in both stability and substrate specificity and may be the perfectenzyme in blood glucose testing.FAD-GDHand GOD belong toglucose-methanol-choline (GMC) oxidoreductases family,and share common conservative motif, such as the FAD binding motif Gly-X-Gly-X-X-Glyand the active center Arg-X-Asn-X-His.The catalytic mechanism of GOD has been illustratedclearly on the basis of its crystal structure. On the contrary, due to its notorious difficulty forthe recombinant expression of FAD-GDH in E.coli, little progresson of structural or catalyticmechanism of FAD-GDH was obtained until now. Therefore, the studies on efficientrecombinant expression, enzymatic characterization and crystallization of FAD-GDH, willaccelerate the elaboration of the catalytic mechanism and expand the application ofFAD-GDH. Itwill facilitateboth the theoretical research andpracticalapplication of FAD-GDH.The previous research showed that inclusion body was formed when the fad-gdh gene ofAspergillus terreus was expressed in E. coli and no enzymatic activity could be detected.Then Pichia pastoris was used to express FAD-GDH, and six recombinant strains were obtained and the optimal one with the productivity of19000U/L was chose to produceFAD-GDH in large scale. After about84h of fermentation, the wet biomass was up to228g/L, the extracellular protein concentration reached0.71g/L and the volumetric FAD-GDHactivity in the culture supernatant wasup to a maximum value of2.6×105U/L. Using cooledisopropanol, FAD-GDH in culture supernatant can be efficiently precipitated with therecovery rate of up to94%. After precipitation, affinity chromatography and one moreisopropanol precipitation was carried out to purify FAD-GDH. The total recovery was65%and the specific activity was541U/mg.The recombinant expression of FAD-GDH in E. coli was further carried out after thatwas finished in P. pastoris. A series of factors including promoters, fusion tags andmolecular chaperones were tested and finally soluble FAD-GDH was successfully expressedin E. coli. With the optimization of inducer and other additives, the optimal expressioncondition was obtained as follows: using pTrc99a as expression vector, FAD-GDH wasco-expressed with molecular chaperone system of DnaK/DnaJ/GrpE in BL21(DE3) strain.When the recombinant strain was cultured in TB medium,3g/L L-arabinose and0.1mMIPTG were used to induce the expression of molecular chaperone and FAD-GDH,respectively. After induction, the volumetric activity and OD specific activity of FAD-GDHwere up to23883±563U/L and1689±40U/L OD unit, which was105-fold of the initialproductivity. On the other hand, several simple carbon sources was used to promote thesoluble expression of FAD-GDH in LB medium. When5g/L of sorbitol was added toculture while other expression condition was kept the same with above, the final volumetricactivity and OD specific activity of FAD-GDH reached22801±401U/L and1679±30U/L OD unit, similar with the yield of TB medium as culture. With one step of affinitychromatography and another step of size exclude chromatography, purified FAD-GDH wasobtained, of which the recovery was44%and the specific activity was604U/mg.Enzymatic characterization of FAD-GDH produced in P. pastoris and E. coli weredetected respectively and compared with each other. The results showed that the optimal pH,stable pH range and substrate specificity were identical between them. Both FAD-GDHs withthe optimal pH of7.5，keep stable at a wide pH range of3.5~9.0. FAD-GDHs harboring arelative high substrate specificity, apart from D-glucose, FAD-GDHs have the relative activityof17.7±1.6%and7.4±0.8%to D-maltose and D-xylose, respectively. The relative activity ofFAD-GDHs toward other monosaccharide or disaccharide is negligible. FAD-GDH expressed in P. pastoris has the optimal reaction temperature at55℃and a long half-life of82minwhen treated at62℃. The Kmvalue and Vmaxof FAD-GDH from P. pastoris were86.4±1.8mM and857±12U/mg, respectively. On the other hand, FAD-GDH expressed in E. coli haslower optimal reaction temperature and shorter half-life, which was50℃and52min under42℃, respectively. The Kmvalue and Vmaxof FAD-GDH from E. coli were86.7±5.3mM and928±35U/mg, respectively. Enzymatic activity testing demonstrated that FAD-GDH is a kindof glucose dehydrogenase, without any activity of glucose oxidase. Two characteristicabsorption peaks at wavelength of378nm and454nm were verified by full wave scanning,which disappeared when the FAD was reduced into FADH2when D-glucose was added. Sizeexclude chromatography was applied to determine the molecular weight of FAD-GDH, theresulted65.6kDa was almost the same as the calculated molecular weight of63kDa, whichverified the active monomer structure of FAD-GDH.Crystal screening of FAD-GDH was carried out with the purified protein obtainedaforementioned. More than1500conditions were screened to get a dozen of crystal conditionsby commercial kits. Based on the results of primary screening, several factors includingconcentration of precipitant, pH value of crystallization reagents and additives and so on wereadjusted so as to get better crystals. The final optimal crystallization condition was as follow:0.8M of sodium citrate,0.1M of Tris pH8.8and0.1M of NaI, the concentration ofFAD-GDH was about30mg/mland the crystallization temperature could be4℃or18℃.With about2to3days of growth, yellow crystals were obtained as polyhedron. Crystalswere freezed with crystallization reagents containing10%of glycerol as cryoprotective regentand kept in liquid nitrogen. Diffraction data of saved crystals was collected at Shanghai lightsource biological macromolecular crystallography line (BL17U)and the final structure ofFAD-GDH was illustrated by molecular replacement method. The refined resolution ofFAD-GDH was2.5.The obtained structure of FAD-GDH was compared with the structure of GOD, whichbelong to the same family. Applying the known information of GOD, the essential aminoacids of FAD-GDH were figure out as Arg504, Asn506, His508and His551, of which wereconfirmed by mutation. With the soft of Autodock, D-glucose was docked into FAD-GDH bymeans of molecular docking to get the compound structure. The compound and the mutationresults supports each other. By comparing the tiny difference around the active centersbetween FAD-GDH and GOD, seven potential sites, including Arg84, Tyr201, Lys336, Tyr340, Leu404, Tyr406and Phe507were chose to mutate by means of site-directedmutagenesis. By detecting their substrate spectrum, three mutants, including Arg84Ala,Tyr340Phe and Tyr406Phe were obtained with narrower substrate specificity. |
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