Membrane-bound Gluconate Dehydrogenase From Serratia Marcescens:Purification,Identification And Characterization

2-keto-D-gluconic acid（2KGA）is an important precursor for synthesizing antioxidant erythorbic acid and its sodium salt,which is widely used in the food,cosmetic,chemical and other fields.Membrane-bound gluconate dehydrogenase（GADH）is a key dehydrogenase involved in the 2KGA biosynthesis.Hence,investigation the membrane bound GADH properties is necessary for further improving the 2KGA production performance.In the study aimed to purifying the membranebound GADH by Triton X-114 two-phase separation and chromatographic techniques from Serratia marcescens JUIM03,and indentifying the membrane-bound GADH by means of SDS-PAGE,Vis-absorption spectrum and MALDI-TOF-MS,and indentifying its oxidized products by means of high performance liquid chromatography（HPLC）and infrared spectroscopy,and then investigating its catalytic characteristics.On the basis,LA-PCR technique was used to clone the membranebound GADH gene,and the structures and functions of GADH protein was predicted by online analysis tools and software.The obtained conclusions are listed as follows:（1）Membrane-bound gluconate dehydrogenase（GADH）with specific activity of 91.43 U/mg,160 purification fold and 3.8 recovery was purified from S.marcescens JUIM03 using ultrasonication,Triton X-114 phase separation,salt fractionation,DEAESepharose fast flow and Hydroxyapatite column chromatography.SDS-PAGE results showed that the membrane-bound GADH of S.marcescens consisted of three subunits,and the large subunit was flavin protein with a molecular mass of 65.0 k Da,and the middle subunit,the small subunit with a molecular mass of 45.0 k Da and 23.0 k Da,separately.The obtained results of Vis absorption spectrum and MALDI-TOF-MS analysis further indicated that GADH from S.marcescens was FAD-dependent membrane-bound GADH.HPLC and infrared spectral indicated the membrane-bound GADH catalyzed gluconic acid to 2KGA.（2）Membrane-bound GADH from S.marcescens JUIM03 had the optimal catalytic temperature of 40°C,and was stable between 30⁴0°C.The membrane-bound GADH had the optimal p H of 5.0 and stable activity at p H between 5.0⁷.0.Na+ had no influence on the activity of membrane-bound GADH,Mg2+ could slightly promote membrane-bound GADH activity in low consistency,while Mn2+,Ni2+,Cd2+,Cu2+,Zn2+ or Fe3+ inhibited membrane-bound GADH activity in different degrees.Mercaptoethanol and SDS could inhibit membrane-bound GADH activity,but EDTA had no influence on the activity of membrane-bound GADH;Organic acids such as glycolic acid,glyoxylic acid and 2KGA had no apparent effect on the activity of membrane-bound GADH,while oxamic acid and oxalic acid had strongly inhibited the membrane-bound GADH;Ethanol,isopropanol,methanol and acetone had significant inhibitory effects on the catalytic activity of membrane-bound GADH.Membranebound GADH of S.marcescens had a strict specificity for D-gluconate oxidation,and Km value for sodium D-gluconate was1.33 mmol/L when reaction p H was set as 6.0.（3）The gad operon was amplified from S.marcescens JUIM03 by LA-PCR technique.The bioinformatics analysis showed that the gad operon has three structural genes gnd S,gnd L and gnd C,and the length of the corresponding gene fragment is 729 bp,1782 bp and 1338 bp,respectively.The gene gnd S shared 98% sequence identity with the corresponding gene encoding the small subunit of gluconate dehydrogenase from S.marcescens SGAir0764,and encoded a transmembrane protein which belongs to gluconate₂-dh3 superfamily.The gene gnd L shared 99% sequence identity with the corresponding gene encoding the large subunit of gluconate dehydrogenase from S.marcescens UMH8,and encoded a transmembrane protein which belongs to GMC_oxred_C superfamily.The gene gnd C shared 99% sequence identity with the corresponding gene encoding the center subunit of gluconate dehydrogenase from S.marcescens UMH8,and encoded a protein which belongs to Cytochrom?C superfamily,without a transmembrane domain.