Heterologous Expression And Fermentation,Purification For FAD-conjugated Glucose Dehydrogenase

Glucose oxidoreductase is the enzyme that catalyzes the oxidation and reduction of glucose,which was developed for using in sensors or test kits.Glucose oxidase is widely used in the clinical biochemical detection of blood sugar.In recent years,it has been discovered that glucose dehydrogenase does not use oxygen as an electron acceptor,which has the potential to replace glucose oxidase.It does not limited by dissolved oxygen and the detection error is smaller.Conjugated tightly with flavin adenine dinucleotide,Glucose dehydrogenase?FAD-GDH,EC1.1.5.9?can be used as a preferred substitution for diagnosing due to its excellent ability of catalyze substrate.This kind of glucose dehydrogenase is mainly derived from filamentous fungi and rarely found in bacteria,which is difficult to recombinant expression and purification.In this study,the gdh gene from Burkholderia cepacia was selected as the research object,mainly involving the heterologous expression of FAD-GDH in E.coli,fermentation regulation,purification process,enzymology studies and try to secretion in B.subtilis WB600.The main contents are as follows:1.The gdh gene was transformed into E.coli.IPTG was used as an inducer.Enzyme activity analysis and SDS-PAGE electrophoresis pattern indicate that soluble expression was achieved.IPTG was replaced by lactose to optimizate induce initial conditions in the shake flask.The gradient feed fermentation of E.coli was carried out to produce FAD-GDH in 7.5 L fermenter under stepped temperature control strategy.Enzyme activity and dry cell weight reached 22200 U·L^-1 and 69.48 g·L^-1.2.The pure enzyme was obtained by three steps:Histrap HP,HiPre TM 26/10Desalting and DEAE anion exchange chromatography.The specific activity was 109U·mg^-1 and the molecular weight was about 60 kDa.The isoelectric point of the enzyme was 5.3.Absorption spectroscopy of full wavelength scanning by microplate reader confirmed that the enzyme was FAD conjugated.Compared with other substances,K_m of enzyme is the lowest of 2.56 mmol·L^-1 when glucose was used as substrate and the k_cat/K_m was 3487.7 mmol·L^-1·s^-1.The enzyme kept relative stable when the pH ranged from 5.5 to8.0,with optimal pH of 7.0.When the pH is higher than 8.0,the enzyme activity decreases rapidly.The T_m of the enzyme measured by differential scanning calorimetry?DSC?was77°C,showing high thermal stability.3.The protease-deficient strain B.subtilis WB600 was used as the host to carry out the FAD-GDH expression.Tandem alteration promoter,signal peptide replaced,and the signal peptide and promoter were combined to investigate the enzyme production.1)The four promoters(P_amyQ',P₄₃,P_gsiB,and P_opuaa)were tandemly connected to the promoter P_HpaIIpaII which was carried by the plasmid.It was shown that the activity of intracellular enzyme of FAD-GDH was highest 2497 U·L^-1 when P_HpaII-P_amyQ'tandem expressed.On the basis of P_HpaII-P_amyQ'to delete of the cre site on the P_amyQ'promoter,the intracellular enzymatic activity was increased to 3626 U·L^-1.Genetic modification reduced the carbon metabolites inhibition of promoter transcription.2)The three double arginine signal peptides?LipA,phoD,YwbN?were used to replace the FAD-GDH self-signal peptide.The signal peptide YwbN contribute to the secretion of the enzyme,and the extracellular/total enzymatic activity was 22%.3)The optimal two-promoter P_HpaII-P_amyQ2 and signal peptide YwbN were combined to construct the recombinant strain WB18.It was find that the activity of extracellular enzyme was only 24%of total enzyme.