Cloning, Expression And Characterization Of A Glucose Dehydrogenase From The Penicillium Sp

FAD-dependent glucose dehydrogenase（FADGDH） form a category （EC1.1.99.10） that is definedas the oxidoreductases capable of oxidizing β-D-glucose to D-glucono，utilizing NADP+or NAD+as theelectron acceptor, and harboring FAD as the cofactor. As the advantages of FADGDH are highthermostabillity, good stability, high turnover rates and high substrate selectivity, more and moreattention is drawn to FADGDH in a few years. It can used in many fields, such as biofuel cell system,enzymatic biosensors, coenzyme regeneration and so on. In a word, the study of FADGDH is ofprofound theoretical and practical significance.This study screened the silt of biomass pool, and got one FADGDH-producing strain Penicilliumsp. ZG1, which identified by18S rDNA and the sharp. At first, this study designed the degenerateprimers based on the conserved motif of FADGDHs alignment results, using touch-down PCR obtainthe gene fragment. And then using TAIL-PCR base on this fragment get the full-length. BLAST thetarget gene gdhz1, the most similarity is61%with FADGDH（XP₀01394544） from Aspergillus，revealed that this gene has certain novelty. Using RT-PCR obtain the cDNA sequence. The length ofgdhz1genome DNA and cDNA was1923bp （2intron）and1776bp. Signal peptide（first18-residueamino acid） and N-Glycosylation sites（68NVS,254NTS,326NIS,378NSS and507NAS） was predictedby SignalP and NetBGlyc. gdhns（do not contain signal peptide） was cloned in pET30a（+） and pPIC9.The recombinant GDHZ1was expressed as inclusion bodies without activity in Escherichia coliBL21（DE3）. The renaturation of the GDHZ1was through4mol/l urea resolution and100folds dilution.Besides, GDHZ1purified by Ni2+column chromatography. GDHZ1expressed in Pichia pastoris GS115with the maximum glucose dehydrogenase activity of0.1U/ml extracellular.The optimum pH and temperature of purified GDHZ1were6.5and40℃. At pH4.0–7.0, GDHZ1remained>80%enzyme activity, it means GDHZ1have a good pH stability. GDHZ1retainning100%activity at40℃for150min,50%activity at50℃for30min. it show GDHZ1hassomethermostabillity. Zn2+, Mn2+, Co2+, Ni2+and SDS can inhibit the enzyme activity. GDHZ1only left40%in the previous metal ions and Chemical reagents（final concentration10mmol/l）, especially, Ni2+inhibit80%activity. The substrate specificity of GDHZ1was investigated using purified protein. GDHZ1showed the highest activity towards glucose, galactose showed40%relative activity to glucose. Thekinetic parameters of GDHZ1were determined in pH6.5,40℃.Km=5.446mmol/l，Vmax=0.1151mmol/l/min, Kcat=29.7s-1, Kcat/Km=5.45l/mmol/s, specific activity was94.14U/mg。Because of thoseadvantages that good stability, high turnover rates、high substrate selectivity and tolerance against ionsand chemical reagents, GDHZ1has a wide future, which GDHZ1can be used in coenzyme regeneration,biofuel cell system, enzymatic biosensors（especially in blood glucose monitoring）.This study first successfully expressed a FAD dependent glucose dehydrogenase gene inEscherichia coli BL21（DE3）and Pichia pastoris GS115in our country. And characterize the enzymatic properties of GDHZ1. Those data can provide theoretical and technical foundation for thedevelopment of application of FAD dependent glucose dehydrogenase.