| Bilirubin oxidase (EC1.3.3.5), is a member of multi-copper oxidase (MCOs) family, which can catalyze the oxidation of bilirubin to biliverdin, so it was named. In this study, the strain producing bilirubin oxidase was isolated from a maize leaves. The medium composition and the conditions of fermentation were optimized by means of response surface methodology (RSM). After fermenting, protein was purified after a series of steps, a single band was obtained, then the enzymatic biochemistry, enzymology, spectroscopy, electromagnetic, electrochemical properties and the applications using the enzyme electrode to detect glucose levels in the blood were studied, the main results are as follows:(1) isolation and identification of bilirubin oxidase producing strainThe strain producing BOD was isolated from diseased leaves of maize by means of conventional microbiological separation techniques and the strain was identified as Bipolaris australiensis, named B. australiensis HD-1by morphology, physiology and rDNA-ITS (accession number JN251106) sequence analysis, which belong to phylum ascomycota, class dothideomycetes, order pleosporales, genus bipolaris and Bipolaris australiensis, having enzyme activity1000U/L in primary culture medium broth. It was a novel strain as so far. A new microbial sources producing BOD was found.(2) optimization of fermentation conditionsMaltose, sucrose, glucose, fructose, soluble starch and trehalose are suitable carbon sources for B. australiensis HD-1to produce BOD by means of single factor test among13kinds of carbon sources. Poly peptone, soy peptone and soybean powder are more suitable nitrogen sources than the other19kinds of compounds containing nitrogen. The enzyme activity would become bigger when the amount of CuSO4was increasing in the culture medium. Three factors, maltose, CuSO4and temperature were all significant ones to impact the amount of producing BOD during fermentation process by means of two-level fractional factorial test and steepest ascent experiment. Then, Box-Behnken Design of response surface methodology was performed succedently to optimize above three factors, the results as followed:maltose,5g/L, CuSO4·5H2O0.32g/L, soy peptone50g/L, temperature is29.8℃, pH6.8, shaking speed is150rpm for6d,2480U/L of enzyme activity was obtained theoretically and after verifying experiment a activity of2180U/L was obtained, which indicated that the response surface method to optimize BOD is a reliable. Enzyme production optimized is2.5times than before optimization.(3) Purification and characterization of the enzyme proteinB.australiensis HD-1After the fermentation broth by ammonium sulfate salting out, chromatography, chromatography on SephadexG-75gel filtration purification and DEAE-Sepharose Fast Flow anion exchange by native-PAGE and SDS-PAGE detection, obtained with bladder red pigment oxidase activity of pure monomeric protein electrophoresis with matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF) method to obtain135amino acids, the use of Mascot NCBI protein database software in line when compared to the results indicate that the protein derived from Helminthosporium tritici-vulgaris (Helminthosporium mold) laccase precursor protein sequence homology of100%. The apparent molecular weight is68KDa and the pI is about4.1.(4) spectroscopy and electromagnetic characteristicsInductively coupled plasma atomic emission spectroscopy (ICP-AES) was measured to find the Cu, Zn and Fe atoms in the protein with the ratio of Cu:Zn:Fe is1:1:4; UV-vis spectroscopy analysis showed that the protein has spectroscopic characteristics of a typical multi-copper oxidase family, namely T1copper in the vicinity of600nn has three nuclear copper center of a strong absorption peak and T2, T3copper composition in the vicinity of330nm with a shoulder shaped peak, the difference is that in the enzyme at340nm and773nm have a strong absorption peak. Electron paramagnetic resonance (ESR) analysis showed that the enzyme in the near neutral (pH7.3),-160℃condition manifested as a silent state, suggesting that the protein may be in a reduced state of Cu2+.(5) enzymatic propertiesDifferent substrates Michaelis constant (Km) values were Km(ABTS)=1.8×10-3(mol/L)(pH3.0) and Km(biiinibin)=2.7×10-5(mol/L)(pH7.5) by means of Lineweaver-Burk method respectively. The enzyme activity is optimum at36℃, the enzyme is stable at low temperatures, the enzyme activity could keep on at high pH range of8～9.5. The enzyme is sensitive for sodium chloride, sodium phosphate, sodium sulfate, organophosphorus pesticides and dissolved oxygen. However, the nitrate sodium and urea were different to this enzyme, low concentrations of them would improve the enzyme activity, by contrary, high concentrations would inhibit enzyme activity. Cu2+and Hg2+both can oxidize bilirubin to biliverdin without BOD, but the products are not unique, the ions of Li+, Na+, K+, Mn2+, Ba2+, Co2+, Mg2+, Al3+, Zn2+and Ca2+could chelate with bilirubin to come into being a compounds, subsequently, the compounds could not be catalyzed by BOD to form biliverdin.(6) electrochemical properties and bioenzymatic applicationsUsing carboxylated multi-walled carbon nano-microtubules/Nafion/BOD modified glassy carbon electrode to study the enzymatic electrochemistry properties by Cyclic voltammetry in citrate-phosphate-buffer (5mM, pH7.3), under aerobic conditions, a pair of well-defined voltammetric response was recorded with a formal potential AEp=107mV (vs.SCE), and the ratio of|ipa/ipc|is0.7, which indicating a quasi-reversible redox reaction was recorded, and a heterologous direct electron transfer phenomenon was occurred between enzyme and electrode.The carboxylated multi-walled carbon nano-microtubules and Nafion membrane solution can effectively increase the surface area of the glassy carbon electrode, increases the adsorption on the electrode the amount of enzyme and electrochemical signals, extending the time of using the enzyme electrode to facilitate direct electron transfer. The electrode can be used to quickly detect trace glucose concentration (0～15μM) by means of LSV method and higher reliability for the enzyme electrode on the clinical application. |
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