Study On Extraction And Purification Of Metallothionein From Oratosquilla Oratoria

Metallothionein?MT?are a group of low molecular mass,ubiquitous and cysteine-rich proteins with a variety of functions that have been isolated from many organisms including prokaryotes,plants,invertebrates and vertebrates.applied research,Through its cysteine thiol group and heavy metal binding ability,it plays an important role in the protection of metal homeostasis and heavy metal toxicity,DNA damage,oxidative stress,antagonistic ionizing radiation,and other metalloproteins and metalloenzymes.Besides,Provides active centers for a wide range of applications in nutrition,health,medicine,bioengineering,aquaculture and environmental monitoring.1.In this study,the extraction and purification preparation process of MT was studied by using Oratosquilla oratoria as raw material.On the one hand,.its provided a reference and theoretical basis for the development,and the application of marine MT resources.On the other hand,it also carried out the mechanism of enrichment of heavy metal elements in Oratosquilla oratoria.the research contents were as follows:Utilize Oratosquilla oratoria to prepare metallothionein.In this paper,the optimization of the response surface methodology was performed in this experiment,with the concentration of buffer,pH,liquid-to-solid ratio and temperature as the experimental factors,versus the extraction capacity of Oratosquilla oratoria MT as the response value.The result shown that the optimal scheme of extraction conditions was obtained,which the extraction time was 1 h,the extraction temperature was 51?,the liquid-solid ratio was 4,the concentration of buffer was 0.02 mol/L,and the value of pH was 7.8.Under the optimization extraction conditions,the experimental yield of Oratosquilla oratoria obtained was 0.232 mg/g,which were almost accorded with the predicted data.2.The crude protein was separated and purified by Sephadex G-50 gel column and DEAE Sepharose Fast Flow anion exchange column.The conditions for separation and purification of MT were obtained.Sephadex G-50 elution conditions:0.02 mol/L Tris-HCl?pH 8.0?elution,flow rate 0.8 mL/min,8 mL/tube,elution volume 450 mL;DEAE Sepharose Fast Flow elution conditions are:First elute with 0.02mol/L Tris-HCl?pH 8.6?combined with 0.02 mol/L Tris-HCl+0.5 mol/L NaCl?pH 8.6?solution,0.8mL/min,8 mL/tube,elution Volume 500 mL.The UV colorimetry at 220 nm was combined with ICP-MS to determine the metal element signal for comparison.The isolate was then desalted by Sephadex G-25 to give high purity sputum MT-1,MT-2.The MT-1 was determined by high performance liquid gel filtration chromatography and Tricine-SDS-PAGE electrophoresis,and its molecular weight was about 15 KDa.Determined by high performance liquid chromatography-fluorescence assay and high performance liquid phase molecular drainage chromatography-ultraviolet assay,the peak area of the target was calculated according to the area attribution method,and the purity of the Oratosquilla oratoria MT-1 was about 91.50%.3.Separation and purification of high-purity Oratosquilla oratoriaMTsubstance,MT element Cu,Zn binding amount,isoelectric point,amino acid composition determination,purity analysis,and then used UV spectroscopy,infrared spectroscopy,circular dichroism,etc.Perform structural characterization.The results showed that ICP-MS showed that MT-1 was mainly in the form of Zn-MT,and also combined with more Cu;the isoelectric point?pI?of MT-1 was 4.1;The cysteine?Cys?in MT accounts for 32.44%of the total amino acids,and does not contain aromatic amino acids.According to UV spectroscopy,the MT-1 solution of Oratosquilla oratoria has a significant absorption peak at 270 nm and no absorption at 280 nm.,which is related to the absence of aromatic amino acids in MT,the absorption peak disappeared at 270 nm after EDTA treatment.Infrared spectroscopy analysis showed that the absorption bands of MT were corresponding to type I irregular coil configuration,MT-1 and The amide band I position of MT-2 is located at 1640.92 cm^-11 and 1642.03 cm^-1,respectively,mainly caused by strong stretching vibration of amide C=O,accompanied by NH bending vibration?shear?;in 1537.32 cm^-11 and 1539.50 cm^-1,it is caused by NH bending vibration and CN moderate stretching vibration.The amide band III is not obvious,but it is accompanied by CN moderate stretching vibration peak.Circular dichroism?CD?analysis showed that the secondary structure of MT-1 and MT-2 was mainly a random coiled conformation with a certain?-fold;and the Oratosquilla oratoria MT-1 and MT-2is relatively close,which is consistent with the results of infrared spectroscopy.After preliminary evaluation of antioxidant activity,the results show that the order of DPPH free radical scavenging capacity is rabbit liver MT-1>rabbit liver MT-2>Oratosquilla oratoriaMT-1>Oratosquilla oratoriaMT-2>VC.The results of this analysis not only supplemented the research gap of MT properties of Oratosquilla oratoria,but also provided a basis for clarifying the structure-activity relationship of Oratosquilla oratoria.