| Cuttlefish is one of the four sea fish in our country,and its nidamental glands are rich in glycoproteins.At present,domestic and foreign researches are focused on the antioxidant properties of polysaccharides in the nidamental gland.Studies have shown that glycoproteins can bind to the peptide target of the carboxyl terminal of the bacterial cell wall,resist the biosynthesis of peptidoglycan,and also inhibit the synthesis of nucleic acids and adhere to the bacterial cell plasma membrane,causing it to cleave,thereby achieving antibacterial purposes.At the same time,the amino acid composition of the protein in the nidamental glands is relatively special,and the content of basic amino acids is relatively high.Therefore,this article taked the nidamental gland of Sepiella maindroni as the research object,optimized the extraction method of glycoprotein,isolated and purified the glycoprotein,studied the antibacterial activity of nidamental gland protein.The structure of glycoprotein was preliminarily identified,which provided a theoretical basis for the development and utilization of glycoproteins from nidamental gland of the Sepiella maindroni in biological preservatives.The main findings are as follows:1.By measuring the basic nutrients of nidamental gland of the Sepiella maindroni,it was found that the sugar,protein and fat contents were 9.54% ± 0.34%,15.38% ± 0.030%,0.90% ±0.020%,respectively.It was concluded that the nidamental gland of the Sepiella maindroni has the characteristics of high protein,high sugar and low fat.2.Based on comparing a variety of glycoprotein extraction methods.The glycoprotein of the nidamental gland was extracted by alkaline extraction method,according to the results of the single factor test,the response surface test was used to optimize the extraction parameters of glycoprotein.The results showed that the actual yield of glycoprotein(dry base)was 8.31% ± 0.18% under the conditions of 0.43mol/L of Na OH,extraction times of 2.8h,extraction temperature of 27?,and liquid to material ratio was 30:1.3.Ion exchange column chromatography was used to purify the crude glycoprotein and its antibacterial properties were studied.The results showed that the crude glycoprotein was effectively separated by ion exchange column chromatography,and two components(component 2and component 3)with antibacterial activity were obtained.The yield of pure glycoprotein in component 2 was 2.12% ± 0.71% and the yield of pure glycoprotein in component 3 was 0.27% ±0.63%.The antibacterial effect of component 2 on Escherichia coli and Staphylococcus aureus was stronger than potassium sorbets(p<0.01),and the minimum inhibitory concentration on Escherichia coli was 20?g/mL,the minimum inhibitory concentration on Staphylococcus aureus was 30?g/mL.Component 3 has a good antibacterial effect on Escherichia coli and Staphylococcus aureus under neutral conditions(p<0.01),but compared with potassium sorbets,Component 3 has no antibacterial effect on Escherichia coli under acidic conditions(p<0.01),the antibacterial effect under alkaline conditions is not as strong as that of potassium sorbets(p<0.01).4.The infrared spectroscopy,glycopeptide bond characterization analysis,gel electrophoresis,mass spectrometry identification,and monosaccharide composition analysis were used to preliminary identify the target glycoprotein structure(component 2).The results showed that there was a characteristic absorption peak of proteins and polysaccharides in component 2,and there were O-glycopeptide bonds in component 2.The active protein in component 2 was a single component.It was shown that there was no protein that exactly matched the component 2 from the existing database,but according to the protein score and peptide coverage,the protein was more likely to be a histone.The analysis result of the monosaccharide composition indicated the component 2 contained mannose,rhamnose,glucuronic acid,galacturonic acid,glucose,galactose,xylose,arabinose,fucose,of which fucose,galactose,mannose,xylose were higher in content. |
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