Fermentation Of Sea Cucumber I-type Lysozyme By The Genetic Strain And Its Product Analysis

Lysozyme,known as N-acetyl-murameptidin hydrolase,is a natural protein that is toxic free,safe and does not remain in the body.It is widely distributed in different organisms and is an alkaline hydrolase capable of hydrolyzin gmucopolysaccharides of microbial cell wall.In 2007,the laboratory isolated and identified the sea cucumber i-type lysozyme gene for the first time and achieved high expression in both eukaryotic and prokaryotic cells.In this study,a Pichia pastoris genetic engineering strain HS3-1,which had been constructed and expressed in the laboratory,was used to ferment in a 5 L fermentor.After the fermentation supernatants were separated by ultrafiltration and nickel ion affinity chromatography,purified by dialysis and freeze-dried.Finally the refined product of the sea cucumber i-type lysozyme was obtained.The lysozyme product was analyzed by high performance liquid chromatography(HPLC)and MALDI-TOF mass spectrometry,molecular dynamics simulations were further used to analyze it in depth.The results of the study are as follows:(1)The initial media for fermentor tank production was 3 L BSM of basal salt components.The fermentation conditions were as follows: pH 6.0,temperature 30 ?,dissolved oxygen 30%,and stirring speed 400 r/min.After finishing fermentation,the yield of lysozyme in fermentation broth was analyzed as 58 mg/L.Finally the refined product of sea cucumber i-type lysozyme was obtained,and its enzyme activity was determined to be 1246U/mg.The lysozyme product was further analyzed by high performance liquid chromatography(HPLC)and the lysozyme product was obtained with the same peak time as the standard product of egg white lysozyme.Meanwhile,there was no miscellaneous peak.The results of MALDI-TOF mass spectrometry showed that the molecular weight of the i-type lysozyme was 14708.7 Da,which was basically consistent with the theoretical calculation value of its amino acid composition.This indicates that the product isolated from the fermentation broth of the genetic strain HS3-1 is truly sea cucumber i-type lysozyme.(2)Through bioinformatics analysis,the sequence of sea cucumber i-type lysozyme was analyzed.Through the primary structural analysis,the composition,quantity andproportion of amino acids of sea cucumber i-type lysozyme were analyzed.The secondary structural analysis shows the proportion of ?-helix,?-folding,?-angular and irregular curl of the lysozyme protein.The position of lysozyme active center and active sites in subunit structure can be clarified by the tertiary analysis.(3)Characteristics of the prepared i-type lysozyme of sea cucumber were analyzed,and the results showed that the optimum temperature of the lysozyme was 35 ?,and there was good thermal stability at a higher temperature of 65~80 ?.The optimum pH was 6.5,and it was stable in the range of pH 5.0 and 8.0.The effect of different temperatures on the conformation of lysozyme protein was analyzed by molecular dynamics simulation.The conformation changes of the enzyme protein at different temperatures were analyzed.The simulation results also showed that it has stable thermal stability,which is consistent with the results of in vitro bacteriostatic test.This study lays a good theoretical foundation for further exploration of the molecular action mechanism of i-type lysozyme in sea cucumber.The thermal stability advantage of the enzyme is very beneficial to the food industry and animal feed processing industry.Therefore,the i-type lysozyme has great application value.