Study On The Fermentation Production And Enzymatic Properties Of Sea Cucumber I-Type Lysozyme

Lysozyme,also known as N-acetylcytosolic protease,is an alkaline hydrolase that can hydrolyze polysaccharides of biological cell wall.It is widely distributed in different organisms.As a natural protein,it is harmless,non-toxic to animals and does not remain in the body.The i-type lysozyme gene of sea cucumber was first isolated and identified in our laboratory in 2007 and its target protein was highly expressed in prokaryotic cells and eukaryotic cells,respectively.In this study,the recombinant Pichia pastoris engineering strain HS3-1,which expressed lysozyme protein of sea cucumber,was constructed in laboratory.Firstly,5 L fermentor was used to ferment the genetic strain HS3-1.The media YPD and BMGY were used in the culture stage of seed,and the basic salt media BSM was used in the fermentation stage of fermentor.The fermentation process parameters were controlled at temperature of 30?,pH6.0,stirring speed 500 rpm/min and DO 30%.First,BSM culture was cultivated about 24hours.After basic glycerol is exhausted,glycerol was added with the rate of 18.15 mL/L/h.When the cell wet weight reached 180-220 g/L,stop to add glycerol for starving the cells 1hour,so that the glycerol in the culture is exhausted.Then methanol was added into the culture to induce the lysozyme protein.The concentration of methanol in the culturewas controlled at 1%and the induced time was 96 h.The results showed that the enzyme yield of fermentation was 58 mg/L.The product was purified by nickel ion affinity chromatography.The enzyme activity was 1238 U/mg.On the basis of fermentation in 5 L fermentor,30 L fermenter was used to amplify the genetic engineering strain HS3-1 of P.pastoris.The purified product was analyzed by high performance liquid chromatography and mass spectrometry.According to the peak time of the standard product,it was inferred that the purified product was i-type lysozyme of sea cucumber.The molecular weight of i-type lysozyme was about 14.7 kDa by mass spectrometry.The optimum reaction conditions of i-type lysozyme were obtained as follows:the reaction temperature was 35?,the thermal stability was better at 25-65?,pH was 6.5,and it was stable in the range of pH 5-8.5.Na⁺can promote enzyme activity.K⁺?Ca²⁺and Al³⁺had little effect on enzyme activity,and Co²⁺,Mg²⁺and Mn²⁺can inhibit it.Through the research of this subject,the preliminary study on the production of sea cucumber lysozyme by using P.pastoris genetic engineering bacteria fermentation and separation and purification,and the purity of the product and the molecular weight of sea cucumber lysozyme were analyzed.The optimum reaction conditions of sea cucumber i-type lysozyme were determined,which laid a foundation for the application of sea cucumber lysozyme.