Optimization Of Fermentation Conditions And Property Analysis Of Sea Cucumber Lysozyme Produced By Recombinant Pichia Pastoris

Lysozyme is widely distributed in different organisms.The enzyme can destroy ?-1,4glycosidic bond of peptidoglycan in bacterial cell wall and lead the cell death.Lysozyme,as a natural protein,has characteristics with non-toxic to human body,easy absorption,no pollution and so on.In 2007,our laboratory was identified the i-type lysozyme gene from sea cucumber for the first time.Further study found that the lysozyme has broad-spectrum antibacterial activity.In this study,the genetic strain Pichia pastoris pPIC9K-SjLys/GS115 was used to produce the lysozyme from sea cucumber Stichopus japonicas(SjLys).The fermentation conditions were optimized for enhancement of lysozyme production.The results showed that the maximum lysozyme yield of 4.88 mg/L was achieved at the conditions of initial pH 6.0,culture temperature 30?,fermentation time 96 h and methanol concentration of 1%.The fermentation culture of SjLys was centrifuged and concentrated by ultra-filtration,then purified by cation exchange and gel filtration chromatography.The specific activity of the purified lysozyme was 826.44 U/mg.Although after optimization of fermentation conditions of sea cucumber lysozyme,the yield of the enzyme has increased,the loss of enzyme purification is severe,causing low production rate.In order to reduce the purification steps and increase the recovery rate of enzyme,the full-length gene sequence of the sea cucumber lysozyme was chosen as the template,adding histidine tags(6×His tag)on its N and C terminus,respectively.And the expression vectors pPIC9K-SjLys-HisN and pPIC9K-SjLys-HisC were constructed.The target gene fragment was linearized by Bgl? and transformed into P.pastoris GS115 by electroporation,respectively.Positive clone were screened by the YPD plate containing1.0 mg/mL~4.0 mg/mL antibiotics G418 and verified by colony PCR.Finally,A genetic strain P.pastoris HS3-1 was obtained with ability to produce the sea cucumber efficiently.After SDS-PAGE and Western blotting analysis,it is confirmed that the sea cucumber lysozyme has been successfully expressed.The yield of lysozyme is 5.12 mg/L.The fermentation culture of SjLys was centrifuged and concentrated by ultra-filtration,then purified by affinitychromatography.The specific activity of the purified lysozyme was 1088.57 U/mg.The further study has done on 5 L fermentor.The genetic strain P.pastoris HS3-1 was used as fermentation strain to produce the sea cucumber lysozyme.3 L of BSM medium was added in the fermentor and the HS3-1 strain was inoculated and cultivated under the condition of 30?,800 r/min,pH 6.0,DO 30%.After glycerin in BSM medium run out,more glycerin was added for 5 h.After that,the culture was starved for 1 h,then fed 1% of methanol for induction of 96 h.The results showed that the lysozyme accounts for 58% of the total protein.The lysozyme yield is 53.2 mg/L.The fermentation culture of SjLys was centrifuged and concentrated by ultra-filtration,then purified by affinity chromatography.The specific activity of the purified lysozyme was 2238 U/mg.The purified SjLys product diaplayed inhibitive effect on the growth of Staphylococcus aureus,Micrococcus lysodeikticus,Vibrio parahemolyticus and Pseudomonas aeruginosa.Through in this study,we successfully got sea cucumber lysozyme expressed by P.pastoris.It also provides a basis for further large-scale industrial production and separation and purification.