Construction And Analysis Of Sea Cucumber Lysozyme Expressed In Saccharomyces Cerevisiae

The i-type lysozyme of sea cucumber was found with the glycosidic enzyme activity and broad spectrum, non-enzymatic antimicrobial activity in the study. It can effectively inhibit the harmful bacteria in the process of aquaculture and food processing by hydrolyzing the β-1,4 glycosidic bond between N-acetylmuramic acid and N-acetyl glucosamine exsit in bacterial cell wall. In this study, the Saccharomyces cerevisiae expression system was used to express the Stichopus japonicus lysozyme(Sj Lys).The His-tag was inserted into the N-terminus of Sj Lys gene and the new plasmid p MD18-His6-Sj Lys was constructed by PCR according to the template p MD18-Sj Lys.According to the analysis by Condon Usage Database in S. cerevisiae, the 69, 77 and 95 arginine codon of Sj Lys protein was optimized to obtain the p MD18-His6-Sj Lys((35)69,77,95)vector by fixed point mutations. The fragments His6-Sj Lys and His6-Sj Lys((35)69,77,95) were connected to the S. cerevisiae expression vector p ESC-LEU by the Spe I/Bgl II digestion,respectively. The recombinant intracellular expression vectors p ESC-LEU-His6-Sj Lys and p ESC-LEU-His6-Sj Lys((35) 69,77,95) were constructed and transformed into S. cerevisiae MKP-0 by lithium acetate, respectively. After inducing for 60 h by 2% galactose, the Sj Lys protein was successfully expressed, and got a highest expression quantity at 72 h by optimizing induction time of galactose which were detected by western blot analysis. Also,the supernatant of the recombinant p ESC-LEU-His6-Sj Lys((35)69,77,95)/MKP-0 cells displayed inhibitive effect on the growth of the Micrococcus lysodeikticus detected by Oxford cup bacteriostasis experiment and SEM observation.Because the intracellular expression quantity of Sj Lys protein in S. cerevisiae was lower,then the secreted expression system of S. Cerevisiae was used to get the Sj Lys protein.According to the template Pichia expression vector p PIC9 K with MFα1 signal peptide, the MFα1 fragment was connected to the p MD18-T by PCR amplification, then the new plasmid p MD18-T-MFα1 was constructed. The MFα1 fragment was inserted into the N terminus of His6-Sj Lys((35)69,77,95) gene by the Not I/Spe I digestion, finally the recombinant exocytosisexpression vector p ESC-LEU-MFα1-His6-Sj Lys((35)69,77,95) was constructed and transformed into S. cerevisiae. Then the new protein expression system of Sj Lys was constructed.Subsequent experiment is still in progress.It was the first time to express the active Sj Lys protein in S. cerevisiae expression system,and proposed a new feasible way to produce the sea cucumber lysozyme.