Heterologous Expression And Large Scale Production Of Alginate Lyase From Vibrio Sp.QY102

Marine Vibrio sp.QY102 isolated from surface of sargassum by the reseachers of Ocean University of China,can efficiently produce alginate lyase which has high enzyme activity,shows the polymannuronic acid specificity and has stability in wide range of pH.With the precious value of the alginate and alginate oligosaccharide discovered,two difficulties have appeared to be settled in the industry manufacturing,one is how to acquire high production of alginate lyase by high-scale fermentation,the other is how to get high purity of alginate lyase by protein purification.This project aims to use Escherichia coli expression system to obtain good yield of the alginate lyase and to get high purity of alginate lyase with appropriate protein purification method.Firstly,we identified the sequence of alginate lyase gene of Vibrio sp.QY102 with degenerate PCR and chromosome walking technology.And then the gene connected to the pET-28a plasmid and then put the recombinant plasmid into HMS174 cells.Finally we got HMS-Alg strains.However,the shaking flask fermentation results showed unstably and low extracelluar enzyme activity in of HMS-Alg strains,we put forward the assumption was that the unstably original signal peptide led to the unstable enzyme activity.So we used a common signal peptide named ompA to replace the original one and rebuilt the HMS-ompA-Alg strains.The 5 L bioreactor fermentation results showed that the average enzyme activity of the extracellular alginate lyase expressed by HMS-ompA-Alg strains which was 2 times of the enzyme activity of HMS-Alg strains,and the extracellular enzyme activity was very stable.In the following protein purification results of 5 L bioreactor fermentation scale,we found the enzyme was obviously degraded during the fermentation process.Therefore,after analyzing the gene sequence and protein structure of alginate lyase,we carried out a series of optimization and transformation of the gene.The first half of the whole gene translated to a kind of F5_F8_TypeC protein family,the second half of the gene translated to alginate lyase.So we cut the first half of the whole gene and reconstructed the HMS-ompA-CF strains.The average extracellular enzyme activity was 375 U/ml,was 3.6 times as much as that of HMS-Alg strains and the enzyme activity was stable.In conclusion,the problems of unstably extracellular enzyme activity and protein degradation of the alginate lyase expressed by HMS-Alg strains have been settled.Finally,the extracellular enzyme activity of 5 L bioreactor fermentation was 1789 U/ml,the yield of the extracellular alginate lyase was about 0.58 g/L,the total production was above 2.0 g.At the same time,we got high purity of the alginate lyase by protein purification.