| Mannase can effectively improve the nutrient utilization of feed by degrading mannan substances.Based on the gene sequence of Aspergillus niger CBS 513.88 on GenBank,this study optimized the beta mannase gene by Pichia pastoris.The complete gene MANsyn was synthesized and connected to the P.pastoris expression vector pAO815.The recombinant plasmid pAO815-MANsyn of beta mannase was transformed into P.pastoris X-33 to express and obtained a strain with enzyme activity finally.The most appropriate temperature is 70?,the most appropriate pH is5.0,and the maximum enzyme activity is 450 U/mL.The enzyme activity of the beta mananase increased by 126%with additional Mg2+to a final concentration of 5mM/L.Then,we successfully constructed the recombinant expression plasmid containing two copies,three copies or four copies genes of beta mananase,and transformed them into P.pastoris respectively.Compared with single copy gene yeast strain,the enzyme activity of two copies genes yeast strain reached 539 U/mL,increased 20%;the three copies gene yeast strain reached 605 U/mL,increased 34%;the four copies gene strain reached 710 U/mL,increased 58%.In this study,a single point mutation of beta mananase gene was further designed and connected to the P.pastoris expression vector pAO815 and transferred into P.pastoris.The results showed that the positive mutation site was Arg208Lys.Moreover,at 37?,the enzyme activity increased by 13 U/mL compared with the single copy gene,about 5%.Then,the four copies gene recombinant strain with highest enzyme activity were fermented in 14 L fermentation tank,after 96 h fermentation the supernatant was obtained.To detect enzyme activity,the mananase was diluted 10000 times,the enzyme activity of mananase reached 10810 U/mL at the optimum temperature70?,the optimum pH 5.0,and the protein production was 4.09 g/L.This study has successfully obtained a high yield mananase strain,which has industrial potential,and provides the basic data foundation for industrialization. |
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