The Study Of Clone And Expression Of β-mannanase

P-Mannanase(EC 3.2.1.78)can randomly cut off β-1,4 mannosidic bonds mannan,and is widely used in fields of medic ine,foodstuff feed,papermaking,textile printing,dyeing,and petroleum exploration.In the study,3 gene fragments were cloned from Bacillus sp.,and expressed in Pichia pastoris GS115 by the means of plasmid pPIC9K.The main research are as follows:1.Search in the NCBI database,the Bacillus β-mannanase gene sequences were obtained for comparisons nd analysis design Cloning primers according to the conservative properties.Finally,3 gene fragments,named as Man-JCC1,Man-JCC2和 Man-JCC18,were obtained and connected to the expression plasmid pPIC9K,and then transformed into Escherichia coli DH5αfor amplification and sequencing.The sequence Alignment results showed homology of 98%,90%,and 99%,respectively.Among them,no signal peptide sequence was detected in Man-JCC2 sequence according to the online signal peptide analysis software SignalP 5.0.Three-dimensional structure of Man-JCC2 constructed using the SWISS-MODEL Workspace online tool indicated that Man-JCC2-encoded protein belongs to the 26 family of glycosyl hydrolase superfamily.2.The cloned sequences connected with the secretory shuttle expression plasmid pPIC9K,were amplified by E.coli DH5α,and then electrotransformed into P.pastoris GS115 for heterologous expression.After 96 hours of continuous induction with methanol,the highest enzyme activity of 539.444 U/mL appeared at 72 h for P.pastoris GS115-JCC2,while no detection for P.pastoris GS115-JCC1,and P.pastoris GS115-JCC18 showed the highest expression activity of 55 U/mL.3.After isolation and purification,the enzymatic properties of recombinant β-mannanase P.pastoris GS115-JCC2 were preliminary studied.The results showed that the recombinant Man-JCC2 protein was about 47.2 kDa,and the optimal reaction temperature is 40℃.Incubating at 50℃ for 2 hours can retain 60%of the initial enzyme activity.When incubation at 60℃ for 2 hours,the enzyme activity remains 36.8%.The optimal reaction pH is 6.0,and the residual enzyme activity should not be less than 65%when stored at pH 3.0-8.0 for more than 1 hour.4.According to the secondary structure prediction and three-dimensional structure simulation of the recombinant enzyme,the site-directed mutations of the recombinant enzyme were carried out by using seamless cloning technology.The mutant sites were inserted into the recombinant vector,and then successfully expressed.The results showed these sites played important role in the Catalytic hydrolysis reaction.