The Modification?immobilization Of ?-xylosidase And Its Expression In Bacillus Subtilis

As an exoglycosidase,beta-Xylosidase has ability to release xylose from the nonreducing ends of xylooligosaccharides.However,because of the low enzymatic activity,the unstable protein structure and the high production cost,the application of?-xylosidase is not available so far.If these problems could be solved,this enzyme would play an important role in a lot of fields including food,medicine,etc.To deal with these problems,three different studies have been performed:1)modification?-xylosidase through a novel strategy of fragment shuffling of metagenomic DNA;2)construction of double host expression vectors which can be used to express genes both in Bacillus subtilis and Escherichia coli;3)Immobilization of?-xylosidase with novel methods.The details of valuable results are as below:First,the?-xylosidase gene BH3683 from Bacillus halodurans C125 was cloned and successfully expressed the enzyme in E.coli,and enzyme characterizations were analyzed.With pNPX being used as substrate,the Km of this?-xylosidase is 1.23 mmol/L;the most suitable reaction temperature is 50?;the most appropriate pH is 8 and when the pH is between 6 and 10,the enzyme activity can be still more than 50%after 30 min incubation in50?.But when the reaction temperature is more than 50?,the thermostability of enzyme will show significant decrease with the rise of temperature.And some Metal ions and organic solvents will also reduce the enzyme activity.Second,Mixure of five soil samples from different sources were used to extract DNAs for analysis of soil microbial diversities.The results shown the species of microorganism are abundant,and the enrichment showed obvious influences on the microbial diversity.Two degenerate primers were designed based on the different sequences of?-xylosidase from GenBank of NCBI,and used for amplification of homologous gene fragments of?-xylosidases in soil.These fragments were used to replace the corresponding fragment of BH3683 by overlap PCR to modify BH3683 gene.A sequence named Xyl-M56 was identified that can express a protein with?-xylosidase enzyme activity.The most suitable reaction conditions for Xyl-M56 was 40?and pH 7 which were different with BH3683,but the stability showed a mild increase:after a 30 min incubation in 60?,Xyl-M56 still has a limited activity;it showed more resistance against high p H;and the resistance against organic solvents showed a significant increase.Third,a plasmid vector with T7 and P_spec promoters,and a reformed RBS was constructed for expression and purification of protein both in B.subtilis and E.coli.This vector including a chloramphenicol resistant gene which can work both in B.subtilis and E.coli.What's more,there is also a CcdB expression gene in this vector which can encode CcdB toxin protein as negative selection marker.The function of the vector was verified by expression and purification of xylanase gene XynII and?-xylosidase gene BH3683 in both B.subtilis and E.coli.Finally,the method of metal–enzyme hybrid was used to immobilize?-xylosidase BH3683 for industrial production.After comparing the enzymatic properties between immobilized enzyme and free enzyme,the most suitable reaction conditions were found to be pH 7 and 50?-55?.The stability of immobilized enzyme showed significant increase under high temperature,the resistance of metal ions and organic solvents also improve a lot.