Cloning, Protein Expression, Characterization And Mutagenesis Of Cold-Active And Halophilic Xylanase

Hemicellulose is the most abundant polysaccharide besides cellulose, and the second large renewable resources in nature. The degradation of xylan which constitutes the major component of hemicellulose has attracted widespread attention. The most critical enzymatic components are endo-β-1,4-xylanase to hydrolyze xylan. These enzymes hydrolyze the internal β-1,4bonds of the main xylan chain to produce xylo-oligosaccharides and xylose. Xylanase have potential applications in a wide range of industrial process covering animal feed, paper, food, energy, textile and so on. There is a great intreast in discovering xylanases with specific characteristic for various applications, such as basophilic, acidophilus, psychrophilic, halophilic, thermophilic, etc.In this study, the xylanase gene xynA was cloned from Zunongwangia profunda which was separated from the deep-sea sediments. XynA was expressed in E.coli (DE3) and its enzymematic properties were analyzed. And random mutation technology was used to improve the salt tolerance. The research results were as follows:1. Endo-β-1,4-xylanase gene(xynA) from the marine microorganism Zunongwangia profunda was identified to consist1125bp and encode374amino acid residues with a molecular weight43.7kDa. GC content of xynA was38.81%and no signal peptide was predicted. The sequence alignment indicated that XynA showed the highest similarity to xylanase from GH10family. Further alignment indicated that XynA contained two highly conserved catalytic residues (Glu169and Glu276) that were crucial for the activity of GH10xylanase. Its product (XynA) showed the highest identity (42.78%) among the characterized xylanases. XynA exhibited the highest activity at pH6.5and30℃, retaining23%and38%activity of the optimal activity at0and5℃, respectively. XynA was not only cold-active but also halophilic, and both its activity and thermostability could be significantly increased by NaCl, showing the highest activity (191%of the activity) at3.0mol/L NaCl and retaining nearly100%activity at5.0mol/L NaCl, compared to the absence of NaCl. In the presence of3.0mol/L NaCl, the Vmax、Km、kcat and kcat/Km of XynA were0.03459mg/mL/min、1.15mg/mL、80.33/s and69.91mL/mg/s, respectively. In the absence of3.0mol/L NaCl, the Vmax、Km、kcat and kcat/Km of XynA were0.05291mg/mL/min、2.98mg/mL、47.26/s、15.87mL/mg/s, respectively, for beechwood xylan compared to no added NaCl. The residual activity of XynA increased from8%(no added NaCl) to55%after1h incubation at45℃. This may be the first report concerning a cold-adapted xylanase from a non-halophilic species that displays the highest activity at a NaCl concentration range from3.0mol/L to5.0mol/L. The features of cold-activity and salt-tolerance suggest the potential application of XynA in food industry and bioethanol production from marine seaweeds.2. A random mutant library was constructed by using error-prone PCR technology to make the salt tolerence of XynA higher.3.5mol/L NaCl was added to the substrate. A mutant XynA-JD27was obtained from about7000mutants successfully. The C-terminal region of XynA-JD27was less than the wild type27amino acids. The optimum pH of the mutant was6.5and the optimum temperature was down to25℃, retaining50%activity of the optimal activity at5℃. The optimum salt concentration was up to3.5mol/L NaCl, and XynA-JD27can keep more than148%activity from1.0mol/L to5.0mol/L NaCl compared to the absence of NaCl. In the presence of3.5mol/L NaCl, the Vmax、Km、kcat and kcat/Km of XynA-JD27were0.0645mg/mL/min,1.51mg/mL and108.9/s and72.12mL/mg/s, respectively. In the absence of3.5mol/L NaCl, the Vmax、Km、kcat and kcat/Km of XynA were0.08288mg/mL/min,3.86mg/mL,69.92/s and18.11mL/mg/s, respectively, for beechwood xylan compared to no added NaCl. These suggested that NaCl can reduce the reaction rate of xylanase, but can enhance the affinity between xylanase and substrate. On the other hand, maybe the C-terminal region of xylanase had some effects on the cold active and salt tolerance. These characteristics make XynA a good candidate for further research on the structure-function relationship.