forms a salt bridge from the c terminus to the body of the enzyme if appropriate a suitable amino acid replacement can be made in the body of the protein so as to enable the formation of a salt bridge an aspartic acid 191d is added to the c terminal serine s190 this results in a salt bridge with arginine at position 58 where wild type lysine has been replaced by arginine k58r by site directed mutagenesis at least one disulfide bridge is formed to stabilise the enzyme in the c terminal part via the u03b1 helix or a region near the u03b1 helix leucine 105 and glutamine 162 are changed to cysteines resulting in disulfide bridge between them l105c and q162c by site directed mutagenesis point mutations are made to increase the stability of t reesei xylanase ii n11d t26r g30h n67r n97r a132r n157r a160r t165n m169h s186r production of mutated and recombinant xynii genes were carried out by the following general procedures t reesei xylanase ii was produced in e coli strains xl1 blue or rv308 using the vector pkktac vtt espoo finland or the vector palk143 roal rajam u00e4ki finland t reesei xynii gene was directly cloned by pcr from the cdna of t reesei to the vector pkktac induction of expression by iptg alternatively the plasmid palk143 was used which contains t reesei xynii gene both of the vectors secrete the xylanase into the medium the vector pkktac by pectate lyase pelb signal sequence and the vector palk143 by amylase signal sequence the.