ABSTRACT
Based on the hyperthermostable family 11 xylanase (EvXyn11(TS)) gene sequence (EU591743), the gene Syxyn11 encoding a thermophilic xylanase SyXyn11 was synthesized with synonymous codons biasing towards Pichia pastoris. The homology alignment of primary structures among family 11 xylanases revealed that, at their N-termini, only SyXyn11 contains a disulfide bridge (Cys5-Cys32). This to some extent implied the significance of the disulfide bridge of SyXyn11 to its thermostability. To confirm the correlation between the N-terminal disulfide bridge and thermostability, a SyXyn11(C5T)-encoding gene, Syxyn11(C5T), was constructed by mutating the Cys5 codon of Syxyn11 to Thr5. Then, the genes for the recombinant xylanases, reSyXyn11 and reSyXyn11(C5T), were expressed in P. pastoris GS115, yielding xylanase activity of about 35 U per ml cell culture. Both xylanases were purified to homogeneity with specific activities of 363 and 344 U/mg, respectively. The temperature optimum and stability of reSyXyn11(C5T) decreased to 70 and 50°C from 85 and 80°C of reSyXyn11, respectively. There was no obvious change in pH characteristics.
Subject(s)
Disulfides/metabolism , Endo-1,4-beta Xylanases/metabolism , Amino Acid Sequence , Disulfides/chemistry , Endo-1,4-beta Xylanases/chemistry , Endo-1,4-beta Xylanases/genetics , Enzyme Stability , Hydrogen-Ion Concentration , Molecular Sequence Data , Mutagenesis, Site-Directed , Pichia/metabolism , Protein Structure, Secondary , Protein Structure, Tertiary , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Sequence Alignment , Sequence Homology, Amino Acid , TemperatureABSTRACT
Twenty-two cases of secondary fungus ball of pulmonary aspergillosis defined by operation were reported. All cases were referred with incorrectly diagnosis to the authors and only 45.3% were correctly diagnosed before operation. The roentgenological and pathological findings were studied. The results show that not familiar with non-typical appearance is the most common cause for incorrect diagnosis.
