Research Of The Aspergillus Niger Xylanase Helix Replacement For The Thermophilic Glucanase Helix

Endo-xylanase [EC 3.2.1.8] can be interrupted the xylan internal of the ?-1,4 glycosidic bond,catalytic degradation of xylan.The enzyme is widely used in the paper,food,feed,textile,and energy industries.Enzyme has been isolated from nature to fungal xylanase activity was the highest,but such enzyme thermal resistance uneven force in line with the actual production requirements.Xylanase will gradually shift to explore the relationship of enzyme structure and function,improve the characteristics of a variety of enzymes,including heat,acid and alkali.Structure with the xylanase of Aspergillus niger xylanase Xyn(upstream of the ? helix,? helix,? helix Preparation,abbreviated as Xyn?-?x-Xyn?,P55329)and Glucanase Glu(abbreviated as Glu?-?g-Glu?,Q60033)bioinformatics analysis and found that the two structures are constituted by a single ? helix and ? pleated sheet grip-like structure of the right-hand half.Also ? helix has great influence on the nature of the family G/11 xylanases and Glu corresponding structural position on the ? helix having a strong thermal stability.Accordingly,the ? helix replacement from Glu should be possible to improve the stability of the enzyme molecules into the Xyn.In the fusion PCR Xyn spiral to be replaced,build two kinds of fusion gene Xyn?-?g-Glu? and Xyn?-?g-Xyn?.Fusion gene respectively connected to the p ET-20 b expression vector was introduced into BL21(DE3)to express the fusion protein.Protein expression by Co2+ resin purification and characteristic analysis.The results showed that:(1)Get two active fusion enzyme Xyn?-?g-Glu? and Xyn?-?g-Xyn?.(2)The optimum temperature of Xyn?-?g-Glu?(Topt)is 53?,than the Xyn(Topt 47?)increased 6?.Under the 50?,the half-life(t1/2)is 20 min than the Xyn(t1/2 17min)increased 3min.(3)The optimum temperature of Xyn?-?g-Xyn? is 40?,than the Xyn(Topt 47?)reduce 7?.Under the 40?,the half-life of Xyn?-?g-Xyn? is 15 min,and the Xyn under the 40 ? incubated for 40 min residual activity is still more than 80%.(3)The Km of Xyn?-?g-Glu? and Xyn?-?g-Xyn?values were 49.7mg/ml,47.4mg/ml than the Xyn(Km 12.1mg/ml)increased by about four times,indicating that two reduce the kinds of fusion enzyme and substrate affinity.The Kcat value of 71.9s-1,41.9 s-1 compared with Xyn(Kcat 589.7 s-1)were reduced by 8 times and 14 times,respectively,the two fusion enzyme catalytic activity reduced.It can be seen that the spiral structure on the stability of the molecule of family G/11 xylanases have an important role as molecular modules,while the helical structure can be generic,in each molecule.The ? helix plays a corresponding role between glucanase and xylanase.The stability of the enzyme molecule coordinated by the various parts of the molecule.And these provide new research ideas for Xylanase molecular modification.