Improvement Of The Thermostability Of Aspergillus Niger Xynlanase By N-Terminal Strucyure Replacement

Endo-xylanases [EC 3.2.1.8] can hydrolyze the ?-1,4-xylosidic linkages,which is widely used in many industries,for example the animal feed,food,pulp bleaching,textile,and energy conversion.The xynlanase from Aspergillus niger(Xyn III)(Gen Bank: ABY77763.1)belong to GH11 family,that not suitable for high temperature of industrial production process.It is very important to transform the thermostability of Xyn III.The spatial structure of Xyn III and the glucanase from Thermotoga maritime(Glu)(Gen Bank: CAA93274.1)were ?-jelly-roll.There are similar structure domains of Xyn III and Glu,that composed by the ?-pleated sheet region of N-terminal??-helix region and the?-pleated sheet region of C-terminal.So,this research wanted to replace the N-terminal of Xyn III with the N-terminal of Glu that exhibits excellent thermostability,expecting to improve the thermal stability of xylanase.The results are as following:(1)Acquisition of recombinant enzymes.The p ET20b-Glu-Xyn as material,the recombinant plasmid p ET20b-GN-X?-XC and p ET20b-GN-G?-XC are constructed by reverse PCR.The recombinant genes are constructed to p ET-22 b by the method of digestion with the restriction enzyme and T4 DNA ligase.The plasmids were transformed into Escherichia coli BL21(DE3),the recombinant enzymes GN-X?-XC and GN-G?-XC were obtained and mostly existed in form of inclusion bodys(IBs).(2)Inclusion bodys refolding by ultrafiltration.In this research,the ultrafiltration renaturation method of inclusion bodys was established.At frist,inclusion bodys dissolved with high concentrations of urea;secondly,the objective protein purification under the condition of denature;thirdly,refolding protein by ultrafiltration.The recombinant enzyme refolding protein can be used in the determination of pure enzyme properties with biological activity.(3)Recombinant enzyme enzymology properties.1)The optimum p H(p Hopt)of GN-X?-XC and GN-G?-XC were 4.6 and 4.8,compared with the wild type Xyn III(p Hopt 3.8)increased 0.8 units and 1.0 units respectively.2)The optimum temperature(Topt)of GN-X?-XC and GN-G?-XC were 50 ?,compared with the wild type Xyn III(Topt 46 ?)increased 4 ? respectively.3)The half deactivation time under 50 ?(t1/2)of GN-X?-XC and GN-G?-XC were173.29 min and 26.66 min,compared with the wild type Xyn III(t1/219.80 min)extended153.49 min and 6.86 min respectively.(4)Recombinant enzyme kinetic parameters.1)The Km values of GN-X?-XC and GN-G?-XC were 3.599 mg·m L-1 and 3.992 mg·m L-1,compared with the wild type Xyn III(Km 3.577 mg·m L-1)increase respectively,suggests that the affinity between two recombinant enzyme with substrate compared with the wild type is reduced.2)The Kcat values of GN-X?-XC and GN-G?-XC were 2.065 s-1 and 1.808 s-1,compared with the wild type Xyn III(Kcat 600 s-1)decreased by 291 times and 332 times respectively,suggests that the catalytic activity of two recombinant enzyme are greatly amplitude reduced compared with the wild type.The N-terminal structure of Xyn III was replaced and the optimum temperature(Topt)and the half deactivation time under 50 ?(t1/2)of recombinant enzyme GN-X?-XC and GN-G?-XC were improved significantly compared with the wild type Xyn III.It provides a new scientific idea to the enzyme molecule modification based on the structure analysis.