Thermostability And Activity Of Aspergillus Niger Xylanase Enhanced By Biterminal Fusion Of Substrate Binding Module C1/C2

Xylanase is widely used in paper,feed and energy industry.One Aspergillus Niger ?-1,4-xylanase Xyn ??X?belongs to the family GH11.Its thermostability needs to be enhanced for application in industry.A carbohydrate-binding module?CBM9?locates at C-terminus of a Thermotoga Maritima Xyn A family GH10.When fused to the C-terminal of Xyn?,the CBM9 improved xylanase activity.When divided into CBM9₁?C1?and CBM9₂?C2?,and respectively fused to the C-terminal or N-terminal of Xyn,the C1 was shown to improve the catalytic activity,and the C2 was shown to improve catalytic activity and thermostability.However,CBM fusion is focused on one terminus of a catalytic module.The study fused the C1 and C2 to the bi-termini of X using the inherent linker peptide to create four kinds of chimeras C1-X-C1?C1-X-C2?C2-X-C1 and C2-X-C2,with an expectation to improve the enzymatic properties by collaboration of the bi-terminal modules.The results are as following:1)Construction of recombinant plasmid : recombinant plasmids p ET20b-C2-X-C1 and p ET20b-C1-X-C2 were constructed using reverse PCR.Reverse PCR was optimized by setting two annealing temperatures and by adjusting ramp to 2 ? /S.Using optimized reverse PCR,recombinant plasmids containing repetitive sequence p ET20b-C2-X-C2 and p ET20b-C1-X-C1 were created.The recombinant plasmids were transformed into BL21?DE3?,positive colonies were screened,and gene accuracies were proved by DNA sequencing.After induction by IPTG,properties of purified enzymes were measured.2)Enzyme properties: the optimal p H?p Hopt?of fusion enzymes C2-X-C2 and C2-X-C1 were 4.2,while the wild-type X was 3.8.Both the C2-X-C2 and C2-X-C1 optimal temperature?Topt?were 45 ?,and both maintained higher than 80% of catalytic activity between 35 ?-50 ?,showing a wide range of temperature adaptation.3)Thermostability: when the enzymes were pre-incubated at 50 ?,the inactivation half-life?t1/2?of C2-X-C1 was 4.27 h,which is 12.7 times that of the X?21.04 min?and 3.7 times that of X-C2?69.3 min?.The t1/2 of C2-X-C2 was 9.4 h,which is 26.8 times that of the X and 8 times that of X-C2.When the enzymes were pre-incubated at 80 ?,the t1/2 of C2-X-C2 was 2.8 h.4)Kinetic analysis: when beechwood xylan was used as the substrate,the Km values ofC2-X-C1 and C2-X-C2 were 3.97 mg/ml and 1.44 mg/ml respectively.Compared with that of the X?2.72?,the C2-X-C2 had a higher affinity for beechwood xylan.The Kcat of C2-X-C1 was183.39s-1,the Kcat of C2-X-C2 was 445.15s-1,which is 1.6 times that of X?280.79?.Compared with the X?19.8 mg/ml?on oat-splt xylan,the Km of C2-X-C1 and C2-X-C2 were18.52 mg/ml and 13.13 mg/ml respectively,showing affinity on oat-splt xylan was significant improved.The Kcat values of C2-X-C1 and C2-X-C2 were 150.62 s-1 and 774.86 s-1 respectively.The C2-X-C2 showed an about5 times hither catalytic activity than the X?150?.Fusing the C1 and C2 to bi-terminal of Xylanase greatly improved the thermostability and the catalytic ability for unsolvable substrate.The bi-terminal fusion enzyme was proved to further improve enzyme properties.It also offers a new way for modification of enzyme properties.