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Journal of bioscience and bioengineering v.123 no.3, 2017년, pp.294 - 299   SCI SCIE
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Engineering a family 27 carbohydrate-binding module into an Aspergillus usamii β-mannanase to perfect its enzymatic properties

Li, J. ; Wang, C. ; Hu, D. ; Yuan, F. ; Li, X. ; Tang, S. ; Wu, M. ;
  • 초록  

    A family 27 carbohydrate-binding module of a Thermotoga maritima β-mannanase (TmCBM27) was chosen from the carbohydrate-active enzyme database by computer-aided design, possessing the lowest binding free energy with mannopentaose. To improve the enzymatic properties of a glycoside hydrolase family 5 β-mannanase from Aspergillus usamii (AuMan5A), two fusion β-mannanases, AuMan5A-F-M and AuMan5A-R-M, were designed by fusing a TmCBM27 into its C-terminus linked with a flexible peptide F (GGGGS) 3 and rigid peptide R (EAAAK) 3 . Two fusion enzyme genes, Auman5A-F-m and Auman5A-R-m, were constructed as designed theoretically by overlapping PCR. Then, Auman5A and two fusion genes were expressed in Pichia pastoris GS115. Three recombinant β-mannanases, reAuMan5A, reAuMan5A-F-M and reAuMan5A-R-M, were purified to homogeneity with specific activities of 230.6, 153.3 and 241.7 U/mg. The temperature optimum of reAuMan5A-R-M was 70 o C, identical with that of reAuMan5A, while its thermostability and melting temperature (T m ) reached 68 o C and 74.9 o C, being 8.0 o C and 8.4 o C higher than those of the latter, respectively. Additionally, the K m values of reAuMan5A-R-M, towards locust bean gum, konjac gum and guar gum, significantly decreased to 0.9, 1.9 and 2.5 mg/mL from 1.7, 3.8 and 4.2 mg/mL of reAuMan5A, while its k cat /K m (catalytic efficiency) values increased to 287.8, 163.7 and 84.4 mL/mg@?s from 171.2, 97.6 and 56.0 mL/mg@?s of the latter, respectively. These results verified that the fusion of a TmCBM27 into the C-terminus of AuMan5A mediated by (EAAAK) 3 linker contributed to its improved thermostability and catalytic efficiency.


  • 주제어

    β-Mannanase .   Carbohydrate-binding module .   Peptide linker .   Thermostability .   Catalytic efficiency .   Computer-aided design.  

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