Engineering a family 27 carbohydrate-binding module into an Aspergillus usamii β-mannanase to perfect its enzymatic properties
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.
유료 다운로드의 경우 해당 사이트의 정책에 따라 신규 회원가입, 로그인, 유료 구매 등이 필요할 수 있습니다. 해당 사이트에서 발생하는 귀하의 모든 정보활동은 NDSL의 서비스 정책과 무관합니다.
원문복사신청을 하시면, 일부 해외 인쇄학술지의 경우 외국학술지지원센터(FRIC)에서
무료 원문복사 서비스를 제공합니다.
NDSL에서는 해당 원문을 복사서비스하고 있습니다. 위의 원문복사신청 또는 장바구니 담기를 통하여 원문복사서비스 이용이 가능합니다.
- 이 논문과 함께 출판된 논문 + 더보기