title: Engineering a family 27 carbohydrate-binding module into an Aspergillus usamii β-mannanase to perfect its enzymatic properties.
abstract: 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)3and 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°C, identical with that of reAuMan5A, while its thermostability and melting temperature (Tm) reached 68°C and 74.9°C, being 8.0°C and 8.4°C higher than those of the latter, respectively. Additionally, the Kmvalues 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 kcat/Km(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)3linker contributed to its improved thermostability and catalytic efficiency.
authors: State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi 214122, China.
authors: Jianfang
authors: J
authors: Li
authors: State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi 214122, China.
authors: Chunjuan
authors: C
authors: Wang
authors: Key Laboratory of Carbohydrate Chemistry & Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China.
authors: Die
authors: D
authors: Hu
authors: State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi 214122, China.
authors: Fengjiao
authors: F
authors: Yuan
authors: State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi 214122, China.
authors: Xueqing
authors: X
authors: Li
authors: State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi 214122, China.
authors: Shihan
authors: S
authors: Tang
authors: Wuxi Medical School, Jiangnan University, Wuxi 214122, China. Electronic address: biowmc@126.com.
authors: Minchen
authors: M
authors: Wu
mesh_terms: D000595:Amino Acid Sequence; D001230:Aspergillus / Q000201:enzymology* / Q000235:genetics; D055162:Biocatalysis; D017076:Computer-Aided Design; D004795:Enzyme Stability; D005685:Galactans / Q000737:chemistry / Q000378:metabolism; D008351:Mannans / Q000737:chemistry / Q000378:metabolism; D009844:Oligosaccharides / Q000378:metabolism; D010843:Pichia / Q000235:genetics; D053149:Plant Gums / Q000737:chemistry / Q000378:metabolism*; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013696:Temperature; D013816:Thermodynamics; D020124:Thermotoga maritima / Q000201:enzymology / Q000235:genetics; D044902:beta-Mannosidase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D005685:Galactans; D008351:Mannans; D009844:Oligosaccharides; D053149:Plant Gums; D011993:Recombinant Fusion Proteins; C048068:mannopentaose; C007894:guar gum; D044902:beta-Mannosidase; C017471:locust bean gum
keywords: Carbohydrate-binding module; Catalytic efficiency; Computer-aided design; Peptide linker; Thermostability; β-Mannanase