Directed modification of the Aspergillus usamii β-mannanase to improve its substrate affinity by in silico design and site-directed mutagenesis.
β-Mannanases (EC 3.2.1.78) can catalyze the cleavage of internal β-1,4-D-mannosidic linkages of mannan backbones, and they have found applications in food, feed, pulp and paper, oil, pharmaceutical and textile industries. Suitable amino acid substitution can promote access to the substrate-binding groove and maintain the substrate therein, which probably improves the substrate affinity and, thus, increases catalytic efficiency of the enzyme. In this study, to improve the substrate affinity of AuMan5A, a glycoside hydrolase (GH) family 5 β-mannanase from Aspergillus usamii, had its directed modification conducted by in silico design, and followed by site-directed mutagenesis. The mutant genes, Auman5A (Y111F) and Auman5A (Y115F), were constructed by megaprimer PCR, respectively. Then, Auman5A and its mutant genes were expressed in Pichia pastoris GS115 successfully. The specific activities of purified recombinant β-mannanases (reAuMan5A, reAuMan5A(Y111F) and reAuMan5A(Y115F)) towards locust bean gum were 152.5, 199.6 and 218.9 U mg(-1), respectively. The two mutants were found to be similar to reAuMan5A regarding temperature and pH characteristics. Nevertheless, the K m values of reAuMan5A(Y111F) and reAuMan5A(Y115F), towards guar gum, decreased to 2.95 ± 0.22 and 2.39 ± 0.33 mg ml(-1) from 4.49 ± 0.07 mg ml(-1) of reAuMan5A, which would make reAuMan5A(Y111F) and reAuMan5A(Y115F) promising candidates for industrial processes. Structural analysis showed that the two mutants increased their affinity by decreasing the steric conflicts with those more complicated substrates. The results suggested that subtle conformational modification in the substrate-binding groove could substantially alter the substrate affinity of AuMan5A. This study laid a solid foundation for the directed modification of substrate affinities of β-mannanases and other enzymes.
State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, Jiangsu, People's Republic of China.
Jianfang
J
Li
Xihuan
X
Wei
Cunduo
C
Tang
Junqing
J
Wang
Mei
M
Zhao
Qingfeng
Q
Pang
Minchen
M
Wu
D001230:Aspergillus / Q000201:enzymology* / Q000235:genetics; D003198:Computer Simulation; D008351:Mannans / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D013696:Temperature; D044902:beta-Mannosidase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
D008351:Mannans; D044902:beta-Mannosidase