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Replacing a piece of loop-structure in the substrate-binding groove of Aspergillus usamii β-mannanase, AuMan5A, to improve its enzymatic properties by rational design

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Abstract

To perfect the enzymatic properties of AuMan5A, a mesophilic glycoside hydrolase (GH) family 5 β-mannanase from Aspergillus usamii, its loop-structure substitution was carried out by rational design and followed by megaprimer PCR. Based on the structural analysis and enzymatic property comparison of various β-mannanases, a piece of loop-structure with seven amino acids between two β-strands (βD and βE) in the substrate-binding groove, named “Loop DE,” was speculated to be correlative to the thermostability and catalytic efficiency of GH family 5 β-mannanases. Therefore, three AuMan5A’s mutants, AuMan5A-Af, AuMan5A-An, and AuMan5A-Th, were designed by substituting a Loop DE sequence (316KSPDGGN322) of AuMan5A with the corresponding sequences of other three family 5 β-mannanases, respectively. Then, the mutant-encoding genes, Auman5A-Af, Auman5A-An, and Auman5A-Th, were constructed as designed theoretically and then expressed in Pichia pastoris GS115. The expressed recombinant AuMan5A-Af (re-AuMan5A-Af) displayed the temperature optimum (T opt) of 75 °C, T m value of 76.6 °C and half-life (t 1/2) of 480 min at 70 °C, which were 10 and 12.1 °C higher and 48-fold longer than those of re-AuMan5A, respectively. Its catalytic efficiency (k cat/K m) was 12.7-fold that of re-AuMan5A. What is more, the site-directed mutagenesis of D320G in AuMan5A-Af was performed. The T opt and t 1/2 of expressed re-AuMan5A-AfD320G decreased to 70 °C and 40 min, respectively, while its k cat/K m was only 35 % of that of re-AuMan5A-Af. These results demonstrated that the mutation of G320 (in AuMan5A) into D320 (in AuMan5A-Af) through Loop DE substitution was mainly responsible for the thermostability and catalytic efficiency improvement of AuMan5A-Af.

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Acknowledgments

The authors are grateful to Prof. Xianzhang Wu (School of Biotechnology, Jiangnan University, Jiangsu, China) for providing technical assistance.

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    Authors and Affiliations

    1. State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, China

      Yun Hai Dong, Jian Fang Li, Chun Juan Wang & Shi Han Tang

    2. Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, China

      Die Hu & Xin Yin

    3. Wuxi Medical School, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, China

      Min Chen Wu

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    Correspondence to Min Chen Wu.

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    This work was financially supported by the National Nature Science Foundation of China (No. 31271811), the Fundamental Research Funds for the Central Universities of China (JUSRP51412B) and the Postgraduate Innovation Training Project of Jiangsu, China (KYLX_1170).

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    Yun Hai Dong and Jian Fang Li contributed equally to this work.

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    Dong, Y.H., Li, J.F., Hu, D. et al. Replacing a piece of loop-structure in the substrate-binding groove of Aspergillus usamii β-mannanase, AuMan5A, to improve its enzymatic properties by rational design. Appl Microbiol Biotechnol 100, 3989–3998 (2016). https://doi.org/10.1007/s00253-015-7224-7

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