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Mitsuaria chitosanase with unrevealed important amino acid residues: characterization and enhanced production in Pichia pastoris

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Abstract

A chitosan plate assay was employed to screen for chitosanase-producing bacterial strains and isolate 141 was found to exhibit high activity. Characterization of this isolate revealed that it belonged to Mitsuaria (designated as Mitsuaria sp. 141). The encoded chitosanase (choA) gene was then cloned by PCR and the deduced amino acid sequence showed 98% identity to a formerly described Mitsuaria chitosanitabida 3001 ChoA (McChoA). Surprisingly, the ChoA encoded by Mitsuaria sp. 141 (MsChoA) appeared to have a much higher optimum temperature compared to McChoA. Site-directed mutagenesis was then employed to generate five MschoA mutant genes encoding MsChoA K204Q, R216K, T222N, R216K/T222N, or K204Q/R216K/T222N. All the ChoA mutants exhibited a much lower specific activity and a lower optimum temperature. The results confirmed that the substitution of three non-conserved amino acids accounts for the major reduction of the enzyme activity in MsChoA. Furthermore, the MschoA gene was cloned for over-expression in Pichia pastoris after coding sequence optimization. One of the P. pastoris transformants with MutS phenotype was found to produce 1,480.2 ± 340.9 U ChoA mL−1 of cell culture by high-cell-density fermentation. This represents the highest yield of recombinant ChoA production that has ever been reported thus far. The recombinant P. pastoris strain should therefore be well suited for industrial-scale production of chitosanase.

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Acknowledgement

This research was supported by the National Natural Science Foundation of China (No. 31100050 and No. 31100096), the National Fund for Talent Training in Basic Science (J1103510) and the National Basic Research Program of China (2009cb724700).

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Authors and Affiliations

  1. State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, 430070, People’s Republic of China

    Nan Peng, Weiling Xu, Fan Wang, Jinlong Hu, Minhui Ma, Yuanliang Hu, Shumiao Zhao, Yunxiang Liang & Xiangyang Ge

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  1. Nan Peng
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  2. Weiling Xu
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  3. Fan Wang
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  4. Jinlong Hu
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  5. Minhui Ma
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  6. Yuanliang Hu
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  7. Shumiao Zhao
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  8. Yunxiang Liang
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Correspondence to Xiangyang Ge.

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ESM 1

Fig. S1. Coding sequence optimization of MschoA gene. a Coding sequence alignment of original (ori) and optimized (opt) MschoA genes, the signal peptide coding sequence was deleted. b Comparison of the average GC-content between original (upper) and optimized (lower) MschoA genes. Fig. S2. Alignments of Mitsuaria choA genes and their encoded enzymes. a Alignment of the MschoA and McchoA genes. b Alignment of the MsChoA and McChoA enzymes. The catalytic region with conserved amino acid residues is indicated (DOC 1486 kb)

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Peng, N., Xu, W., Wang, F. et al. Mitsuaria chitosanase with unrevealed important amino acid residues: characterization and enhanced production in Pichia pastoris . Appl Microbiol Biotechnol 97, 171–179 (2013). https://doi.org/10.1007/s00253-012-3901-y

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