Abstract
Bacillus velezensis RB.IBE29 is a potent biocontrol agent with high chitinase activity isolated from the rhizosphere of black pepper cultivated in the Central Highlands, Vietnam. Genome sequences revealed that this species possesses some GH18 chitinases and AA10 protein(s); however, these enzymes have not been experimentally characterized. In this work, three genes were identified from the genomic DNA of this bacterium and cloned in Escherichia coli. Sequence analysis exhibited that the ORF of chiA consists of 1,203 bp and encodes deduced 45.46 kDa-chitinase A of 400 aa. The domain structure of chitinase A is composed of a CBM 50 domain at the N-terminus and a catalytic domain at the C-terminus. The ORF of chiB includes 1,263 bp and encodes deduced 47.59 kDa-chitinase B of 420 aa. Chitinase B consists of two CBM50 domains at the N-terminus and a catalytic domain at the C-terminus. The ORF of lpmo10 is 621 bp and encodes a deduced 22.44 kDa-AA10 protein, BvLPMO10 of 206 aa. BvLPMO10 contains a signal peptide and an AA10 catalytic domain. Chitinases A and B were grouped into subfamily A of family 18 chitinases. Amino acid sequences in their catalytic domains lack aromatic residues (Trp, Phe, Tyr) probably involved in processivity and substrate binding compared with well-known bacterial GH18 chitinases. chiB was successfully expressed in E. coli. Purified rBvChiB degraded insoluble chitin and was responsible for inhibition of fungal spore-germination and egg hatching of plant-parasitic nematode. This is the first report describing the analysis of the chitinase system from B. velezensis.
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Data availability
Sequences in this study have been deposited in the DNA Data Bank of Japan database under accession numbers LC649802–LC649804.
Abbreviations
- GlcNAc:
-
N-Acetyl-D-glucosamine
- GH:
-
Glycoside hydrolases
- AA10:
-
Auxiliary activities family 10
- CBM50:
-
Carbohydrate-binding module family 50
- LB medium:
-
Luria–Bertani medium
- PCR:
-
Polymerase chain reaction
- bp:
-
Base pai
- ORF:
-
Open reading frame
- SDS–PAGE:
-
Sodium dodecyl sulfate–polyacrylamide gel electrophoresis
- aa:
-
Amino acid
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Acknowledgements
This work was funded by the Ministry of Education and Training under grant number B2020-TTN-04. The authors wish to thank Dr. Ngoc Luong Nguyen (School of Science, Hue University) for his generous sharing of the pColdII vector (Takara, Japan) and E. coli BL21-CodonPlus (DE3)-RIPL cells (Agilent, USA).
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DMT conceived the study, designed the experiments, analyzed, and wrote the manuscript. DMT, TUH, THN, TOD, QVN, and ADN performed experiments. ADN involved in the interpretation of the data and critical reading of the draft. All authors reviewed and approved the final manuscript.
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Tran, D.M., Huynh, T.U., Nguyen, T.H. et al. Molecular analysis of genes involved in chitin degradation from the chitinolytic bacterium Bacillus velezensis. Antonie van Leeuwenhoek 115, 215–231 (2022). https://doi.org/10.1007/s10482-021-01697-2
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DOI: https://doi.org/10.1007/s10482-021-01697-2