Objective: To investigate the biochemical characteristics of the chitosanase from Fusarium solani, and its application in chitooligosaccharides production, and express the chitosanase gene (csn) in a Saccharomyces cerevisiae industrial strain.
Methods: The chitosanase cDNA (EU263917) was amplified by reverse transcription-mediated PCR (RT-PCR). A His-chitosanase fusion protein was expressed in E. coli DE3. This protein was purified and characterized. Thin-layer chromatography (TLC) and high performance liquid chromatography (HPLC) were used to analyze the hydrolysates of this enzyme when it acted on 85% deacetylated chitosan. In addition, the csn cDNA was also fused to the inulinase (INU1A) signal sequence from Kluyveromyces marxianus. The fusion sequence was transferred into S. cerevisiae industrial strain N-27.
Results: The purified chitosanase had a specific activity of 2.5 U/mg. The enzyme showed the maximum activity at 50 degrees C, which was different from the first reported chitosanase that from F. solanif. sp. Phaseoli. TLC and HPLC results indicated that most of the hydrolysis products were chitooligosaccharides with a polymerization below 10, and no monomers were detected. Furthermore, chitosanase activity was detected in the culture medium of the recombinant S. cerevisiae cultures, and the volume activity reached 50.2 mU/mL. This result indicated that the recombinant protein was secretively expressed in S. cerevisiae.
Conclusion: The chitosanase from F. solani 0114 is an endochitosanase. Because of its special characteristics, this enzyme is very useful in chitooligosaccharides production, and the construction of recombinant S. cerevisiae made a step towards the large-scale production of this enzyme.
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| Evidence ID | Analyze ID | Gene/Complex | Systematic Name/Complex Accession | Qualifier | Gene Ontology Term ID | Gene Ontology Term | Aspect | Annotation Extension | Evidence | Method | Source | Assigned On | Reference |
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| Evidence ID | Analyze ID | Gene | Gene Systematic Name | Phenotype | Experiment Type | Experiment Type Category | Mutant Information | Strain Background | Chemical | Details | Reference |
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| Evidence ID | Analyze ID | Gene | Gene Systematic Name | Disease Ontology Term | Disease Ontology Term ID | Qualifier | Evidence | Method | Source | Assigned On | Reference |
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| Evidence ID | Analyze ID | Regulator | Regulator Systematic Name | Target | Target Systematic Name | Direction | Regulation of | Happens During | Regulator Type | Direction | Regulation Of | Happens During | Method | Evidence | Strain Background | Reference |
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| Site | Modification | Modifier | Source | Reference |
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| Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Allele | Assay | Annotation | Action | Phenotype | SGA score | P-value | Source | Reference | Note |
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| Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Assay | Annotation | Action | Modification | Source | Reference | Note |
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| Complement ID | Locus ID | Gene | Species | Gene ID | Strain background | Direction | Details | Source | Reference |
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| Evidence ID | Analyze ID | Dataset | Description | Keywords | Number of Conditions | Reference |
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