[Characterization of a chitosanase from Fusarium solani and its expression in an industrial strain of Saccharomyces cerevisiae].
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.


CONCLUSIONS
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.
Liu
Huaiwei
H
State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, China. Huaiwei.liu@hotmail.com
Bao
Xiaoming
X
D003001:Cloning, Molecular; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005670:Fusarium / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008970:Molecular Weight; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism
D005656:Fungal Proteins; D006026:Glycoside Hydrolases; C019868:chitosanase