Based on the phenotypic characteristics and 18S rRNA gene sequence analysis, the fungus was identified as a member of the genus Penicillium
. Up to now, other reported Penicillium
strains which could produce chitosanase were P. islandicum
, P. spinulosum
and P. chrysogenum
(Fenton and Eveleigh, 1981
; Ak et al., 1998
; Rodríguez-Martín et al, 2010
Two highly conserved regions, NMDIDCD and YGIWGD were found from GH75 family chitosanases according to the alignment of the deduced amino acid sequences of some fungal chitosanases. Consequently, a pair of degenerate primers, DFP and DRP were designed to amplify a single fragment of 270 bp, and then the fully csn gene fragment was further cloned by I-PCR. The deduced amino acid sequences DIDCD of CSN were 100% similar to the corresponding conserved region of fungal chitosanases which have been classified into family GH75. It should be noted that CSN showed no similarity to bacterial chitosanases, suggesting a different evolutionary origin between fungal chitosanase and bacterial counterpart.
Although CSN exhibited high homology with the chitosanases of T. stipitatus ATCC 10500 and P. marneffei, they were different in molecular weight. CSN of Penicillium sp. D-1 consists of 250 amino acids, while the chitosanases of T. stipitatus ATCC 10500 and P. marneffei ATCC 18224 were composed of 459 amino acids and 1070 amino acids, respectively. As shown in the phylogenetic tree of the GH75 chitosanases (Figure ), CSN of Penicillium sp. D-1 was different from the chitosanases of T. stipitatus and P. marneffei by clustering into the outlying clade of orthologous chitosanases.
Figure 5 Phylogenetic sequences of GH75 chitosanases from 10 fungal proteins. The Phylogenetic tree was constructed with Mega 4. Neighbor-joining was used with 1000 random bootstrap replication. Alignment of the GH75 domain amino acid sequences used Clustal X (more ...)
To ensure that the cloned csn gene could encode a functional chitosanase, the csn gene encoding the mature protein without the signal sequence was heterologously overexpressed in E. coli. As shown in Figure expression of the CSN chitosanase resulted in large amounts of insoluble recombinant protein, and Sarkosyl and Triton-100 were used to solubilize the inclusion body. The Sarkosyl is thought to help solubilize the protein by partially denaturing it, and the subsequent addition of Triton-100 allows the renaturation of the protein. The active CSN was purified using Ni2+-NTA column after the denatured condition and then refolded. This is a simple and efficiently purification methods for active protein from inclusion body.
Although the calculated molecular mass of the mature protein was 24.6 kDa, the approximate size of the His-tagged protein was 36 kDa as showed by the SDS-PAGE electrophoresis. This discrepancy is apparently related to the acidic nature of the protein (16.9% glutamate and aspartate compared to 8.7% lysine and arginine and histidine). The predominance of acidic residues in most proteins results in their abnormal behavior during SDS-PAGE (Izotova et al., 1983
As previously reported, chitosanase from P. islandicum was a moderately acidiphilic enzyme, with the pH optimum at 4.5 to 6.0 and optimum temperature at 45°C. The optimum catalyzing condition for chitosanase from P. spinulosum is pH 5.0 and 55°C. Correspondingly, Penicillium sp. D-1 produced an acidiphilic chitosanase, showing maximum activity at pH 4.0 and 48°C. Thus, it is concluded that the chitosanase CSN of strain D-1 prefers lower pH and higher temperature to optimally hydrolyze substrate.
In conclusion, a novel gene belonging to GH75 chitosanase was cloned from a newly isolated Penicillium sp. D-1, and was successfully expressed in E. coli BL21 (DE3). To our knowledge, this is the first report on expression and characterization of Penicillium chitosanase. Biochemical and molecular characteristics showed that CSN should be a novel chitosanase which could be considered as a potential candidate for producing chitooligosaccharides from chitosan.