鏈黴菌Streptomyces griseobrunneus S3菌株所產生ChiA幾丁質分解酵素分子特性及與其抗真菌性之關係

Abstract

The chitinolytic Streptomyces spp. are worldwide distributed beneficial microbial resources being actively explored as bio-agent for controlling plant diseases. The application of Streptomyces griseobrunneus strain S3 (SGS3) is one of the typical examples, its application as biofungicide for the control of various soil borne fungal diseases has been shown to be successful. And its effectiveness of disease control was found to due at least in part to the production of bountiful chitin and glucan hydrolytic enzymes. A family 19 chitinase gene chiF was cloned and shown to be one of the primed attributes contributing to the antifungal activity of SGS3 (Yang, 2005). In the present investigation, a full length chiA gene was cloned from SGS3 and its biochemical characteristics and biological significances especially the possible involvement in antagonistic activity of the test bacterium were explored. The cloned gene appeared to be 1668 bps in length which encoded a 555-amino acid composed protein molecule with molecular weight around 61.7 kDa. The sequence analysis by blast software of NCBI indicated that the cloned SGS3 chiA mainly consisted a carbonhydrate binding domain (CBM) at the N-terminal and a chitin catalytic domain on the C-terminal. The pairwise sequence comparison indicated that it shared a 84 % and 92 % identity to chiA of S. coelicolor A3(2) (GenBank accession no. BAB86375) and chiI of S. griseus (GenBank accession no. BAA75642) respectively. The encoded enzyme thus appeared to belong to chi18bA (subfamily B of family 18 chitinase A) as that suggested by Saito (2000). A promoter region belonging to group B was identified, in adjacent to the promoter region were some repetitive regulatory sequences relating to chitin induction and glucose repression. The repetitive sequences found close to the -35 upstream of promoter sequence appeared to be discrete as compared to Streptomyces group B promoters known to date. The full length of chiA has been transformed to Escherichia coli expression vector pET26b(+), and the recombinant ChiA protein was produced by over-expression of the transformant bacteria and purified by an AKTA FPLC system using with Superdex 200 HR 10/30 column. The serial analysis on the hydrolytic activity including that by application of specific fluorochrome-linked oligo-chitin substrates, by electrophoresis zymogram investigation, by viscometry, by reducing sugar assay and by high performance liquid chromatography (HPLC) of the reaction products, indicated clearly the studied SGS3 ChiA was endo-chitinase with certain exo-enzyme activity. The analysis was performed with the use of recombinant SGS3 ChiF purified from a SGS3 chiF transformant E. coli as a comparison. From the data herein presented, the enzyme appeared to cut a 4-methylumbellifery linked chitobiose (4-MU-GlcNAc2) into a chitobiose (GlcNAc2) and a free fluorochrome, and a 4-MU-GlcNAc3 into a GlcNAc2, a chitose monomer GlcNAc and the free fluorochrome. With the provision of GlcNAc6, the primary early product appeared to be GlcNAc2 and GlcNAc4, whereas the final product was primarily the disaccharide GlcNAc2. The hydrolytic activity of SGS3 ChiA appeared to differ greatly to that of SGS3 ChiF. The later was known to have endo- and exo-chitinase activity, and with the provision of GlcNAc6, the data herein presented indicated that the primary early product was GlcNAc2, GlcNAc3 and GlcNAc4, whereas the final product was primarily GlcNAc2.and GlcNAc. The activity of SGS3 ChiA was shown to have an optimum pH at 5.0, optimum temperature at 50 ℃, and was promoted by the presence of dications Mg2＋ and Ca2＋; the compared SGS3 ChiF was shown to have optimum pH at 5.0, an optimum temperature at 40 ℃, and the activity was not affected by the above shown dications. Also, both enzymes were shown to work best on artificial soluble chitin, fair on colloidal chitin and chitosan, and very weak on glyco-chitin. To illustrate the role of both enzymes in the antagonistic activity against fungal pathogen, a chitin binding domain truncated SGS3 ChiA designated as chiAΔCBM was constructed and cloned to E. coli for preparation of ChiAΔCBM recombinant protein. And by immunization of rabbit, the ChiA specific polyclonal antibody was prepared. The in situ detection of chitinase expression during the mycoparasitism of SGS3 was performed by differential staining fluorescent microscopy with the use of FITC labeled ChiAΔCBM specific IgG and rhodamine labeled ChiF. The results obtained revealed that upon the infection of SGS3 on Rhizoctonia solani AG4, the colonization of the spore biomass on the fungal thallus was consistently accompanied with the expression of both ChiF and ChiA. It was worth noting however that the expression signal of ChiF detected from the mycelial extension of SGS3 was still quite strong, but that of ChiA appeared to be trivial. The results seemed to downplay the role of ChiA in the antagonistic effectiveness of the target bacterium.