Biochemical Properties And Functions Of Chitinases And Chitin-binding Protein From Pseudoalteromonas Sp. DL-6

Chitin, the most abundant renewable resource in ocean, is the second largest biomass on the earth after cellulose. In nature, microorganisms have evolved synergistic chitinase (chi, EC3.2.1.14) systems to address the recalcitrance of chitin and degrade the polymer into chito-oligosaccharides, N-acetylglucosamine (GlcNAc) as well as their derivatives, which are value-added and enhance immunity, antitumor, hypocholesterolemic, antifungal and antihypertensive activity, have potential applications in medical, agricultural, industrial and food industry and agricultural production. Marine environment is generally characterized by low temperature, high salinity, poor light penetration and oligotrophy. Marine organisms have evolved to adapt the extreme environment, and thus present gene resources to be explored. Therefore, studies of the biochemical property and degradation mechanism of chitinases secreted by marine bacteria would help to elucidate mechanism underlying chitin metabolism and utalize the enzyme resource.In this thesis, using colloidal chitin as the sole carbon source, a marine bacterium with high productivity of chitinase was isolated from samples collected in Bohai Bay in Dalian of Liaoning Province, China. According to physiological and biochemical characteristics and 16S rDNA sequence, it was identified as Pseudoalteromonas sp. DL-6, which produced different types of chitinases identified by the zymogram analysis and activities determined by degrading the fluorogenic substrates by the crude chitinases.Two chitinases genes chiA and chiC and one chitin-binding protein gene CBP58 were cloned by PCR from the genomic DNA of Pseudoalteromonas sp. DL-6. The expression plasmids pET28a-ChiA, pET23b-CBP58 and pET28a-ChiC were constructed and successfully expressed in E. coli BL21(DE3) with products as fusion proteins. The recombinant proteins ChiA, CBP58, and ChiC were purified by the Ni-NTA chromatography, and 33.74 mg ChiA,19.04 mg ChiC and 11.9 mg CBP58 were harvested from 1 L culture broth with concentration factors of 7.32 and 7.18 and recovery yields of 63.25% and 79.37%, respectively, for ChiA and ChiC.Enzymology study of the recombinant proteins suggested ChiA and ChiC showed high catalytic activities, even at a low temperature of 4 ?, and exhibited maximal activities at 20? and 30?, respectively. The Kcat/Km of ChiA and ChiC at 4? with colloidal chitin as the substrate was 0.56 and 1.83. ChiC also showed salt-tolerant, and exhibited more than 60% maximal activity in the presence of 5 M NaCl. ChiA and ChiC could hydrolyze other substrates including chitosan and Avicel, indicating their broad substrate spectrum. Oligomers with different degrees of polymerisation were detected when chitin was hydrolysed by ChiA, which suggested ChiA is an endo-chitinase. However, (GlcNAc)2 was the only product released during the hydrolysis catalyzed by ChiC, indicating it is an exo-chitinase.Scanning electron micrographs of chitin treated by CBP58 indicated that CBP58 could break the polymer chains. The substrate binding study showed that CBP58 bound preferably to a-chitin and colloidal chitin, less preferably to ?-chitin and (3-chitin nano-whiskers, and poorly to avicel. In addition, CBP58 acted synergistically with chitinases to efficiently degrade chitin and release chito-oligosaccharides. The psychrophilic bacterium, Pseudoalteromonas sp. DL-6 evolved a synergistic enzyme system to degrade recalcitrant chitin, which can be explored for bioconversion of chitin to oligosaccharides, and in the meantime for elucidating mechanism underlying chitin metabolism in oceans.