Studies On The Chitinolytic Pathway And Its Related Thermophilic Enzymes From The Hyperthermophilic Archaeon Pyrococcus Horikoshii OT3

Chitin is the second most abundant organic compound on our planet, and the degradation of this biomass is an important process in the recycling of nutrients in the environments. By analysis of the genome features of the hyperthermophilic archaeon P. horikoshii 0T3, A novel chitinolytic pathway was identified in this archaeon. In this study, the thermophilic enzymes encoded by the conserved ORFs involved in this pathway were characterized. It was concluded that there is a unique chitin degradation pathway in the hyperthermophilic archaeon in the genera Pyrococcus, and the biochemical properties, mechanism and thermostability, and the catalytic mechanism of some thermophilic and thermostable enzymes were studied in detail. These thermophilic chitin degrading enzymes have potentials for industrial application in the fields of biotechnology. This thesis includes several parts as follows:1. The chitinolytic pathway from the the hyperthermophilicarchaeon P. horikoshiiThe PH0511(exo-β-D-glucosaminidase), PH0499 (diacetylchitobiose deacetylase), PH0495 (glycerate kinase) genes were amplified by PCR and cloned into the plasmid pET15b, expressed in E. coli BL21-codonPlus (DE3)-RIL, and purified by affinity chromatography and ion exchange chromatography. The biochemical properties of these thermophilic enzymes were studied by gel filtration chromatography, thin layer chromatography (TLC), spectrofluorimetry and radioisotope-labeling examination method.By characterization of the thermophilic enzymes involved in the chitinolytic pathway, a novel chitin degradation pathway in this archaeon was proposed. In this pathway, diacetylchitobiose deacetylase (Dac_ph) is able to deacetylate one acetyl group of GlcNAc₂ or chitooligosaccharides to form GlcN-GlcNAc or partly deacetyled chitooligosaccharides. By the concerted reaction with the exo-β-D-Glucosaminidase (BglA_Ph), it eventually converts GlcNAc₂ or chitooligosaccharides into GlcN.Although the chitinase genes are absent in the P. horikoshii genome, by the concerted action of exo-β-D-Glucosaminidase, diacetylchitobiose deacetylase and other glycosidase in vivo such asβ-mannose (PH0501), The archaeon could transform the chitin related heteropolysaccharides in benthal circumstance into GlcN, followed by the glycolysis pathway. The role of glycerate kinase is probably involved in the glycolysis pathway.2. Characterization of the thermophilic enzymes involved in the chtinolytic pathway from the hyperthermophilic archaeon P.horikoshii2.1 Exo-β-D-GlucosaminidaseThe exo-β-D-glucosaminidase from the hyperthermophilic archaeon P. horikoshii was found to be a thermostable homodimer and reducing agent and heat-treatment had little effect on its homodimeric structure in SDS-PAGE. The temperature and pH optima of the enzyme were determined to be 90°C (over 20 min) and 6.0 respectively. It has a half-life of 9 h at 90°C and is the most thermostable glucosaminidase described up to now. The purified protein can hydrolyze the nonreducing terminal glycosidic bond of chitosan oligosaccharides or chitosan, forming chitosan oligosaccharides one residue shorter or chitosan. The activity was not inhibited by organic solvents ethanol, 2-propanol, DMSO, PEG-400, denaturing agents SDS, urea, guanidine hydrochloride and bivalent metal ions Mg²⁺, Mn²⁺ Co²⁺, Ca²⁺ Sr²⁺, Ni²⁺. A modified spectrofluorimetry method to measure glucosaminidase activity was introduced to study kenetic properties of the wild type and site-directed mutant enzymes, and the results showed that the catalytic mechanism of the enzyme is not the classical catalytic mechanism of glycosyl hydrolase family 35. The C terminal about 100 amino acids is probably indispensable for the thermostability of the enzyme.2. 2 Glycerate kinase (forming 2-phosphoglycerate)The glycerate kinase (GK_ph) from the hyperthermophilic archaeon P. horikoshii was found to be a homodimer based on SDS-PAGE (47 kDa) and gel filtration chromatography (100 kDa) analysis. The temperature and pH optima of the enzyme were 45°C and 7. 0 respectively and about half of the maximal activity remained at 100°C. The enzyme was highly thermostable with almost no loss of activity at 70°C, 80°C and 90°C for 12 hours. A radioisotope-labeling examination method was initially used for the enzymatic activity detection, and the enzyme was found to catalyze the formation of 2-phosphoglycerate using D-glycerate as the substrate, Mg²⁺, Mn²⁺, Co²⁺, Ni²⁺ as bivalent metal ions and ATP, GTP, CTP, UTP, ADP and Ppi as phosphoryl donors. The enzyme exhibited unique phosphoryl donor specificity with maximal activity towards pyrophosphate. Monovalent metal ions and reducing agents could stimulate the activity of the enzyme. Based on sequence alignment and structural comparison, it was assigned to group I of the trichotomy of glycerate kinases.2. 3 Diacetylchitobiose deacetylaseThe gene (Dac_ph, PH0499) from the hyperthermophilic archaeon P. horikoshii was amplified by polymerase chain reaction, cloned into expression vector pET15b, and expressed in E. coli BL21-codonPlus(DE3)-RIL A soluble fraction of Dac_ph (31.6 kDa) was obtained as shown by SDS-PAGE. TLC analysis showed that Dac_ph is able to deacetylate one acetyl group of GlcNAc₂ and GlcNAc. By the concerted reaction with the exo-β-D-Glucosaminidase (BglA_Ph), it is also able to convert GlcNAc₂ into GlcN. So it is nominated a diacetylchitobiose deacetylase.2.4 Exo-chitinase and endo-chitinaseThe exo-chitinase and endo-chitinase from the hyperthermophilic archaeon P. furiosus were studied. TLC analysis indicated that the endo-chitinase could cleaved internal 1,4 glycosidic linkages in chitooligosaccharides randomly, while endo-chitinase could cleave off chitobiose from the nonreducing end of chitin or other chitooligomers.3. The applications of the thermophilic enzymes involved inthe chitinolytic pathwayBy characterization of the thermophilic enzymes involved in the chitinolytic pathway, the applications of these enzymes in biotranformation and biodegradation were discussed. Exo-chitinase and endo-chitinase could be explored to degrade chitin effectively and produce chitooligosaccharide. The exo-β-D-glucosaminidase may be exploited to be useful in biotechnological applications, such as production of glucosamine (optimizing the HC1 hydrolysis method), degradation or structural analysis of chitosan or partly acetylated chitooligosaccharides and peptidoglycan with glucosamine residues at the nonreducing end; by the concerted reaction with the exo-β-D-glucosaminidase and diacetylchitobiose deacetylase, the chitooligomers could be purposefully degraded. Glycerate kinase has potentials in applications for the enzymatic catalysis of glycerate to synthesize 2-PGA.