Study On The Screening, Identification, And Optimization Of Fermentation Conditions Of A Chitinase-producing Strain Hd002, And The Purification And Characterization Of The Chitinase Secreted By It

Chitin, the second most abundant macromolecular polysaccharide in nature after cellulose, is also the second most abundant organic compound containing nitrogen on the planet after proteins. It is consists of 0-1,4-linked units of N-acetylglucosamine (GlcNAc). Chitin is widely found in the exoskeleton of shrimp, crabs and insects and in the cell wall of fungi and plants. More than 10 billion tons of chitin is produced each year. Chitin is the important raw material in food, pharmaceutical, chemical and other areas. But chitin can not be dissolved in acid, alkali and ordinary organic solvents, which severely limits its application.There are three methods in the degradation of chitin, which are chemical, physical, and enzymatic, respectively. In the process of chemical degradation of chitin, there are harsh reaction conditions, severe corrosion of the equipment, poor reaction stability, heterogeneous products, much by-products, low yield, low quality of the products, high cost, serious environmental pollution, etc. Therefore, the application of chemical methods is limited. In the process of physical degradation of chitin, there are uniform products, less by-products, high quality of the products, no pollution. The physical degradation is an ideal method. But it is mainly used in the degradation of chitosan; there is less report on chitin degradation. The method of enzymatic degradation of chitin is to use enzymes to degrade chitin to oligosaccharides or monosaccharide. The enzymes degrading chitin can be divided into specific and non-specific enzymes. Non-specific enzymes include cellulase, lysozyme and lypase; specific enzymes are chitinases. The reaction of enzymatic degradation of chitin is mild; the products are homogeneous. There is a very good prospect of enzymatic degradation of chitin because of the high yield and less pollution. Chitinases are found in a wide range of organisms including bacteria, fungi, actinomycetes, as well as plants and animals. Bacterial chitinases can be used in the hydrolysis of chitin in the environment. Chitin is hydrolyzed into oligosaccharides and monosaccharide, which are the nutrition for the bacteria. Fungal chitinases can hydrolyze cell wall in the proliferation process of fungi, which play an important role in morphogenesis of fungi. Arthropod chitinases are involved in the process of the normal molting process of arthropod, which play an important role in morphogenesis of arthropods. Plants contain chitinases, so that plants can resist the invasion of pathogenic fungi and pests. Chitinases are capable of hydrolyzing chitin to its oligomers and monomer though specifically cuttingβ-1,4 glycosidic bonds. Chitinases have many potential applications. For example, they can be used in the control of plant-pathogenicfung and pest, production of functional chitooligosaccharides, handling of chitin waste, production of isolation of fungal protoplasts, and environmental protection. Hence, the production of chitinases has received considerable attention during recent decades. However, with respect to low amount of chitinases production by strains screened form nature and the relatively long period of chitinases production, there is not a suitable candidate for production of chitinase on an industrial scale. Therefore, it is very important to get chitinases with high activity.The aim of this research is to screen and select an effectively chitinase-producing strain from the soil by seaside and to investigate conditions affecting chitinase production. In this study, preliminary identification of the strain, enzyme purification, enzymatic properties, and enzymatic substrate are also studied.Six strains with ability to degrade chitin are isolated during a screening program. An effectively chitinase-producing strain HD002 is screened for further study based on the high-activity and is initially identified as Massilia sp. through 16S rDNA partial sequence analysis along with physiological and biochemical characteristics. Strain HD002 is able to produce high levels of extracellular chitinase in a simple medium containing chitin as sole carbon source.The effects of medium composition and physical parameters on chitinase production by strain HD002 are studied. Through the optimization of fermentation conditions, the ability of strain HD002 to produce chitinase is strengthened. The optimal compositions of the medium for the strain to produce chitinase are as follows (%, w/v):(NH4)2SO4 0.5%, K2HPO4 0.07%, KH2PO4 0.03%, MgSO4·7H2O 0.1%, colloidal chitin 1.0%. When strain HD002 is incubated at 30℃for 192 h in the liquid medium with initial pH6.0, the maximal activity of the chitinase in the fermentation liquid could reach 1.314 U/mL. This study shows initial pH, carbon and nitrogen sources playing important roles in enzyme production.The strain HD002 is cultured under the optimal fermentation conditions. After the treatment with centrifugation, the chitinase of the strain is extracted by 70% saturation of ammonium sulfate from the fermentation supernatant. The crude enzyme powder is gotten after dialysis and freeze-drying. Through DEAE-Sepharose Fast Flow anion exchange chromatography, dialysis, freeze-drying, enzyme powder I is received. Through Sephadex G-75 gel filtration chromatography and freeze-drying, enzyme powderⅡis received. After the three steps, the enzyme is purified 7.3 folds, the yield is 31.1%. SDS-PAGE shows single band for the enzyme powderⅡ, which proves the chitinase purified. The molecular weight of the chitinase is estimated by SDS-PAGE to be 60.2 kDa. The mass spectrometry is also used to analyze the molecular weight of the enzyme, which shows it is 59.0 kDa. There are little differences between the two measurement methods. Thus, the molecular weight of the enzyme is estimated to be 60.2 kDa.Through the study of the enzyme characterization, the optimum reaction temperature of the chitinase is 55℃. The enzyme has thermal stability when the temperature is below 45℃. There is almost no loss of enzyme activity, when it is incubated in the water bath (45℃) for 3 hours. While incubated in the water bath (65℃) for 30 minutes, the enzyme activity has been basically lost. The optimum pH of the enzyme is 5.0, but between pH4.6-7.2, there are no significant differences in enzyme activity. Colloidal chitin is the optimum substrate. Metal ions have great influence on the enzyme activity. The enzyme is extremely inhibited by Cu2+ (1 mmol/L) and Fe3+ (1 mmol/L), especially Fe3+, whereas both of Ca2+(1 mmol/L) and Na+(1 mmol/L) promote the enzyme activity.The result of thin-layer chromatography shows that the degradation products of the chitinase hydrolyzing colloidal chitin. Thus, the enzyme is endochitinase or exochitinase, not N-Acetyl-β-D-Glucosaminidase.