Chitin (poly-β-(1→4)-N-acetyl-D-glucosamine) is a natural polysaccharide found particularly in the shells of crustaceans such as crab and shrimp, the cuticles of insects, and the cell walls of fungi. It is the second most abundant polysaccharides receiving worldwide interest for its industrial, agricultural and medical applications. However, its application is quite restricted for its limited affinities with solvents due to the strong intermolecular hydrogen bonding and rigid crystalline structure. Chitosan, its deacetylated derivative, is soluble in aqueous medium in the presence of a small amount of acids such as AcOH, lactic acid, HC1 and so on, but precipitates at neutral or high pH regions. Consequently, various studies were conducted to make water-soluble derivatives of chitin or chitosan by partial deacetylation, acetylation or other chemical modification (Kurita, 2001). The latter has the possibility of losing the original physicochemical and biochemical activities, and bring some toxicity by changing the fundamental skeleton of chitin and chitosan. It is known that chitosan with about 50% degree of N-deacetylation (DD), produced by homogeneous acetylation of chitosan, was soluble in water for its random distribution of N-acetyl groups on the amino groups (Kurita, Kamiya & Nishimura, 1991; Aiba, 1991), the lower the molecular weight, the higher the solubility (Kubota, Tatsumoto, Sano & Toya,2000). These methods seem more tedious and expensive than that by deacetylation of chitin. However, heterogeneous deacetylation concentrated in the amorphous region of chitin to give block-type distribution of acetyl groups along the polymer chain (Kurita, Sannan & Iwakura, 1977), which resulted from its partially swollen with sodium hydroxide solution and thus led to locally impotent deacetylation (Ottoy, Varum, & Smidsord, 1996). Sannan, Kurita, & Iwakura (1976) reported a homogeneous deacetylation of chitin, but it was time-consuming (over two days) and procedure-complicated. In this thesis, a novel rapid preparation of water-soluble chitosan with relatively large molecular weight is studied, and its immobilized enzymatic degradation and hydrolytes were investigated as well. The brief work is described as followed:1 .Chitin from shrimp was systematically deacetylated using a NaOH treatment through forced penetration by vacuum with control of the reaction time. The degree of deacetylation was monitored using potentiometric titration, FT-IR and ~1H-NMR. The half deacetylated chitosans with degree of deacetylation (DD) of 49-57% reproducibly prepared behaved viscous and translucent in water. After degradation by pectinase, they were soluble in aqueous solution at all pH. Scanning electronic microscopy (SEM) shows a distinct change of deacetylated chitin by vacumm penetration for about 1 hour with respect to the powdered chitin material as a control. The chitin particles after deacetylation were turned into microfibrils with a diameter of around 1 micron, which facilitate homogeneous deacetylation. Powder X-ray diffraction (XRD) displayed adecreasing of crystalline index (CrI) as a function of DD under the same conditions of deacetylation. The d-spacing increase of the (020) plane indicated that there were expansions of the crystal lattices with increasing DD and/or decreasing molecular weight of chitosan and thus resulted in water-soluble derivatives with less intermolecular force, which was agreed to that from FT-1R. All the results show that the molecular weight and DD are collectively responsible for the solubility in the condition of random deacetylation of acetyl groups, which resulted from the intermolecular force.2. Solubility of half deacetylated chitosan can be improved by degradation or depolymerization of its precursor, with acid or enzymatic commonly used. Enzymatic degradation can be easily controlled, monitored and the products can be obtained without any modifications which is normally seen with acid hydrolysis. Chitosanase and chitinase are the specific enzymes for such degradation, but its cost, unavailability and specificity limit its usage. Pectinase and cellulose are known as the nonspecific chitinsanolytic enzymes which behaved higher hydrolytic activity to chitosan among other nonspecific enzymes. In present work, it is of necessity to select an enzyme more specific to half deacetylated chitosan between the two enzymes. The following work should be finished at first: determination of degree of deacetylation and optimization of the dinitrosalicylic acid (DNS or DNSA) method which is used in the determination of reducing ends that released during the assay of enzymatic activity.2.1. Establishment of a XRD method and an Enzymatic method for the determination of degree of deacetylation as a comparison with Potentiometric titration method, FT-IR and 'H-NMR.A novel method to determine degree of deacetylation (DD) of a-chitin and Chitosan with the range of 17%-94%DD using X-ray diffractometer (XRD) is proposed in the present work. It was calibrated using ' H-NMR for chitosan and FT-IR for chitin material, as a comparison to potentiometric titration method. The results showed a good linear correlation between the Crlo2o from XRD and the calibrated DD value. The calibration equation is y = -0.753x+104 (r2=0.9924). This method is found to be simple, rapid and sample reusable compared to other known techniques like IR and titration methods.Another improved method is an improved potentiometric titration method with a pretreatment of enzymatic depolymerization. The results show that this pretreatment reduced the viscosity of chitosan solution significantly, and in turn eliminated the adhesion problem with the electrode. Pectinase has the optimum hydrolytic activity at about pH 3.5-4.0, which facilitate the solubilization of chitosan compared to cellulose (pH6.0). On the hand, the pectinase solution added is very little (1:1000), hardly interfere with the determination. Therefore, it is a relatively efficient and low-cost method for the frequently routine test.2.2. Advanced DNS method3,5-dinitrosalicylic acid (DNS or DNSA) method is widely utilized in many fields for the determination of reducing sugars. But the detection wavelength was different among papers, such as 480nm, 482nm-484nm, 513 nm, 520nm, 530nm, 540 and 550nm. This paper aimed to validate the wavelength range based on improvement of this method. The results show that pretreatment with 10 mol I"1 NaOH at a ratio of 1:10 to a sample of reducing sugar, may lower the minimumchromogenic concentration, which was 0.28mmol 1"' for glucose (Glc), 0.32mmol L"1 for galacturonic acid (GalUA), 0.46mmol I"1 for glucosamine (GlcN), and 0.17mmol 1"' for N-acetylated glucosamine (GlcNAc), within the wavelength range from 482 nm, 482nm, 480nm and 484nm respectively, to 520nm. The minimum chromogenic concentrations within these ranges are all above their LOQs (limit of quantitation), which means the former is more important than the latter. Precision test shows that, for higher sensitivity, we could select shorter wavelength near the minimum wavelength around the maximum absorption; and for larger range of concentration of reducing sugar, we could select longer wavelength around 520nm with least interference and relative standard deviation (R.S.D). Moreover, measurement at the inflexion about 500nm, may lose some sensitivity, but gain relatively lower measurement errors. According to a t-student test with the improved method, sodium acetate buffer, and sodium hydrogenphosphate buffer hardly interfere with the determination. In addition, glucosemine showed torlerance to citric acid and sodium hydrogenphosphate buffer, and sodium borate buffer.In addition, the absorptivity of GlcN is different from that of GlcNAc. Therefore, for the comparison among the chitosan hydrolytes with different degree of deacetylation, there needs some parameter correction according to the two equations of the calibration curves. In another way, the reduction rate of viscosity can also be used to evaluate the hydrolytic acitivity of chitosanolytic enzymes. The latter is used in this thesis. Consequently, pectinase shows susceptibility to chitosan with about 60%, and cellulase shows higher activity to chitosan with higher DD. We can concluded that pectinase can be used to depolymerize the half deacetylated chitosan, and that excess acetyl may result blockage to the pectinase which make it possible to use chitin particles as an immobilized carrier.Enzymatic immobilization by covalent bonding to the Eupergit carrier and chitin respectively is used to prepare lower molecular weight of half deacetylated chitosan.with pectinase and cellulase, separately. The results indicated that the immobilized enzyme prepared by Eupergit gained lower activity recovery compared to that by chitin, but is more appropriate to the continuous preparation in column reactor due to its higher rigidity and mechanical strength. The immobilizations of pectinase and cellulase separately with respect to the activity recovery and stability were optimized. The optimum condition of pectinase is that 1 mg protein of pectinase in pH 6.0, 1.25mol I"1 Na2HPO4-NaH2PO4 buffer solution is immobilized with lg Eupergit carrier, which can get high stability and activity retention.Lower molecular weight of chitosan can be relatively soluble, therefore, the fractionation was made to make chitosan with various molecular weight to meet the requirement of its application. Generally, chitosan can be precipitated in organic solvent as alcohol. In the consideration of itsslightly poor solubility in alkali medium, NaOH and alcohol were used to consume least alcohol, and thus cut the cost.The biological activity of half deacetylated chitosan was reported to have many application that benefit for human's health. In this paper, the fractionated chitosan was used in the screening of angiotensin-converting enzyme (ACE) inhibition, and the result show that the inhibition of chitosan to ACE was increased with the increase of the degree of deacetylation and the decrease of the molecular weight..In general, deacetylation by forced penetration with concentrated NaOH was feasible, rapid, low-cost and high efficient. The solubility of half deacetylated chitosan was determined by its molecular weight. |