| Summary: | 碩士 === 國立臺灣大學 === 食品科技研究所 === 91 === Chitooligosaccharides, manufactured by the hydrolysis of chitosan, are low molecular weight oligosaccharides formed with 2~10 glucosamines byβ-1,4 glycoside linkage. The chitooligosaccharides possess bioactive functions, including activation of macrophage, antitumor and antibacterial activities. It is also known that high degree polymerized (DP) oligomers, such as chitohexaose and chitoheptaose, have better biological function compared to other chitooligosaccharides. The objectives of this study were to investigate the processing conditions for manufacturing high DP chitooligosaccharides by enzymatic hydrolysis of chitosan in membrane reactor as well as in reverse micelle reactor using the chitosanase yielded from Bacillus cereus NTU-FC-4.
It was found that when crude enzyme was used in the membrane reactor, and the system operated at E/S ratio = 0.06 unit/mL；reactor volume = 500 ml；residence time = 50 min, a product containing chitooligosaccharides from DP2 to DP7 could be obtained. However, when the crude enzyme was purified by a cation exchanger and then used in the same system, the product only contained DP3 to DP7. In addition, the chitooligosaccharides concentrations in the product yielded from the purified enzyme were in general higher than that yielded from the crude chitosanase.
The chitosanase could be solubilized in the reverse micelles at 20℃ when the aqueous phase contained 200 mM of NaCl and the organic phase contained 20％ AOT. Using the mixture of chitooligosaccharides as substrate and the system was operated at E/S ratio = 0.06 unit/mL and Wo = 14.8, the phenomenon of transglycosylation was observed. The optimal Wo for the highest transglycosylation activity, however, was found at 11.86, and the product contained chitohexaose, chitoheptaose, chitooctasose and chitononaose. Results of this study demonstrated that high DP chitooligosaccharides could be formed due to the reaction of transglycosylation in the reverse micelle reactor.
In order to investigate the reactive mechanism of chitosanase on chitooligosaccharides, various standard solutions of chitooligosaccharides were reacted with chitosanase in a batch reactor. It was found that the major product of the enzymatic hydrolysis of chitotetraose was chitobiose, and glucosamine and chitotriose were only produced in a minor amount. When the chitopentaose was hydrolyzed the major products were chitobiose and chitotriose. And the major hydrolysis product of chitohexaose was chitotriose. It was also found that this enzyme had relatively higher hydrolysis activity toward chitopentaose and chitohexaose, less active toward chitotetraose, and inactive toward chitotriose and chitobiose. The above observed phenomena may be useful in explaining the chitooligasaccharides profiles of the products yielded from the membrane and reverse micelle reactors operated at various conditions.
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