利用Bacillus cereus NTU-FC-4水解不同粒徑之蝦殼以生產N-乙醯幾丁寡醣

Abstract

N-乙醯幾丁寡醣是由2~10個N-乙醯葡萄糖胺以β-1,4醣苷鍵結而成的低分子量寡醣分子，具有抗菌、抗腫瘤及組織修復等活性，其中又以聚合度較高的N-乙醯幾丁六醣與七醣效果較顯著。傳統製備N-乙醯幾丁寡醣是由幾丁質水解而來，並且幾丁質通常是利用大量的酸鹼溶液處理蝦殼後而得，有造成環保污染的問題。本論文嘗試以研磨技術降低蝦殼顆粒大小，利用Bacillus cereus NTU-FC-4與其所產生之幾丁質酶直接作用於未經酸鹼處理過之蝦殼來生產N-乙醯幾丁寡醣，藉以達到綠色環保生產技術的目的。 本論文研究膠態幾丁質、葡萄糖、幾丁聚醣粉末、葡萄糖胺或N-乙醯葡萄糖胺做為培養Bacillus cereus NTU-FC-4之碳源，結果發現以膠態幾丁質作為碳源可獲得最高的幾丁質酶活性，約為0.45 unit/mL，可知本菌株為誘導型幾丁質酶生產菌。將蝦殼區分為蒸煮與未蒸煮，將之研磨成不同粒徑用以誘導Bacillus cereus NTU-FC-4產生幾丁質酶，酵素活性測定結果顯示，無論蝦殼有無蒸煮處理，皆在10 mm、1.5%濃度下可誘導Bacillus cereus NTU-FC-4產生最多量幾丁質酶，蒸煮過之蝦殼誘導出之幾丁質酶活性約為0.48 unit/mL；未蒸煮者之幾丁質酶活性約為0.45 unit/mL。另外，本研究發現以上述基質培養2天時可得到最多的酵素量，因此收集此時之酵素進行後續之酵素水解實驗，用以製備N-乙醯幾丁寡醣。 利用Bacillus cereus NTU-FC-4與其所產生之粗酵素水解不同粒徑之蝦殼，研究結果中發現，利用Bacillus cereus NTU-FC-4直接處理蝦殼，可獲得不同聚合度(NAG)1-6之N-乙醯幾丁寡醣，且其N-乙醯幾丁寡醣總量較酵素水解者高，然而隨培養時間增加，各寡醣含量沒有隨之增加。酵素水解之實驗發現，水解產物以N-乙醯幾丁單醣 (NAG)與N-乙醯幾丁二醣 (NAG)2為主，中間產物有N-乙醯幾丁四醣 (NAG)4、N-乙醯幾丁五醣 (NAG)5與N-乙醯幾丁六醣 (NAG)6，但是含量皆不高。不過，本研究發現，最小粒徑0.038~0.044 mm之蝦殼所產生之N-乙醯幾丁單醣 (NAG)與N-乙醯幾丁二醣 (NAG)2含量較高，證明越小粒徑之蝦殼愈有利酵素水解作用，可產生高聚合度的N-乙醯幾丁寡醣。

N-acetyl-chitooligosaccharides are composed of 2–10 N-acetyl-glucosamine residues with β-1,4-linkage to form low molecular weight oligomer. The N-acetyl-chitooligosaccharides have numerous bioactive functions, including anti-bacterial, antitumor, and tissue repair properties. It is also known that high degree polymerized oligomers, such as N-acetylchitohexaose and N-acetylchitoheptaose, have better biological function compared to other N-acetyl-chitooligosaccharides. Traditionally, chitin are produced from crustaceans (like shrimp shell) by a strong acid and base chemical procedure, and then hydrolysis of the chitin is carried out to produce N-acetyl-chitooligosaccharides. However, the use of strong acid and base makes this procedure environment unfriendly. The objective of this study was to incorporate medium milling technology to reduce the particle size of shrimp shell, thus it can be used directly for the production of N-acetyl-chitooligosaccharides. Various carbon sources, inculding colloidal chitin, glucose, chitosan powder, D-glucosamine and N-acetyl-D-glucosamine, were used to cultivate Bacillus cereus NTU-FC-4 to induce enzyme. It was found that the highest activity (0.45 unit/mL) of the chitinase produced by the Bacillus cereus NTU-FC-4 was obtained when the cultivation was carried out in the medium containing colloidal chitin, suggesting that Bacillus cereus NTU-FC-4 was an inductive chitinase producer. The steamed or non-steamed shrimp shell was processed by medium milling technology to reduce particle size, then, the suspension containing shrimp shell particle was used to cultivate Bacillus cereus NTU-FC-4. The results indicated that the chitinase produced by the Bacillus cereus NTU-FC-4 was about 0.48 unit/mL when the microorganism was cultivated in the medium containing 1.5% of 10 mm steamed shrimp shell. And the chitinase produced by the Bacillus cereus NTU-FC-4 was about 0.45 unit/mL when cultivated in the medium containing 1.5% of 10 mm non-steamed shrimp shell. Besides, it was also found that 48 hours of cultivation time could induce the highest amount of enzyme. Using shrimp shell of different particle sizes to cultivate Bacillus cereus NTU-FC-4 directly, it was found that the hydrolysate contained various chitooligomers, including monomer to hexamer. By hydrolyzing shrimp shell of different particle sizes by crude chitinase from Bacillus cereus NTU-FC-4, the main hydrolysate were N-acetylglucosamine and N-acetylchitobiose, and the intermediates were N-acetylchitotetraose, N-acetylchitopentaose, and N-acetylchitohexaose. It is interesting to note that the N-acetyl-chitooligosaccharides concentrations in the product yielded from the microorganism were generally higher than that yielded from the crude chitinase. In addition, the smaller the shrimp shell particle used, the higher the concentration of N-acetylglucosamine and N-acetylchitobiose could be obtained during crude chitinase hydrolysis. Results of this study demonstrated that particle size reduction was an effective method to facilitate enzymatic hydrolysis of shrimp shell.