利用脂肪酶催化合成或自中草藥分離新穎性唾液酸衍生物及其對脂多醣的結合力分析

['唾液酸(Sialic acid, SA)又稱為N-乙醯神經胺酸（N-acetylneuraminic acid, Neu5Ac）屬於具有高度可變化學結構的醣類家族。在真核生物中，含SA的結構分子參與許多重要的生理和病理過程，如細胞間的黏附、病毒或細菌與目標細胞的結合等。因此，SA及其衍生物在保健食品和製藥工業中具有廣泛的應用。而革蘭氏陰性菌是常見的敗血症病原體，位在細菌外壁上的脂多醣 (Lipopolysaccharide, LPS)，其結構包含了具有親水性的多醣組成，以及疏水性的脂質組成，即脂質A (lipid A)，脂質A毒性會引起強烈的免疫反應。 SA已被證明能夠中和LPS毒性，但SA為親水性不具疏水性結構，為了提高SA對LPS的親和力，本研究中利用脂肪酶進行SA酯化或轉酯化反應，以產生新的唾液酸酯類衍生物，提升唾液酸的疏水性。使用SA及油酸做為反應物進行兩相酯化反應，加入重組脂肪酶Candida rugosa lipase 1~4 (CRL1~4)進行催化反應，結果並未發現產物形成，SA也沒有減少。使用不同鏈長脂肪酸酯和SA於有機溶劑中進行單相轉酯化反應，使用冷凍乾燥的重組CRL1~4、市售CRL或Novozymes Eversa Transform (即Thermomyces lanuginosus lipase, TLL)催化反應亦未有產物產生。當SA與vinyl acetate進行兩相轉酯化反應時，使用固定化Novozymes 435可達到90.5 %的最高轉化率，其次為重組脂肪酶CRL1、CRL4、及Novozymes TLL依序為53.7 ％、34.5 ％和28.2 %。當SA與vinyl propionate反應時，使用固定化Novozymes 435可達到92.5 %的最高轉化率，其次為CRL1及CRL4，轉化率依序為75.3 %及63.0 %。當SA與vinyl laurate反應時，固定化Novozymes 435、CRL1和CRL4的轉化率分別為67.2 ％、49.6 %、35.8 ％。使用vinyl palmitate作為基質，Novozymes435、CRL1及CRL4催化之轉化率分別為51.9 ％、68.4 %及17.0 %。由此可知，不同酵素對相同受質有不同的催化活性，而且同一種酵素對不同鏈長度脂肪酸酯受質，也有不同的催化活性，同時，相較於混合了多個isoforms的CRL商業化酵素，個別重組CRL isoforms的催化效率較佳。以前的研究表明中草藥LS含有唾液酸衍生物，透過加熱萃取和鹽酸水解，並使用HPLC進行分析和收集，得到一種可能為唾液酸衍生物LS-2。使用表面電漿共振技術 (Surface Plasmon Resonance, SPR)，我們比較了唾液酸和新型唾液酸衍生物LS-2對LPS的親和性。由於利用商業化SPR晶片所製作的SA固定化晶片，對LPS的靈敏度極低，故自行製作出一種對LPS有高靈敏度的Cys-SA 晶片，測得SA 與LPS的結合常數為KA =6.82 x 1010 M-1，顯示此晶片適合用來偵測LPS，而使用自行製作的Cys-LS-2 晶片測得LS-2對LPS的親和性比唾液酸高，結合常數為KA=1.10 x 1013 M-1，本研究結果可以應用於工業化生產唾液酸衍生物，擴展唾液酸衍生物的應用潛力。']

關鍵字

唾液酸；脂肪酶；兩相轉酯化反應；唾液酸衍生物；脂多醣

並列摘要

['Sialic acids (SA) (N-acetylneuraminic acids, Neu5Ac) represent a family of sugar molecules with highly variable chemical structure. SA-containing structures in eukaryotic systems play important roles in various physiological and pathological processes. These special features enable them to fulfill several important biological functions such as cell-cell adhesion and target-cell binding of viruses or bacteria. Therefore, SA and its derivatives have broad applications in health food and the pharmaceutical industry. Lipid A is a lipid component of an endotoxin and its toxic effects can induce a strong immune response. SA has been proved to be able to neutralize LPS toxins, but there are no hydrophobic structures in hydrophilic SA. To improve the SA’s binding affinity for LPS, the lipophilicity of SA could be increased by esterification or transesterification. In this study, the lipase-catalyzed esterification or transesterification of SA was performed to produce novel SA ester derivatives.Recombinant Candida rugosa lipase 1~4 (CRL1-4) were used to catalyze the esterification of SA and oleic acid in two-phase system. However, the substrate SA did not decrease and no product formed. Transesterification in organic solvent (one-phase system) was performed using various esters with different chain-length fatty acids and SA as substrates, and catalyzed by lyophilized recombinant CRL1-4, commercial CRL or Novozymes Eversa Transform (Thermomyces lanuginosus lipase, TLL).Still no products were formed. When transesterification in two-phase system was performed using vinyl acetate and SA as substrates, Novozymes 435, recombinant CRL1 and CRL4 achieved conversion rates of 90.5%, 53.7% and 34.0%, respectively.When similar transesterification reactions were performed using vinyl propionate, Novozymes 435, recombinant CRL1 and CRL4 achieved conversion rates of 92.5%, 75.3% and 63%, respectively. Usingvinyl laurate as substrate,the conversion rates were 67.2% for Novozymes 435, 49.6% for CRL1 and 35.8% for CRL4. Using vinyl palmitate as substrate, the conversion rates were 51.9% for Novozymes 435, 68.4% for CRL1 and 17.0% for CRL4. The results showed that different enzymes have different catalytic activities toward the same substrate, and a specific enzyme also has different catalytic activities toward different fatty acid ester substrates. In addition, individual CRL isoform showed higher catalytic activity than commercial CRL that contains mixture of different lipase isoforms. Previous studies have indicated that Chinese herbal medicine LS contains SA derivatives. In this study, a possible SA derivative LS-2 was obtained by hot-water extraction, hydrochloric-acid hydrolysis and then was analyzed and collected by HPLC. Using surface plasmon resonance (SPR), we compared the LPS-binding affinity of SA and the novel SA derivatives. Because the SA-immobilized SPR chip which is made from commercially available chip showed very low affinity for LPS, therefore, we used our in-house Cys-SA and Cys-LS-2 chips to assay the LPS-binding affinity. It was found that the Cys-SA chip had a binding constant (KA) of 6.82 x 1010 M-1, and showed high sensitivity for detecting LPS. The Cys-LS-2 chip had a binding constant (KA) of 1.10 x 1013 M-1,and showed that LS-2 had higher affinity for LPS than SA. This study could be potentially applied in industry for the production of SA derivatives and expand the applications of SA derivatives.']

並列關鍵字

Sialic acid ； Lipase ； Two-phase transesterification ； Sialic acid derivatives ； Lipopolysaccharide

參考文獻

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