轉葡糖苷酶之生產、固定化及其應用於轉化熊果素之策略探討

Strategies for enhanced production of arbutin using enzymatic conversion of transglucosidase

許理絜(Li-Jie Syu)

指導教授 ： 魏毓宏

元智大學/工程學院/生物科技與工程研究所/碩士(2011年)

摘要

轉葡糖苷酶 (transglucosidase)，具有糖基催化反應及化學結構修飾的能力。尤其，轉葡糖苷酶之醣基化反應可輔助生物體在體內直接合成寡醣類，或在體外利用糖苷水解酶形成之糖苷鍵，以間接合成異麥芽寡糖或其他糖苷產物。此外，轉葡糖苷酶亦具有將水不溶性複合物轉化成水溶性物質，以改善其溶解度、穩定度之能力，進而使轉換而成之物質具備生理活性，因此被廣泛應用於製藥、食品、化妝品領域中。基於前述特質，以麥芽糖 (maltose)為醣基供給者，對苯二酚 (hydroquinone)為醣基接受者，轉葡糖苷酶 (transglucosidase)會將麥芽糖結構上的一個葡萄糖基分子轉移到對苯二酚結構上，以轉化合成α型熊果素 (arbutin)；熊果素為一可應用於美白及防曬的化妝保養品。因此，本研究擬利用Escherichia coli BL21以生產轉葡糖苷酶，並藉由固定化轉葡糖苷酶，以強化轉葡糖苷酶之穩定性及合成熊果素之轉化率與回收率。研究初步顯示：以LB 為基礎培養基培養Escherichia coli BL21，並經乳糖 (lactose)誘導24小時，其轉葡糖苷酶之活性與產物熊果素轉化率及濃度皆可達最高值，分別為0.012 U/mg、22%及10 ppm；若於菌體培養時加入10 mM Mn2+，可再提高轉葡糖苷酶之活性，轉化出的熊果素能達134 ppm。
經由French破胞處理所得的粗萃酵素，以80% 硫酸銨沉澱、兩次His-tag column的純化步驟，可將轉葡糖苷酶 (transglucosidase)之活性提高到0.39 U/mg 。其次，研究發現轉葡糖苷酶 (transglucosidase)於40℃、pH7.0的反應條件下，有最高的相對活性。
研究進一步証實：以1%海藻酸鈉 (Alginic acid)及0.5% EDC交聯劑固定化轉葡糖苷酶 (transglucosidase) 於40℃的耐受性測試中，酵素活性之半衰期 (half-life)可延長至26天，相較於未固定化之游離轉葡糖苷酶酵素半衰期4天為佳，顯示轉葡糖苷酶經固定化後，的確有助於穩定維持酵素之活性。

關鍵字

轉葡糖苷 ； 酶 ； 熊果素 ； 酵素固定化 ； 酵素活性半衰期

並列摘要

III

Strategies for enhanced production of arbutin using
enzymatic conversion of transglucosidase
Student：Li-Jie Syu Advisors：Yu-Hong Wei
Graduated School of Biotechnology and Bioengineering
College of Engineering
Yuan Ze University

Abstract
Transglucosidase is an enzyme that can catalyze the transglycosylation
and modified the chemical structure. Transglycosylation also can assist the
synthesizing of oligosaccharides in vivo. Literatures shown that
transglucosidase has transform water-insoluble compounds into
water-soluble to improve their solubility, ability, and make the substance
with physiological activity for the reason that used in pharmaceutical, food,
cosmetics. Based on the above characteristics, maltose as a donor and
hydroquinone as an acceptor, the transglucosidase will transferred a
glucose unit from the maltose and turn in the hydroquinone to synthesis of
α-arbutin. Arbutin is a potent Tyrosinase inhibitoy, whitening agent,
sunscreen agent and antioxidant. This study aims to use the recombinant
Escherichia coli BL21 to produce arbutin by the enzymatic conversion of
transglucosidase. Preliminary study shows the recombinant E. coli BL21
IV

can produce higher transglucosidase activity (0.012 U/mg), conversion rate
(22%) and arbutin production (10 ppm) in Luria-Bertani medium by lactose
induce after 24 h cultivation. Furthermore, the addition of Mn
2+
to the LB
fermentation broth markedly enhanced arbutin production. Arbutin
concentration of 134 ppm was obtained when the LB medium was
supplemented with 10 mM Mn
2+
.
Beside, crude transglucosidase from the French and ammonium sulfate
was added to the transglucosidase-cotaining supernatant at 80% saturation,
dialysis, and then purification by His-tag column that increased the enzyme
activity to 0.39 U/mg. Moreover, transglucosidase stability can be
improved by the immobilization of transglucosidase using various
conditions. Under an immobilization strategy with 1% alginic acid and
0.5% crosslinking (EDC), transglucosidase obtained a half-life of 26 d.

並列關鍵字

Transglucosidase ； Arbutin ； Immobilization ； Half-life

參考文獻

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