異體表現β-櫻草糖苷酶與β-葡萄糖苷酶及其增進茶葉香氣之應用

Abstract

茶葉香氣是由揮發性有機化合物 (volatile organic compounds, VOCs)組成，而部分VOCs會在茶葉中形成β-櫻草糖苷 (β-primeverosides) 及β-葡萄糖苷 (β-glucosides)的糖苷鍵結揮發物 (glycosidically bound volatiles, GBVs) ，以存在於液胞中。在烏龍茶液的製造階段中，由於殺菁階段會造成內生性酵素失去活性，因此烏龍茶葉中仍有大量的 GBVs未被酵素水解，而外加β-櫻草糖苷酶 (β-primeverosidase)及 β-葡萄糖苷酶 (β-glucosidase) 可能具有增加烏龍茶葉或茶湯香氣的潛力。本實驗選殖並表現出茶葉中的糖苷水解酶─β-櫻草糖苷酶及 C. sinensis glycosyl hydrolase 1 β-glucosidase 1 (簡稱 CsGH1BG1)，並將其加入茶湯及茶葉製程的揉捻階段中，以頂空固相微萃取法輔助氣相層析串聯質譜儀 (headspace micro-extraction gas chromatography-mass spectrometry, HS-SPME GC-MS) 測定揮發性化合物含量差異。將β-櫻草糖苷酶及 CsGH1BG1構築至可轉譯出麥芽糖鍵結蛋白的 malE 基因下游的pMAL-c5x 質體，並以大腸桿菌表現系統異體表現會產生出分子量大小分別為 99.9 kDa 及 96.3 kDa 的 MBP-β-櫻草糖苷酶及 MBP-CsGH1BG1，再以澱粉樹酯管柱進行純化。為了證明重組蛋白的酵素活性，以 para-nitrophenol glucopyranosides (pNP-glucoside) 測定重組蛋白活性，結果顯示，MBP-β-櫻草糖苷酶及 MBP-CsGH1BG1 皆與控制組無顯著差異。然而，以茶葉水萃物進行酵素活性分析的結果中，MBP-β-櫻草糖苷酶處理組相較於控制組，在芳樟醇及香葉醇的含量皆有顯著提升，分別增加 78 % 及 88%，此結果證明MBP-β-櫻草糖苷酶的酵素活性。另一方面，為了評估外加糖苷水解酵素在烏龍茶製程中之應用性，將 MBP-β-櫻草醣苷酶及兩種商用酵素，Aromase及 β-葡萄糖苷酶 (com-β-GD) 的酵素液噴灑在揉捻階段的茶葉上進行發酵，結果顯示，重組蛋白 β-櫻草醣苷酶的處理組與控制組相比，揮發性化合物的含量無顯著變化，然而在 10 mg/mL com-β-GD的處理組中，葉醇 ((Z)-3-hexenol) 及苯甲醛 (benzaldehyde) 的含量有顯著提升 (p <0.05)，且苯甲醛之相對含量為對照組增加88 %，證明外源糖苷水解酵素應用在部分發酵茶製程具備可行性。本研究異體表現出具有β-櫻草糖苷酶酵素活性的MBP-β-櫻草糖苷酶，並開發出外源糖苷水解酵素應用在部分發酵茶製程的方法。

Tea aroma was comprised of volatile organic compounds (VOCs) which would transform into glycosidically bound volatiles (GBVs), and β-primeverosides and β-glucosides were two major GBVs in tea plants which were stored in the vacuole. Due to the high temperature of rolling stage during oolong tea manufacturing process, the endogenous enzymes were inactivated and resulted in a large amount of non-hydrolyzed GBVs remaining in dried oolong tea leaves. Therefore, exogenous β-primeverosidase and C. sinensis glycosyl hydrolase 1 β-glucosidase 1 (CsGH1BG1) might have the potential to enhance the aroma of oolong tea leaves and their infusion. In this study, β-primeverosidase and CsGH1BG1 (a β-glucosidase in tea plants) were cloned, identified and expressed. The recombinant proteins were applied in the tea extracts and rolled leaves during the production of oolong tea, and the variation of volatile compounds was analyzed by a head space solid-phase microextraction coupled with gas chromatography-mass spectrometry (HS-SPME GC-MS). The inserts of β-primeverosidase and CsGH1BG1 were constructed with pMAL-c5x plasmid, which contained malE gene encoding maltose binding protein (MBP), and 99.9 kDa of MBP-β-primeverosidase and 96.3 kDa MBP-CsGH1BG1 was expressed by Escherichia coli expression system, and then purified by amylose resin affinity column. To determine the enzyme activities, MBP-β-primeverosidase and MBP-CsGH1BG1 were treated with para-nitrophenol glucoside and showed no activity. The other enzyme assay by using tea extracts was applied, and MBP-β-primeverosidase was capable of increasing the contents of linalool and geraniol about 78 % and 88%, respectively. These results showed that MBP-CsGH1BG1 barely had enzyme activity of β-glucosidase. Since MBP-β-primeverosidase had the ability to increase the volatile compounds in tea extracts, indicating that MBP-β-primeverosidase might have β-primeverosidase activity. On the other hand, to evaluate the applicability of exogenous enzyme treatments during oolong tea manufacturing process, MBP-β-primeverosidase and two commercial enzymes, Aromase and β-glucosidase from almonds (com-β-GD), were sprayed on the tea leaves in the rolling step prior to fermentation. The results showed that the treatment of MBP-β-primeverosidase showed no influence on the volatile compounds in tea leaves. However, the relative content of (Z)-3-hexenol and benzaldehyde increased in the treatment of com-β-GD, and the latter increased about 88%, suggesting that the utilization of exogenous glycoside hydrolase during oolong tea manufacturing process was practicable. In conclusion, the recombinant MBP-β-primeverosidase was expressed and have β-primeverosidase activity. Furthermore, the method of exogenous glycoside hydrolase during oolong tea manufacturing process was established in this study.