大腸桿菌及乳酸鏈球菌表現重組甜味蛋白質之改進及其發酵生產

Abstract

甜味是現今人們飲食當中不可缺少的一環，早期的甜味劑主要來自於自然界中的單糖及寡糖,但由於過量的攝取糖類會導致齵齒、糖尿病、高血壓等疾病。因此為了追求低卡路里、安全無虞、甜度高的甜味劑，分別從各種植物中分離出天然甜味劑，其中又以brazzein分子量低、pH值穩定、耐熱性佳、甜度高且風味與蔗糖相近，可望成為取代人工代糖的純天然甜味劑。大腸桿菌(Escherichia coli) 是遺傳工程及商業上最常使用於表現異源蛋白質之革蘭氏陰性菌，而乳酸鏈球菌(Lactococcus lactis)自古至今應用於發酵食品、食品保存、乳製品已有數千年的歷史，且為美國食品藥物管理局（US Food and Drug Administration, FDA）認定安全級（generally recognized as safe, GRAS）菌株，在遺傳工程中則作為蛋白質表現宿主，其食品級且不產生內毒素、具有較佳的蛋白質分泌能力、胞外蛋白酶活性低等特性，被認為是具有潛力的微生物蛋白質生產工廠。 本研究以大腸桿菌及食品級乳酸鏈球菌表現重組甜味蛋白質，首先構築大腸桿菌表現之質體為以異丙基-β-D-硫代半乳糖苷誘導之pET-brazzein質體，乳酸鏈球菌構築則nisin誘導之MNICE及酸誘導型pHI及MpHI表現質體。使用大腸桿菌轉型株於低溫發酵誘導表現，取一公升發酵液之菌體部分純化重組甜味蛋白質，經由濃縮透析可得到15.4 mg之產量，並可以品嚐出甜味，其相對甜度以濃度80~100ug/ml之重組甜味蛋白質最為接近8%蔗糖水溶液。而乳酸鏈球菌表現系統中則由於使用NICE系統成本較為昂貴，較不適合做大量發酵生產，因此另外以nsr基因構築食品級表現系統，食品級表現系統之nisin耐受性試驗結果顯示可以nisin濃度1~3μg/ml作為食品級表現系統之抗生素濃度，然而食品級表現系統表現不佳，故以酸誘導系統大量發酵表現及純化重組甜味蛋白質，結果顯示融合可穩定mRNA之UTL的MpHI系統確實可提升重組甜味蛋白質之表現量，且蛋白質鑑定確認上清液中確實含有重組甜味蛋白質。而純化方面以陽離子層析純化管柱純化所得之重組甜味蛋白質則因訊號過於雜亂而無法確認，實際品嚐也沒有甜味，推測可能因取24小時之上清液而此時菌體lysis，造成純化不完全所致。本實驗亦以批式大量發酵及連續式發酵配合pH值調控的方式以達到提升菌體及重組甜味蛋白質之效果。結果顯示無論是以氨水調整pH值或是加入新鮮培養基，菌體生長情形與重組甜味蛋白質表現量相較於小量發酵皆無顯著性差異。而蛋白質表現量則以有額外置換添加培養基發酵方式之第8小時具有最佳之產量，約為222.5 μg/ml。

Sweetness is an indispensable flavor of the people’s dietary. Traditionally the nature resources of sweeteners are mainly the monosaccharides and oligosaccharides. However, the excessive intake of carbohydrates lead to caries, diabetes and hypertension in recent diets. Therefore safer, low-calorie, high sweetness, natural sweeteners were isolated from a variety of plants to substitute the tradition sweetners. Among them brazzein has the attractive features such as small size (53 amino acid residues), stability over wide ranges of temperature and pH, and the similarity of sweetness to sucrose, which made it a potential artificial sweetener. Escherichia coli is the most commonly used host in genetic engineering and a commercially heterologous proteins expressing host. Lactococcus lactis has been used in fermented foods, food preservation and dairy products for thousands of years; and regarded as GRAS (generally recognized as safe) by FDA (Food and Drug Administration). Therefore, L. lactis has been used as a food grade host in heterologous protein expression, moreover characteristics such as no endotoxin, better protein secretion ability and low activity of extracellular protease led L. lactis a potential host for microbial protein production factory. In this study, Escherichia coli and Lactococcus lactis are used as host to express the recombinant sweet protein rbrazzein. In the first part, Isopropyl β-D-1-thiogalactopyranoside (IPTG)-indicibe pET-brazzein was constructed to express the recombinant sweet protein rbrazzein by E. coli. And the recombinant sweet protein rbrazzein-expressed plasmid for L. lactis including nisin-inducible and acid-inducible (pHI, MpHI) system were constructed as well. The optimized recombinant sweet protein rbrazzein was purified from fermented E. coli transformants and yielded rbrazzein about 15.4 mg. The purified rbrazzein tasted sweet and the relative sweetness of sweet protein concentration of 80 ~ 100 ug/ml is almost close to 8% sucrose solution. Among L. lactis expression systems the NICE system is not suitable for fermentation due to the expensive cost. The constructed food grade expression system can be selected at nisin concentration of 1 ~ 3μg/ml but unstable in expression. The modified acid-inducible system (MpHI) which fused the UTL infront of promoter showed better expression without any antibiotic. We tried to purified the rbrazzein from the 24 hr culture supernatant of MpHI system by cation exchange chromatography. However the purified protein were not so pure as analyzed by MALDI-TOF, possibly due to the cell lysis at 24 hr culture. Finally the batch type and fed-batch type fermentation of MpHI system were preceded. The highest 222.5 μg/ml rbrazzein was achieved after 8 hr induction of fed-batch culture.