Improvement of recombiant sweet proteinbrazzein expression and fermentation by Escherichia coli and Lactococcus lactis

• Main Authors: Lee-Hao Cheng, 鄭力豪
• Other Authors: 葉娟美
• Format: Others
• Language: zh-TW
• Published: 2012
• Online Access: http://ndltd.ncl.edu.tw/handle/01418033374455259293

碩士 === 國立中興大學 === 食品暨應用生物科技學系所 === 100 === 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.