Optimization of recombinant sweet protein brazzein expression, purification and functional analysis by Bacillus licheniformis

• Main Authors: Chien-Ya Hung, 洪千雅
• Other Authors: 葉娟美
• Format: Others
• Language: zh-TW
• Published: 2013
• Online Access: http://ndltd.ncl.edu.tw/handle/75773580110235271610

碩士 === 國立中興大學 === 食品暨應用生物科技學系所 === 101 === The sweetener plays an important role in our daily diet. However, the excessive intake of carbohydrates lead to health risk. In recent years, the search for low-calorie sweeteners to substitute sugar is highly demand. The sweet-tasting protein has potential as low-calorie sweeteners that can be used to replace sugars in food, beverage, and medicines. Among them, brazzein is an attractive candicate sweetener because of its small size(53 amino acid residues), high sweetness, sugar-like taste, and good stability at high temperature and wide pH ranges. Bacillus subtilis and Bacillus licheniformis are gram positive and endospore-forming bacteria. They have been regarded as GRAS (generally recognized as safe) by FDA (Food and Drug Administration). They have the ability to secrete protein and can be used as good hosts for heterologous protein expression. In this study, B. subtilis and B. licheniformis are used as hosts to express the recombinant sweet protein rbrazzein. Plasmids with different signal peptides were constructed to observe the secretion of rbrazzein by different protein secretion pahway. The results indicated that rbrazzein couldn''t be secreted by B. subtilis. However, the rbrazzein is successfully expressed by B. licheniformis dual plasmid system. The optimized rbrazzein purification achieved approximately 10 mg purified rbrazzein from 36-hour fermented B. licheniformis culture.The rbrazzein was examined to confirm the disulfide bond existence. The purified rbrazzein was 266 times sweeter than sucrose on a weight basis, and about 4957 times sweeter than sucrose on a molar basis. It is also with good heat stability between pH 5 to pH 9. These results demonstrated that B. licheniformis expression system is useful to produce active recombinant sweet protein brazzein and can be useful as potent sweetener producing system and applied in food industry