Extracellular production of riboflavin-binding protein, a potential bitter inhibitor, by Brevibacillus choshinensis.
Riboflavin-binding protein (RBP) is a glycophosphoprotein found in hen eggs. We previously identified the extraordinary characteristic of RBP in reducing bitterness. For a more detailed study on the mode of action and industrial application of this characteristic, we investigated the microbial production of recombinant RBP (rRBP). We constructed a chicken RBP gene expression vector by inserting the RBP cDNA in pNCMO2, the Escherichia coli-Brevibacillus choshinensis shuttle vector. B. choshinensis HPD31 transformants produced 0.8g/l of processed and unglycosylated RBP in a soluble form in the culture supernatant. However, the expressed RBP was partially dimerized and monomeric RBP was purified by two step anion-exchange and gel-filtration chromatographies. The purified rRBP elicited bitterness reduction against quinine and caffeine, although it largely lost its riboflavin-binding ability. These results indicated that glycosylation and riboflavin-binding ability are not essential for the bitterness reduction of RBP. In addition, we assessed the usefulness of the Brevibacillus system for the expression and secretion of RBP as a new type of bitterness inhibitor.
Maehashi
Kenji
K
Department of Fermentation Science, Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya-ku, Tokyo 156-8502, Japan. maehashi@nodai.ac.jp
Matano
Mami
M
Saito
Makiko
M
Udaka
Shigezo
S
D000818:Animals; D001671:Biochemistry / Q000379:methods*; D001951:Brevibacterium / Q000378:metabolism*; D018929:Cell Culture Techniques; D002645:Chickens; D005110:Extracellular Space / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000494:pharmacology*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012256:Riboflavin / Q000378:metabolism; D013649:Taste / Q000187:drug effects*; D013997:Time Factors
D026901:Membrane Transport Proteins; D011994:Recombinant Proteins; C019079:riboflavin-binding protein; D012256:Riboflavin