Int. J. Mol. Sci. 2007, 8(6), 513-525; doi:10.3390/i8060513
Article

Branched-chain Amino Acid Biosensing Using Fluorescent Modified Engineered Leucine/Isoleucine/Valine Binding Protein

1 Department of Biotechnology, Graduate School of Engineering, Tokyo University of Agriculture & Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan 2 Department of Technology Risk Management, Graduate School of Technology Management- Tokyo University of Agriculture & Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan
* Author to whom correspondence should be addressed.
Received: 20 December 2006; in revised form: 23 April 2007 / Accepted: 12 May 2007 / Published: 13 June 2007
(This article belongs to the Section Molecular Recognition)
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Abstract: A novel fluorescence sensing system for branched-chain amino acids (BCAAs)was developed based on engineered leucine/isoleucine/valine-binding proteins (LIVBPs)conjugated with environmentally sensitive fluorescence probes. LIVBP was cloned fromEscherichia coli and Gln149Cys, Gly227Cys, and Gln254Cys mutants were generated bygenetic engineering. The mutant LIVBPs were then modified with environmentallysensitive fluorophores. Based on the fluorescence intensity change observed upon thebinding of the ligands, the MIANS-conjugated Gln149Cys mutant (Gln149Cys-M) showedthe highest and most sensitive response. The BCAAs Leu, Ile, and Val can each bemonitored at the sub-micromolar level using Gln149Cys-M. Measurements were alsocarried out on a mixture of BCAFAs and revealed that Gln149Cys-M-based measurementis not significantly affected by the change in the molar ratio of Leu, Ile and Val in thesample. Its high sensitivity and group-specific molecular recognition ability make the newsensing system ideally suited for the measurement of BCAAs and the determination of theFischer ratio, an indicator of hepatic disease involving metabolic dysfunction.
Keywords: Periplasmic binding proteins (PBPs); biosensor; fluorescence; leucine / isoleucine / valine-binding protein (LIVBP); branched-chain amino acid (BCAA)

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MDPI and ACS Style

Chino, S.; Sakaguchi, A.; Yamoto, R.; Ferri, S.; Sode, K. Branched-chain Amino Acid Biosensing Using Fluorescent Modified Engineered Leucine/Isoleucine/Valine Binding Protein. Int. J. Mol. Sci. 2007, 8, 513-525.

AMA Style

Chino S, Sakaguchi A, Yamoto R, Ferri S, Sode K. Branched-chain Amino Acid Biosensing Using Fluorescent Modified Engineered Leucine/Isoleucine/Valine Binding Protein. International Journal of Molecular Sciences. 2007; 8(6):513-525.

Chicago/Turabian Style

Chino, Sakura; Sakaguchi, Akane; Yamoto, Rie; Ferri, Stefano; Sode, Koji. 2007. "Branched-chain Amino Acid Biosensing Using Fluorescent Modified Engineered Leucine/Isoleucine/Valine Binding Protein." Int. J. Mol. Sci. 8, no. 6: 513-525.

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