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Art. 3
Art. 3
Talanta
journal homepage: www.elsevier.com/locate/talanta
a r t i c l e i n f o a b s t r a c t
Article history: A new kinetic method is proposed for the simultaneous determination of 3-hydroxybutyrate (3HB) and
Received 11 June 2009 3-hydroxyvalerate (3HV) based on the different rate of the 3-hydroxybutyrate dehydrogenase-catalysed
Received in revised form reactions of these compounds with coenzyme NAD+ . A flow injection system with two reactors of
21 September 2009
immobilised 3-hydroxybutyrate dehydrogenase and dual detection is used. The concentrations of NADH
Accepted 25 September 2009
produced after two different reaction times are measured by fluorometry or spectrophotometry and mul-
Available online 3 October 2009
tivariate linear calibration is applied for quantification. Concentrations of 3HB and 3HV between 1 × 10−6
and 1 × 10−4 M can be determined at an average sampling frequency of 20 h−1 . In contrast to usual meth-
Keywords:
Biopolymer
ods, the proposed here makes possible the discrimination of 3HB and 3HV without previous separation
3-Hydroxybutyrate so that usual extraction with chlorinated solvents and/or chromatographic separation is not required.
3-Hydroxyvalerate The method is of interest in a wide variety of fields concerning PHAs, as it can provide information on
Flow injection the degradation rate and mechanism, composition and structure of these polymers. Its applicability has
Immobilised enzyme been proved through the determination of 3HB and 3HV in the digests of some chemically degraded
3-Hydroxybutyrate dehydrogenase commercial PHAs.
© 2009 Elsevier B.V. All rights reserved.
1. Introduction ical hydrolysis is also a crucial stage of methods for indirect analysis
of PHAs.
Polyhydroxyalkanoates (PHAs) [1,2] are a large group of nat- Although more than 150 different monomers of hydroxyalka-
ural polymers that are synthesized by numerous bacteria under noic acids can be combined to give PHAs, only very few of them
unfavourable growth conditions. Tailor-made PHAs, with con- are actually used. The commonest PHA is poly(3-hydroxybutyrate)
trolled composition, structure and properties, are industrially (PHB), a homopolymer of (R)-3-hydroxybutyric acid (3HB). The
produced with genetically modified bacteria. PHAs have ther- high cost and some disadvantages of PHB have lead to the pro-
moplastics properties but unlike synthetic plastics, they are duction of copolymers of 3HB and other hydroxyalkanoic acids.
biodegradable and biocompatible. Most current applications of The most widespread commercial products are the copolymers of
PHAs are based on these features and are effected in the environ- 3HB and (R)-3-hydroxyvaleric acid (3HV) with variable properties
mental and medical fields [3,4]. PHAs are ecological plastics that are depending on the percentage of poly(3-hydroxyvalerate) (PHV).
used in packaging and coating as substitutes for non-biodegradable Experimental products include also blends of PHAs (homopoly-
petrochemical polymers. Their main medical applications are the mers, copolymers or both) and blends of PHAs with other
fabrication of biodegradable body implants and the control of biodegradable polymers. As PHB and PHV are by far the common-
drug delivery, this being used also in pharmacy, agriculture and est components of PHAs, 3HB and/or 3HV are generally among
veterinary. Moreover, the degradation of PHAs is a source of opti- the ultimate degradation products. The quantification of these
cally active monomers of hydroxyalkanoic acids [5] that can be monomers can provide information not only on the degradation
used to synthesize chiral compounds such as aminoacids, vitamins, rate and mechanism but also on the composition and structure of
antibiotics, pheromones, perfumes or biodegradable solvents. PHAs PHAs. Indirect quantification of PHB, PHV and their copolymers is
can be degraded by enzymes (biodegradation) but also by chem- commonly performed after alcoholysis with methanol, ethanol or
ical or thermal processes. From an applicative point of view, the propanol in sulphuric or hydrochloric acids. The polymers are quan-
degradation kinetics is essential since the degradation rate or the titatively transformed into the esters of 3HB and/or 3HV which can
resultant products can determine the potential applications. Chem- be extracted with chloroform or other solvents and determined
by gas chromatography with flame ionization (GC–FID) or mass
spectrometry (GC–MS) detection [6,7]. In these methods, the recov-
eries of PHB and PHV are not complete, due to the partition of the
∗ Corresponding author. Tel.: +34 923294483. esters between the aqueous and organic phases. Alternatively, the
0039-9140/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.talanta.2009.09.049
C. García de María, K.B. Hueso Domínguez / Talanta 80 (2010) 1436–1440 1437
was injected, and b was the slope of the proposed equation). Rela-
tive standard deviations (n = 10) between 0.8% and 1.5% were found
for the peak heights of samples 1.0 × 10−5 M. The average sampling
frequency was 20 h−1 .
For samples containing both 3HB and 3HV, the peak heights hF
and hS were predicted by multiple linear regression, this being
only valid when the total concentration of 3HB + 3HV was below
1 × 10−4 M, approximately. The partial regression coefficients of the
multilinear equations (Table 2) were coincident with the individual
coefficients found for samples of only 3HB or 3HV. This indicated
that the signals of 3HB and 3HV were additive so that the fluoro-
metric or spectrophotometric signals of any mixture of 3HB and
3HV could be calculated by the addition of the signals given by the
equivalent individual samples containing only 3HB or 3HV. Apart
from not being predictable by multiple linear regression, negative
deviations from the additivity were observed for the signals of mix-
tures with concentrations of 3HB + 3HV higher than 1 × 10−4 M,
particularly when the 3HB:3HV molar ratio was close to unity. This
suggested some kind of interaction between 3HB and 3HV, proba-
Fig. 2. Effect of pH on the catalytic activity of 3-HBDH. Fluorescence signals for bly due to the competition of both substrates for the catalytic site
5.0 × 10−5 M 3HB and 3HV at different pHs. on the immobilised enzyme.
Table 2
Calibration equations for the determination of 3HB and/or 3HV.
Table 3 5. Conclusions
Analysis of chemically degraded commercial PHAs. Concentrations estimated, added
and found in the analysed samples.
The described enzymatic method makes possible the direct
Sample Concentrationa , M × 105 determination and discrimination of 3HB and 3HV. In contrast
Estimated Added Foundb to the methods commonly used for these compounds, previous
separation by extraction with chlorinated solvents and/or chro-
HB HV HB HV HB HV
matography is not required. The method is of interest in a wide
BP-1 5.23 0.69 – – 5.2 ± 0.2 0.8 ± 0.2 variety of fields concerning PHAs, as it can provide information on
BP-1 1.00 – 6.2 ± 0.2 0.7 ± 0.2
the degradation rate and mechanism, composition and structure
BP-1 – 1.47 5.3 ± 0.2 2.1 ± 0.2
of these polymers. Its applicability has been proved through the
BP-2 3.23 0.77 – – 2.9 ± 0.1 0.7 ± 0.2 determination of 3HB and 3HV in the digests of some chemically
BP-2 1.00 – 3.9 ± 0.1 0.6 ± 0.2
degraded commercial PHAs.
BP-2 – 1.47 3.0 ± 0.1 2.0 ± 0.2
BP-2 1.00 1.47 3.9 ± 0.1 2.2 ± 0.2
Acknowledgments
BP-3 2.56 0.62 – – 2.1 ± 0.1 0.7 ± 0.2
BP-3 1.00 – 3.0 ± 0.1 0.9 ± 0.2
BP-3 – 1.47 2.3 ± 0.1 2.1 ± 0.2 This work was supported by the Spanish Ministerio de Ciencia
BP-3 1.00 1.47 3.2 ± 0.1 2.3 ± 0.2 e Innovación (Ref.: BQU2002-02332). Karina B. Hueso Domínguez
a
Values refer to the injected solutions. thanks the Spanish Ministerio de Ciencia e Innovación for the grant
b
Mean values of three determinations with 95% confidence limits. BES-2003-1012.