Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy
Synthesis of gold–cellobiose nanocomposites for colorimetric measurement of cellobiase activity
Graphical abstract
Introduction
Lignocellulose is the most abundant renewable resource, and the enzymatic hydrolysis of lignocellulosic biomass has led to an increasing interest since it can be used to produce environment-friendly biofuels and chemicals [1], [2], [3], [4], [5]. The hydrolysis of β-1,4-glycosidic linkages of cellulose depends on the degradation capacity of cellulase, which is produced by cellulolytic microorganisms, and usually contains three components: β-1,4-endoglucanase (EC 3.2.1.4), cellobiohydrolases (EC 3.2.1.91) and cellobiase (EC 3.2.1.21). Low activity of cellobiase may restrict the conversion of cellobiose to glucose, and the accumulation of cellobiose will cause feedback inhibition to the cellulase reaction. Therefore, the role of cellobiase in the hydrolysis of cellulose is significant, and improving cellobiase activity is the key to raising the saccharification yield [6]. Thus, significant efforts are being made to develop technologies for improving the production of cellobiase [2], [7], [8], [9]. The activity of cellobiase is an important parameter since it could characterize the performance and efficiency of those methods. As such, development of the specific methods for the detection of cellobiase activity has become crucial.
A widely used method to assay cellobiase activity is the protocol recommended by the International Union of Pure and Applied Chemistry (IUPAC) [10]. One international unit of cellobiase activity is the amount of enzyme that forms 2 μM of glucose per minute from cellobiose, and glucose was determined by using a 3,5-dinitrosalicylic acid (DNS) colorimetric assay method. The enzyme blanks are necessary when glucose is present in the diagnostic process [10], [11]. In addition, some methods based on chromophore, fluorescent group release or product measurement are able to perform such task [12]. A simple and novel method for the efficient detection of cellobiase activity is still necessary since most of the current methods require tedious sample preparation, or the chemicals involved in such procedures are expensive or toxic [11], [13].
Gold nanoparticles (AuNPs) exhibit attractive optical properties, friendly biocompatibility with biomolecules, and easily controllable size-distribution [14], [15], [16], [17], the size and relative distance between nanoparticles could modulate its surface plasmon resonance (SPR). Taking advantages of these characteristics, AuNPs has been widely used as optical labels for biospecific interaction analysis [18], [19], [20]. On the other hand, polysaccharides have a series of virtues such as non-toxic, special structure characteristics, and biodegradability. Moreover, the commercial availability makes them to be convenient choices for the synthesis of nanomaterials. It has been reported that the reductive polysaccharides could be used as the reducing or stabilizing agent for producing AuNPs [21], [22]. The synthesis of gold-polysaccharides nanocomposites has also attracted much attention due to their specific properties and application in biotechnology [23].
In this research, a colorimetric probe based on the synthesis of gold–cellobiose nanocomposites (GCNCs) is proposed for the detection of cellobiase activity. Cellobiose acted as the controller of nucleation or stabilizer in the formation of AuNPs, and 6-Mercapto-1-hexanol (MCH) was modified on GCNCs to promote the sensitivity. The SPR of MCH–GCNCs correspondingly changed with the activity of cellobiase due to the specific interaction between cellobiase and cellobiose, and the change in SPR of cellobiase treated MCH–GCNCs was detected by UV–visible spectrometer. The absorbance ratio of treated MCH–GCNCs (A650/A520) was used to estimate the cellobiase activity. The linear ranges, sensitivity, and accuracy of the colorimetric probe were also investigated. This detection method has advantages of easy operation and non-toxic. It also showed satisfactory sensitivity and selectivity. It is believed that this research could expand the application field of gold nanocomposites.
Section snippets
Materials
Cellobiose, chloroauric acid (HAuCl3·3H2O), MCH, glucose oxidase from Aspergillus niger, laccase from Trametes versicolor and superoxide dismutase from Bacillus stearothermophilus were purchased from Sigma–Aldrich. Sodium borohydride (NaBH4) was purchased from Sinopharm Chemical Reagent Co., Ltd. All the other chemicals were of analytical grade or the highest purity commercially available. Ultrapure water (18.2 MΩ) was obtained from a Milli-Q purification system and used throughout the
Characterization of GCNCs
The UV–visible absorption spectra of the GCNCs solutions are shown in Fig. 1. Different concentrations of cellobiose were used to prepare GCNCs, and all spectra exhibited an absorption band around 520 nm. It is a typical SPR band for AuNPs, suggesting the formation of AuNPs [21], [22], [25]. The intensity of the absorption band varied with the concentration of added cellobiose. When the concentration of cellobiose increased from 0.005% to 1%, the intensity of absorption band first decreased, and
Conclusions
In the present study, the gold–cellobiose nanocomposites were prepared, and a colorimetric probe based on the SPR property of GCNCs was developed for the measurement of cellobiase activity. The cellobiase activity was linearly related to the absorbance ratio A650/A520 of treated GCNCs solution, ranging from 3.0 to 100.0 U L−1. The selectivity of the probe was also illustrated. The authors believe that the proposed method could provide an alternative tool for the cellobiase activity detection due
Acknowledgments
This study was financially supported by the National Natural Science Foundation of China (51378190, 51039001, 50808073, 51308076 and 51278176), the Environmental Protection Technology Research Program of Hunan (2007185), the Young Teacher Growth Program of Hunan University, New Century Excellent Talents in University (NCET-13-0186), Changsha Planning Project of Science and Technology K1207026-31 and the Program for Changjiang Scholars and Innovative Research Team in University (IRT-13R17).
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