A comparison of HPLC and spectrophotometrical methods to determine the activity of ferulic acid esterase in commercial enzyme products and rumen contents of steers
Introduction
Ferulic acid esterase (EC 3.1.1.73; FAE) is an inducible enzyme capable of hydrolysing the ester bond between the polysaccharide main chain of xylans and the monomeric or dimeric ferulates present in plant cell walls (Christov and Prior, 1993, Topakas et al., 2005). It has attracted much attention relative to understanding of their potential applications in pulp and paper processing as well as the feed, pharmaceutical and food industry. In recent years, researches about FAE have increased in the feed industry and relative to animal production. FAE activity has been reported in microorganisms that utilize plant cell wall carbohydrates (Bonnin et al., 2001), and in mammalian cells and plants (Sancho et al., 1999, Andreasen et al., 2001). Several of the enzymes responsible for FAE activity have been purified and studied for homogeneity, characterization and cloning (Donaghy et al., 2000, Kroon et al., 2000), while protein sequences and three-dimensional structures are also available (Kroon et al., 2000, Hermoso et al., 2004). As a subclass of the carboxylic acid esterases (EC 3.1.1.1), FAEs have been classified into types A, B, C and D based on substrate utilization data and amino acid sequences (Crepin et al., 2004). Methyl ferulate (MFA), one of the synthetic substrates that can be hydrolysed by the four types of FAE (Crepin et al., 2004), was often used to measure the FAE activities by high performance liquid chromatography (HPLC) or spectrophotometry at different wavelengths, respectively (Ralet et al., 1994, Bonnin et al., 2001, Vafiadi et al., 2006). However, no direct comparison of the different methods has been published. We therefore decided to make a direct comparison of the different methods to determine if the different methods gave similar results.
As agricultural by-products, crop straws and bran middlings are often used as feeds for ruminants and, in China their proportion in a ration may be up to 800 g/kg. The cell wall of these feedstuffs is largely composed of cellulose, hemicellulose and lignin. But its digestibility is not more than 50%. This might be due to the linkages between lignin polymers and polysaccharides in the cell wall, commonly formed as substituents by hydroxycinnamic acids, such as ferulic and p-coumaric acid (Ralph et al., 1992, Reid, 2000), which provide cell wall integrity and resist enzymatic degradation. The linkage between two ferulic acid (FA) molecules on adjacent chains has been identified, and this seems to be one of the most important cross-linkages in plant cell walls and. Indeed it was compared to ‘spot welding of a steel mesh frame’ (Iiyama et al., 1994) because of its effects on plant cell wall mechanical properties. Therefore, besides main-chain degrading enzymes such as cellulases and xylanase (EC 3.2.1.8, XYL), side-chain degrading enzymes, such as FAE and acetyl esterase (EC 3.1.1.6, AE) in the rumen, might be important to better understand the digestion of crop straws and bran middlings in order to improve their net energy value. Some researchers have shown that ruminal microbes can produce FAE (Borneman et al., 1990), but FAE activity in the rumen has to date only been estimated in buffaloes (Paul et al., 2004).
The objective of this study was twofold. The first aim was to provide a methodological recommendation to determine FAE activity in rumen content samples based on comparison of HPLC and spectrophotometric assays in commercial enzyme products. The second aim was to measure the FAE activity in the rumen of steers fed different diets to provide a reference for possible addition of exogenous FAE enzyme products to rations of ruminants.
Section snippets
Enzyme products and its pretreatment
The seven commercial enzyme products used in the study were gifts from various commercial manufacturers. Feed-grade xylanase A, Feed-grade multienzyme cocktail E and Feed-grade cellulase G were from Ningxiner Co. Ltd. (Beijing, China), Ferulic acid esterase J and Cellulase K were from Biocatalysts Co. Ltd. (Wales, UK), Cellulase L was from Xinfa Co. Ltd. (Zhejiang, China), and β-glucanase U was from Xiasheng Co. Ltd. (Ningxia, China). When available as a dry powder, enzyme solutions were
Comparison of HPLC and spectrophotometrical methods to determine the activity of ferulic acid esterases
The protein concentration and activities of FAE and other fibrolytic enzymes in the seven commercial enzyme products are in Table 2. The level of FAE activity among enzyme preparations varied dramatically. The activity of FAE detected by HPLC ranged from 0 in Feed-grade cellulase G and Cellulase K to 3.530 U in Ferulic acid esterase J. Ferulic acid esterase J had a high FAE activity, but the activity of other fibrolytic enzymes were relatively low. In contrast, Feed-grade cellulase G had no
Comparison of HPLC and spectrophotometrical methods to determine activity of ferulic acid esterases
A variety of methods have been developed to determine FAE activity using natural or synthetic substrates such as feruloylated oligosaccharides (Bonnin et al., 2001), de-starched wheat bran (O’Neill et al., 1996), methyl and ethyl ferulates (Vafiadi et al., 2006). As the simplest substrate, MFA was more frequently used for assaying esterase activity, and it was also applied in our study. As all four types of FAE can release FA from MFA, the esterase containing FAE activity determined in our
Conclusions
HPLC and SPs to determine the activity of ferulic acid esterase in seven exogenous commercial enzyme products were compared. HPLC analysis is recommended because it is more sensitive and the results are less confusing, but it is an expensive, labour intensive and time-consuming method. Low cost, rapid, and efficient spectrophotometric assay by microplate reader at 340 nm might be an alternative choice especially for enzyme producers in product quality control and assurance. The
Acknowledgements
The study was supported by funds from the State Natural Science Foundation (proj.30400315) and the Grant (200803033B050401) of the Science & Education Department of the Ministry of Agriculture in China. The authors also express their great thanks to Prof. Seerp Tamminga at Wageningen University for giving constructive revision comments in the manuscript preparation.
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