A comparison of HPLC and spectrophotometrical methods to determine the activity of ferulic acid esterase in commercial enzyme products and rumen contents of steers

Abstract

The aim of this study was to compare different methods to determine ferulic acid esterase (FAE) activity in commercial enzyme products and to measure the FAE activity in the rumen of steers fed different diets (A: 5 kg forage and 4 kg concentrate; B: 5 kg forage and 5 kg concentrate; C: 5 kg forage and 6 kg concentrate). FAE activities in seven commercial enzyme products were determined by high performance liquid chromatography (HPLC) at a wavelength of 320 nm and by spectrophotometric assays at wavelengths of 335 and 340 nm. FAE activities varied dramatically among the seven exogenous enzyme preparations. Correlation between the enzyme activities determined by HPLC and spectrophotometric assays was high (r > 0.98), but consistency among the different methods was poor, especially with the spectrophotometrical method (SP) at 335 nm. Therefore, the SP by microplate reader at 340 nm with standard substrate of methyl ferulate (MFA) was recommended for rapid analysis of large numbers of samples. Rumen fluid of steers had low, and different, ferulic acid esterase activity at different times of the day. Furthermore, high diurnal variation occurred for the ferulic acid esterase and other fibrolytic enzymes that were measured. The activity of FAE and other fibrolytic enzymes differed along with the diet compositions and feed intake. Diet C had the highest mean fibrolytic enzyme activity in the rumen, and diet A had the lowest activity of FAE and birchwood xylanase (BX). High correlations occurred between activities of carboxymethyl cellulase (CMC), BX, acetyl esterase (AE) and those of FAE, both with all data and within each diet. As an accessory enzyme, ferulic acid esterase should not be neglected in studies on furthering understanding of biodegradation of plant cell wall in rumen, and impacts of exogenous enzyme products.

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.