A rapid and simple method for the determination of organic acids in proteolytic enzymes by capillary electrophoresis with indirect ultraviolet detection

Highlights

• Acetic and gluconic acids are added to enhance stability and activity of proteases.

• A CE method with indirect UV detection is developed for high content protein samples.

• A hydroalcoholic microextraction is optimized to extract both organic acids.

• Both organic acids are accurately determined in a commercial protease sample.

Abstract

The use of organic acids (e.g. acetic acid and gluconic acid) as additives during protease production is regarded as one of the simplest alternatives to increase enzyme stability and activity in many industrial processes. However, no methods have been described for the determination of organic acids in proteases and their contents have not been established yet. In this work, a novel, rapid and simple method for the determination of organic acids in proteolytic enzymes by capillary electrophoresis (CE) with indirect ultraviolet (UV) detection has been developed. Under the optimized conditions, the method was validated in terms of linearity, limit of detection (LOD), limit of quantification (LOQ) and intra-day and inter-day repeatability. Later, a sample pretreatment based on a hydroalcoholic microextraction was carefully optimized to obtain good recovery and repeatability and determine acetic and gluconic acids in a commercial protease sample. The complete procedure was validated using the standard-addition calibration method, finding matrix effects on the studied compounds. Finally, acetic acid and gluconic acid were quantified at 80 mg/Kg (0.0080% (m/m)) and 69 mg/Kg (0.0069% (m/m)) in the protease sample, respectively.

Introduction

Protein hydrolysis can be used in a wide range of applications, from proteomic studies to cleaning and food biotechnology processes, and can be carried out by chemical or enzymatic processes [1], [2], [3], [4]. While chemical processes (e.g. alkaline and acid hydrolysis) tend to be less eco-friendly, difficult to control and yield products with modified amino acids [5], [6], enzymatic hydrolysis can be performed under milder conditions, hence avoiding the extreme environments required for chemical treatments [6], [7]. Additionally, enzymes present substrate specificity, which allows the development of protein hydrolysates with better defined chemical and nutritional characteristics [7].

Proteases (also termed peptidases) are one of the most important groups of industrial enzymes and their designs range from small and simple catalytic units (around 20 kDa) to sophisticated protein-processing and degradation machines (0.7–6 MDa) [6], [8]. Novel protease applications in industrial processes are constantly being introduced, but autolysis of proteases still remains the major limitation in enzyme production, which may lead to chain instability, low reaction rate and low substrate susceptibility [9], [10]. Several methods have been described in the literature to increase enzyme stability, which include enzyme immobilization, cross-linking with chemicals or chemical modification of the amino acid side-chains [11], [12], [13]. Among them, the introduction of organic acids (e.g. acetic acid, gluconic acid and their salts, etc.) into the production medium is one of the simplest alternatives to increase enzyme stability and activity. These molecules are believed to provide additional points of hydrogen bonding with the enzyme surface, decrease dehydration and/or provide thermodynamic barriers to unfolding [14], [15]. Despite its use in enzyme production is well-known, no dedicated methods have been described for the determination of organic acids in proteases and their contents have not been established yet. Novel analytical methods in this field should provide further information about the content of organic acids during protease production, which could be used in quality control to ensure maximum enzyme stability and activity, as well as to track enzyme production.

Different techniques have been employed for organic acid determination, traditionally gas chromatography (GC) [16], [17] and, more recently, liquid chromatography (LC) [18], [19], [20], [21]. However, these techniques present some deficiencies. When using GC, organic acids need to be derivatized to make them volatile. LC, besides needing in general organic solvents, is time-consuming and limited by the narrow linear dynamic range and the susceptibility to matrix interferences. Capillary electrophoresis (CE) with direct [22], [23], [24], [25] or indirect [26], [27], [28], [29], [30], [31], [32], [33], [34] ultraviolet (UV) detection has also been demonstrated, but to a lesser extent, despite the well-known benefits of this high-performance microscale electroseparation technique [35]. CE provides complementary and, very often, better separations than hydrophobicity-driven reversed-phase LC. Additionally, analyses can be performed using smaller amounts of sample and reagents, no organic solvents are necessary, separation times are considerably low and it offers good repeatabilites [35]. CE has proven to be a good choice for the determination of organic acids in several beverages [22], [23], [24], [25], [28], [29], [30], [31], microbial fermentation process samples [32], engine coolants [33] and ionic liquids from biomass hydrolysates [34], where no more than a simple dilution is needed. In addition, CE and capillary isotachophoresis have also been reported for determination of anions of organic acids as counterions of basic drugs [36], [37], [38]. However, to the best of our knowledge, the use of CE for the analysis of organic acids in more complex matrices containing a high protein content (such as enzymes, or specifically proteases) has never been reported.

In this work, we have developed a CE method with indirect UV detection for the determination of acetic acid and gluconic acid in proteases. Since most organic acids lack of a strong UV chromophore, the use of indirect UV detection offers an excellent alternative [26], [27], [28], [29], [30], [31], [32], [33], [34], without the need for any derivatization step or the use of a contactless conductivity detector [39], [40], [41]. After validating the method with standards, we optimized an appropriate sample pretreatment method for the extraction of organic acids from a commercial protease sample before CE-UV. The complete procedure was then validated using the standard-addition calibration method, before the accurate quantification of the detected organic acids.