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  • Introduction The annual global market

    2020-07-06

    Introduction The annual global market of industrial SR 1555 hydrochloride is reported to be billions of USD [1], [2], [3], [4], [5], [6], [7], and impacts commercial sectors that include energy, animal feed, household products, food processing and pharmaceuticals [1], [2], [5], [6]. Enzymatic processing is even recognized as a critical component for sustainable chemical manufacturing [8]. For this reason significant effort is made to discover new enzymes [9], [10] as well as to improve the stability, specificity, or efficiency of existing enzymes [7], [11]. These endeavors require analytical approaches to characterize the enzyme performance in order to advance manufacturing. The purpose of this paper is to shed light on the capabilities of capillary electrophoresis for the evaluation of enzyme performance. In order to understand how the technique is adapted to different enzyme and substrate systems, fundamental principles of the method are described. The process of converting capillary electrophoresis separations into Michaelis-Menten constants (KM) is also delineated. Applications reported from 2012 to 2017 are summarized. Areas of focus include the calculation of KM, inhibition studies, optimization of enzyme turnover and approaches to screen or compare different enzymes. Different strategies for enzyme immobilization for in-capillary analyses are described. Future directions in this field, especially in strategies for in-capillary sequencing as well as structural identification are addressed.
    Background
    From 2012 to 2017 approximately fifty KM determinations were reported in the literature that utilized capillary electrophoresis. These reports, summarized in Table 1, were predominantly studies of hydrolases or oxoreductases, although transferases, lyases, and isomerases were also investigated. Separations were based on differences in the charge-to-size ratio of the substrate and product for most reports. Several reports evaluated enzyme specificity for enantiomers and as a result, additives that separated chiral substrates were included in the background electrolyte. The primary method of detection was UV–visible absorbance detection, which is a universal detection method applicable to most analytes. In addition, the linear range of absorbance detection, typically between 50 and 500 µM, is appropriate for the reported KM values. Enzyme assays were performed both off-line and on-line, depending upon the conditions required of the enzyme reaction and the constraints of the assay. These aspects of enzyme analyses are addressed in greater detail in the sections that follow.
    Evaluating enzyme inhibitors using capillary electrophoresis
    Enzyme immobilization techniques
    Emerging techniques and future directions
    Acknowledgements This material is based upon work supported by NIH Grant No. R01GM114330. CLC acknowledges a National Science Foundation IGERT fellowship, DGE #1144676.
    Introduction Enzymes are macromolecular biological catalysts that accelerate or catalyze chemical reactions. Like all catalysts, enzymes increase the rate of a reaction by lowering its activation energy and are known to catalyze a wide range of biochemical reactions, not only in the food industry. They are used in numerous industrial productions, playing important roles in the fields of foods, feed, pharmaceuticals, dyes, water treatment, textiles, cosmetics, leather and biofuels, among others [1, 2]. In recent years, the industrial applications of enzymes have exploded. Compared with conventional procedures based on acid-base and high temperature, enzymatic hydrolysis offers high efficiency and specificity. In addition, enzymatic reactions can be carried out under milder conditions with less pollution and fewer by-products. Moreover, enzymes themselves are relatively nontoxic and can be modified by various means [3, 4]. A worldwide survey on the sales of enzymes ascribed 31% of sales to food enzymes, 6% to feed enzymes and the remaining to technical enzymes [5]. However, enzymes have a relatively low stability under extreme conditions and have an extravagant expense for commercial use. To overcome these drawbacks, there is great interest in enhancing enzyme activity, stability, re-usage capacity and enzymatic efficiency. Generally, the methods of promoting enzymatic reactions are focused on three aspects: enzyme modification, substrate pretreatment, and enhancement of enzyme-substrate combination. These improvements can be attained via chemical, physical and genetic methods. As shown in Table 1, there are various techniques available to improve enzyme characteristics. Among these methods, ultrasound is one of the most important techniques.