br Conflicts of interest br

Conflicts of interest
Acknowledgements This research was supported by grants from National Research Foundation, Korea (NRF-2017RIDIA3B04033313) and Yeungnam University (216-A-061-042).
Introduction Kluyveromyces lactis is a yeast that produces one of the ß-galactosidases (ß-gals) most widely used in neutral milk products [1,2]. The enzyme, commonly known as lactase, is produced on an industrial scale by selected yeast strains containing extra copies of the LAC4 gene [3]. Since the ß-gal is located in the cytoplasm of yeasts, cell disruption is necessary to release the enzyme into solution. This step is a critical operation because conditions of disruption may have a profound effect on the later processing and overall efficiency of the commercial process [4,5]. The ideal method of cell disruption would be one that releases the protein selectively, in high yield, and with minimal micronization of cell debris in order to facilitate the processing of the crude extract. Permeabilization of Fmoc-Lys(Boc)-OH sale under mild physical-chemical conditions provides some of these advantages when compared to the methods of mechanical rupture by homogenization or ball-milling typically used on industrial scale [6]. The objective of permeabilization is to disrupt the cell envelope and the internal membranes in order to release a desired protein [7]. Several permeabilization methods have been used to obtain ß-gal from different Kluyveromyces species. In these instances, cells were permeabilized by electrical fields [8], toxic solvents such as toluene or chloroform [[9], [10], [11]], or high ethanol concentrations [12,13]. The characteristics of such systems would likely limit their commercial application [5]. This yeast species have also been permeabilized with organic solvents [[14], [15], [16]] and detergents like Triton X-100 [17], cetyl trimethylammonium bromide [18], and digitonin [19]. Because in those instances the permeabilization was carried out in order to prepare whole-cell biocatalysts for lactose hydrolysis or for in-situ determination of the ß-gal activity, the details of the enzyme release were not investigated. An effective chemical for yeast permeabilization would appear to be the anionic detergent N-lauroylsarcosine (sarkosyl). This detergent is an inexpensive chemical that is widely used for the solubilization of recombinant proteins from inclusion bodies and in many other biological applications [20,21]. This detergent was furthermore highly efficient in permeabilizing K. lactis [22] and Kluyveromyces marxianus [23] as well as other yeasts such as recombinant Saccharomyces cerevisiae and Saccharomyces pombe strains expressing lactase [24] and baker ’s yeast for measuring catalase activity [25] or to accelerate autolysis for the recovery of intracellular enzymes [26]. Because sarkosyl is biodegradable and exhibits a low toxicity, the compound has been authorized for use in personal-care pharmaceutical products and in food films [27,28]. These characteristics, makes sarkosyl a potential permeabilizing agent for use in the downstream-processing of enzymes used in the food industry [5]. In the present report, we have used sarkosyl permeabilization for the release of ß-gal from K. lactis cells and developed a simple process for obtaining a partially purified, stable β-gal preparation.
Materials and methods
Results and discussion
Conclusions The results presented here demonstrate that the permeabilization of cells of K. lactis by exposure to sarkosyl is an effective method for releasing ß-gal from the yeast's cytoplasm. Sarkosyl permeabilization, moreover, constitutes an improved method for the release of yeast ß-gal over other permeabilization systems that use toxic solvents or high alcohol concentrations and longer incubation times to release the enzyme [[9], [10], [11], [12], [13]]. The method was more selective than mechanical disruption; furthermore, the lack of significant cell fragmentation facilitated the clarification of the extract by a low-speed centrifugation and the subsequent diafiltration and ultrafiltration used to partially purify and concentrate the crude cell-free extract. A simple formulation obtained by mixing the concentrate with glycerol proved to be stable under refrigeration. The preparation described here may be considered a technical-grade lactase that can be used in different processes—such as GOS production, the elaboration of dulce de leche (a sweet, viscous Argentine milk product similar to butterscotch) or other products where the presence of accompanying unrelated activities (e. g., proteases or arylsulfatases) would not constitute a quality problem for a final milk product during long-term storage [41]. The concomitant invertase activity would not be a problem in most applications, but can be a drawback in sugary milk products. Conversely, the presence of invertase may give rise to applications in which the sweetness of the preparation needs to be improved without an excessive increase in the sugar content of the final product [42]. Although this study deals with the ß-galactosidase of K. lactis, the procedure could be used as well for the recovery of other intracellular enzymes from various yeast species.