Extracellular activity of proteases from Yarrowia lipolytica IPS21 as a function of the carbon and nitrogen source

The yeast was isolated from soil samples. The soil samples were collected in spring from the area of the former “BORUTA” dye factory in Lodz, Poland (51°50’35.4”N 19°23’33.5”E). The purified isolate was subjected to Sanger sequencing and identification at Nexbio in 2022 (Wroclaw, Poland). The yeast isolates were identified on the basis of the 18S rRNA gene sequence. The sequence obtained was aligned with nucleotide sequences available in the NCBI database using BLAST (Supplementary material 1). Y. lipolytica IPS21 from the pure culture collection of the Lukasiewicz Research Network - Lodz Institute of Technology was maintained on “modified YPG” medium: yeast extract – 15.0 g, sodium chloride – 5.0 g, agar – 25.0 g per litre of distilled water, stocks at 4 °C.

A “modified YPG liquid medium” containing: yeast extract – 15.0 g (BTL Sp. z o.o.) and NaCl – 5.0 g (Chempur) was used as the basic liquid medium in this study. Subsequently, one of the additional carbon sources was introduced into the medium: glucose (Chempur), saccharose (Chempur), oat (from the oat crop of 2022 from a farm in the Lodz province), glycerine (Eurochem BGD Sp. z o.o.), pectin (P.P.H.U Eccodin Sp. z o.o.), semolina (MW FOOD Sp. z o.o.), chrome-tanned leather shavings – CTLS (tannery in Lodz, Poland).

The inoculation liquid medium contained: yeast extract - 15.0 g; NaCl – 5.0 g; distilled water to 1 L, pH 7.0. Inoculation cultures were inoculated with yeast suspension from ‘modified YPG’ slants. A slant was used to prepare the inoculum medium, which was added with 10 mL of 0,85% saline. The whole was then transferred to Erlenmeyer flasks, comprising 30 ml of the ‘modified YPG liquid medium’. Cultures were carried out in 100 mL Erlenmeyer flasks containing 40 mL of the inoculation medium at 140 rpm for 24 h at 30.0 °C using a Biosan ES-20 environmental shaker incubator (Biosan).

The liquid medium contained: yeast extract - 15.0 g; NaCl – 5.0 g; different carbon sources - equivalent to 15 g of carbon sources, distilled water up to 1 L, pH 7.0. The production cultures were inoculated with cell material taken from the inoculation medium. To 100 mL flasks filled with the production medium, 0.4 mL of inoculum was added at OD=1. Cultures were grown at 210 rpm for 72 h at 30.0 °C on a rotary shaker type Biosan ES-20 environmental shaker-incubator (Biosan) GmbH, Germany.

The liquid medium contained: 3 different amounts of yeast extract – 5.0 g, 10.0 g; 15.0 g, CTLS – 15.0 g, NaCl – 5.0 g; distilled water up to 1 L, pH 7.0. The production cultures were inoculated with cell material taken from the inoculation medium. To 100 mL flasks filled with the production medium, 0.4 mL of inoculum was added at OD=1. Cultures were grown at 210 rpm for 72 h at 30.0 °C on a rotary shaker type Biosan ES-20 environmental shaker-incubator (Biosan) GmbH, Germany.

The weight of dry yeast biomass was measured, after filtering the cells under pressure using Whatman GC/F filters (100 mm in diameter), and then washing the cells with acetone, hexane (3:1), and sterile water. The cells were dried to a constant weight at 80 °C. The results are expressed in g g^-1 of carbohydrate substrate.

The Lowry method for protein quantitation was used according to the literature method [18] .

The pH change was measured every 24 hours using an Elmetron CP411 pH meter. Measurements were made for a 5 ml post-culture liquid sample.

Proteolytic activity was assessed according to the literature [19, 20]. The reaction mixture contained 200 μL of medium supernatants and 200 μL of 5 % azocasein (w/v). The azocasein was dissolved in 0,1 M Tris HCl. The samples were then incubated at 40 °C for 40 min. To inhibit the reaction, 1 % (w/v) trichloroacetic acid (TCA) was added. The ratio of TCA to the reaction mixture was 3 to 1. After 10 min, samples were centrifuged (4000 rpm, 10 min). In the last step, the supernatant (1000 μL) was neutralized by adding 1000 μL of 1.0 mol L^-1 NaOH. Absorbance at 440 nm was measured on a UV/Vis spectrophotometer (Rayleigh UV-9200). One unit of proteolytic activity was defined as an increase of 0.01 in 40 min at A440 absorbance.

Amino acids in the supernatants were determined by HPLC/DAD/FLD. All formulations tested were subjected to acid hydrolysis at 105 °C for 24 hours [21]. The derivatization procedure was applied. All samples were filtered through 0.45 μm membrane filters: Acrodisc 13 mm syringe filters, GHP (Waters, Milford, MA, USA). A Shimadzu Prominence-i LC-2030C, pump, autosampler injector, Nova-Pak C18, 4 μm column (150×3.9 mm, Shimadzu, Kyoto, Japan) was used for amino acid analysis. The column was thermostated at 37 °C with a flow rate of 1.0 cc/min and 10 μL (amino acid concentration 5-200 pmol) was injected. A gradient mobile phase was used for the chromatography. The mobile phase consisted of eluent A (prepared from Waters AccQ-Tag Eluent A concentrate by adding 200 cc of concentrate to 1 L of distilled water), eluent B (60 % acetonitrile).

Metals (Ca, Cr, Fe, K, Mg, Na, P, S) were determined in biomass and supernatants using atomic emission spectrometry with an inductively coupled plasma technique (ICP/AES). Samples of 0.2 g (biomass) and 5 g (supernatants) were placed in a Teflon vessel and 7 mL of HNO₃ (65 % wt, Chempur, Piekary Slaskie, Poland) was added. They were then mineralized using a Magnum II microwave mineralizer (Ertec, Wroclaw, Poland). The sample was then transferred to a 25 mL volumetric flask. The contents of the tested metals (Agilent ICP-OES 5110 spectrometer Santa Clara, USA) in the samples were read from the standard curves prepared from the ICP standard.

Statistica 10.0 (13) was used for mathematical and statistical analyses. Differences were determined by analysis of variance (ANOVA) and the Tukey-Kramer test, also with ± standard errors (SD). The probability level was set at 0.05 for three determinations.

The results for Y. lipolytica IPS21 biomass production and total protein obtained from different carbon sources are shown in Table 1. The highest biomass production was observed on glycerol (0.465±0.3 g g^-1 of carbohydrate substrate) and glucose (0.452±0.11 g g^-1 of carbohydrate substrate). These data are consistent with literature reports that Yarrowia yeast prefers glycerol and glucose for growth [22,23]. The lowest biomass production was obtained with CTLS as a carbon source (0.204±0.09 g g^-1 carbohydrate substrate). The lower biomass production is due to the more complex structure of the protein waste, in which the availability of carbon atoms for efficient biomass formation is greatly reduced. The CTLS used in the study contains collagen cross-linked with a chromium complex [24]. The effect of the change in the carbon source on the increase in biomass was observed, but not on the amount of protein determined.

The amount of total protein, ranging from 109±1.99 to 130±2.33 μg mL’¹ (Table 1), is not statistically different among the variants tested. This is probably due to the fact that biomass is a complex mixture of different components such as polysaccharides, lipids and polypeptides [25]. Other studies have shown that high levels of extracellular proteins (proteolytic enzymes) are not necessarily associated with high biomass production [25]. Getting the right amount of extracellular protein secreted by the cell is more important. This is consistent with what we observed with protease activity - Figure 1. The highest value of protease activity was obtained for the CTLS variant as a carbon source of 800 U mg^-1 of protein (Fig. 1); when the production of biomass was low (Table 1). CTLS waste proved to be the most favorable substrate for enzyme biosynthesis among the carbon sources used in the experiments. The lowest enzyme activity was observed in the variant with glycerol or saccharose and CTLS. For both variants, it was slightly below 100 U mg ^-1 protein. In this case, the metabolism was “switched” to polyol production due to the presence of glycerol as a carbon source in the medium. [26]. The ability of Y. lipolytica to produce erythritol on glycerol-containing media has been extensively described in the literature [27]. When saccharose was added to the medium with CTLS (Fig. 1), the protease activity decreaSed too. Similar data were obtained by Akpinar et al. (2011). They examined media supplemented with skimmed milk, glutamine and glucose. Adding glucose or glutamine to skimmed milk had a negative effect on the overall metabolic transformations of the Yarrowia strain, which was reflected in decreased synthesis of proteolytic enzymes [28]. Similar results were reported by Ogrydziak et al. (2003). They showed that the secretion of proteases and ribonucleases from yeast cells of the genus Yarrowia can be inhibited by some additional sources of carbon and nitrogen in the medium [29].

Fig. 1.

A number of environmental factors are known to influence the process of protease biosynthesis [13,30]. One of the most important factors in the production of proteolytic enzymes is the pH of the medium [31]. In many of the variants tested, a pH above 8.00 was achieved (Figure 2). Our results confirm that a pH above 7.00 may be optimal for alkaline protease synthesis (Figure 1 and Figure 2) [28]. The lowest pH of the medium was determined for saccharose, glucose, and glycerol variants. The yeast Y. lipolytica hydrolyses saccharose to D-glucose and D-fructose. These sugars are incorporated into the glycolysis pathway. The by-products of glycolysis lower the pH of the medium. In turn, the metabolism of glycerol can lead to the formation of citric acid, which also lowers the pH of the medium. Our results confirm previous reports that multifactorial optimization of enzyme biosynthesis conditions is necessary to produce high levels of proteases in culture supernatants [32]. It is not sufficient to check only one of the parameters. We showed that changing the sugar source had a greater effect on the proteolytic activity of Y. lipolytica IPS21 than the pH. This may be due to the fact that the pH of the medium was not under control during the process, which is normally used to produce industrial enzymes [31].

Fig. 2.

In the research, the highest proteolytic activity was found for the CTLS variant and much lower for the yeast extract variant, thus it was decided to compare the amounts of amino acids in the respective supernatants. Casein or yeast extract was reported as the main source of nitrogen and carbon for maximum protease production by yeast [13]. By comparing the retention time with a standard sample, 22 amino acids were identified. The results of the experiments are presented in Table 2. The total amount of amino acids identified in the supernatant was the highest in the CTLS medium with Y. lipolytica IPS21 – 2.78 g 100 g^-1 of supernatant. The increase in selected amino acids in these samples may indicate the intensification of the degradation of CTLS by Y. lipolytica or the secretion of proteases into the medium [33, 34, 35]. The lowest total amount of amino acids identified were found in a variant with the yeast extract medium with Y. lipolytica IPS21 – 0.12 g 100 g^-1 of supernatant. The yeast Y. lipolytica IPS21 can use amino acids as a substrate for biomass formation [36]. Compared to the literature data [13,31], the amount of extract in our medium was lower than in other studies. Almost all amino acids were incorporated into the biomass.

In the second set of studies, it was decided to test the effect of reducing the amount of yeast extract in the CTLS medium. In the first series, the highest protease activity was obtained in the medium containing CTLS and yeast extract. Yeast extract is the product of yeast cells, containing amino acids, lipids, vitamins, minerals, and other soluble components. It is considered to be a good source of nitrogen. The biomass production of the yeast Y. lipolytica IPS21 and the total protein at different levels of yeast extract in the CTLS medium are shown in Table 3.

The highest biomass production was determined after the addition of 0.4 g of yeast extract – 0.375±0.16 g g^-1 as a carbohydrate substrate. However, the test with 0.6 g of yeast extract in the CTLS medium showed the highest total protein 112.4±6.55 μg mL’¹ (Table 3). The results obtained are in line with previous work [37,38], showing the different adaptation processes in yeast and bacteria exposed to environmental stress, such as changes in carbon sources. The protease activities of the IPS21 yeast were also determined – Figure 3. The highest proteolytic activity of 941.0±21.55 U mg^-1 protein was observed in the variant with 0.4 g of yeast extract in the CTLS medium. The potential of Yarrowia to produce proteolytic enzymes was affected by changing the amount of yeast extract. A factor in the reduction of protease production may have been too little or too much yeast extract in the CTLS medium.

Fig. 3.

The differentiation of these results shows the importance of the carbon source in the process of the formation and secretion of proteolytic enzymes from the cell, which is consistent with the literature [29]. The data obtained show how important it is to have a ratio of carbon and nitrogen sources in a medium with Y. lipolytica IPS21. A small change can significantly slow down the secretion of enzymes.

Yeast cells, through their inherent mechanisms, can easily adapt to, tolerate, and detoxify metal-contaminated environments. Yarrowia strains can be used to accumulate different metals [39, 40]. It was therefore decided to determine the concentrations of selected metals in biomass and cells. The presence of the metals in the samples was related to that of metals in the medium substrates. The results for the metal content of selected samples regardless of the amount of yeast extract in the medium indicate that metals accumulate primarily in the biomass and not in the supernatant (Table 4).

Significantly different amounts of elements were found in the biomass. The biomass of the yeast Yarrowia IPS21 seems to be a useful waste from the point of view of the elemental composition due to the high content of Ca 3.16 g kg^-1, K 5.16 g kg^-1· and P 16.02 g kg^-1. These elements are essential for proper plant growth and development [43]. The biomass obtained in the process of protease production could also be used as a biostimulator or soil conditioner [44]. The literature also describes the possibility of the absorption of dyes from textile effluents by yeast. Adsorption on biomass may be one of the basic mechanisms for removing pollutants from textile wastewater in aerobic biological treatment systems [41, 42]. In future research biomass of yeast IPS21 could be used to adsorb dyes from textile effluents.