Analysis and application of a suite of recombinant endo-β(1,3)-d-glucanases for studying fungal cell walls

Background The fungal cell wall is an essential and robust external structure that protects the cell from the environment. It is mainly composed of polysaccharides with different functions, some of which are necessary for cell integrity. Thus, the process of fractionation and analysis of cell wall polysaccharides is useful for studying the function and relevance of each polysaccharide, as well as for developing a variety of practical and commercial applications. This method can be used to study the mechanisms that regulate cell morphogenesis and integrity, giving rise to information that could be applied in the design of new antifungal drugs. Nonetheless, for this method to be reliable, the availability of trustworthy commercial recombinant cell wall degrading enzymes with non-contaminating activities is vital. Results Here we examined the efficiency and reproducibility of 12 recombinant endo-β(1,3)-d-glucanases for specifically degrading the cell wall β(1,3)-d-glucan by using a fast and reliable protocol of fractionation and analysis of the fission yeast cell wall. This protocol combines enzymatic and chemical degradation to fractionate the cell wall into the four main polymers: galactomannoproteins, α-glucan, β(1,3)-d-glucan and β(1,6)-d-glucan. We found that the GH16 endo-β(1,3)-d-glucanase PfLam16A from Pyrococcus furiosus was able to completely and reproducibly degrade β(1,3)-d-glucan without causing the release of other polymers. The cell wall degradation caused by PfLam16A was similar to that of Quantazyme, a recombinant endo-β(1,3)-d-glucanase no longer commercially available. Moreover, other recombinant β(1,3)-d-glucanases caused either incomplete or excessive degradation, suggesting deficient access to the substrate or release of other polysaccharides. Conclusions The discovery of a reliable and efficient recombinant endo-β(1,3)-d-glucanase, capable of replacing the previously mentioned enzyme, will be useful for carrying out studies requiring the digestion of the fungal cell wall β(1,3)-d-glucan. This new commercial endo-β(1,3)-d-glucanase will allow the study of the cell wall composition under different conditions, along the cell cycle, in response to environmental changes or in cell wall mutants. Furthermore, this enzyme will also be greatly valuable for other practical and commercial applications such as genome research, chromosomes extraction, cell transformation, protoplast formation, cell fusion, cell disruption, industrial processes and studies of new antifungals that specifically target cell wall synthesis. Supplementary Information The online version contains supplementary material available at 10.1186/s12934-021-01616-0.


Introduction
Fungal cells are surrounded by a thick and rigid structure, namely the cell wall. The function of this structure is to protect against environmental changes, such as osmotic or temperature stress, that can cause cell

Open Access
Microbial Cell Factories *Correspondence: ribas@usal.es; cortes@usal.es Instituto de Biología Funcional y Genómica Zacarías González, 2. CSIC and Universidad de Salamanca, 37007 Salamanca, Spain alterations. However, despite its robustness, the cell wall is an extremely dynamic structure with a great plasticity and phenotypic diversity [1][2][3]. The cell wall composition directly affects its function; therefore, it is fundamentally important to identify the components involved and determine their contribution to this structure. In addition, the architecture and composition of the cell wall is specific for each fungal species, and the composition can change depending on the site of the cell wall, during the cell cycle or in response to environmental disturbances [2,3].
The fungal cell wall is predominantly composed by polysaccharides [4,5], and there are different methods based on either chemical or enzymatic degradation that can be used to analyze its composition. The main examples for chemical degradation are alkali solubilization [6,7], acid hydrolysis [8,9], periodate oxidation [10], borohydride reduction [6,11], Smith degradation [12], permethylation [9] and carboxymethylation [13]. However, these methods are more laborious to carry out than those based on enzymatic degradation and only provide information about the type of bonds between the monosaccharides forming the polymers, but not about the amount and type of polymers forming the cell wall [14]. On the other hand, the use of carbohydrate-degrading enzymes greatly facilitates the analysis and quantification of cell wall polysaccharides [15]. Both methods are complimentary, and as such the combination of chemical and enzymatic analyses together with the usefulness of radioactive labeled cell walls is the most comprehensive method for quantifying the different cell wall polymers [16]. Additionally, other strategies that have provided valuable information on the composition and construction of the cell wall generally focus on the amount and type of bonds between monosaccharides [9,17,18].
We have previously described a simple and accurate method for analyzing the cell wall polymers of the fission yeast Schizosaccharomyces pombe by enzymatic and chemical analyses of radioactive labeled cell walls [16,[19][20][21][22][23]. Briefly, this protocol consists of 14 C-glucose labelling and fractionation of cell wall polysaccharides by using specific chemical and enzymatic procedures (Fig. 1). This allows for the quick and accurate quantification of the main cell wall polymers: α-glucan, β(1,3)d-glucan, β(1,6)-d-glucan and galactomannoproteins [22,23]. Although this protocol has been established by using fission yeast cell walls, it can be easily adapted for analyzing the cell wall of other fungal species [24][25][26][27][28]. This protocol involves enzymatic degradation of the cell wall with the enzymatic complex Zymolyase 100T (Seikagaku Biobusiness Corporation) and the recombinant endo-β(1,3)-d-glucanase Quantazyme (MP Biomedicals), whose activities are essential for quantifying the amount of α-glucan and β(1,3)-d-glucan in the cell wall, respectively [22,23]. To accomplish this, both enzymatic complex and recombinant enzyme must contain very precise and defined enzymatic activities, free from contaminants or unknown activities in the first case, and a completely specific and efficient activity in the second [15].
Endo-β(1,3)-d-glucanases have also been important for the genomic analyses of processes such as induced cell wall stress, altered cell wall integrity pathway and altered response to cell wall synthesis inhibitors [48][49][50][51][52]. In addition, endo-β(1,3)-d-glucanases have been frequently used for the analyses of cell wall composition, as well as enzymatic product required in the study of antifungal drugs and the discovery of new antifungals that specifically target the synthesis of cell wall polysaccharides [46,[53][54][55][56][57][58][59] Despite all of the applications and uses of highly specific endo-β(1,3)-d-glucanases, Quantazyme was the only commercially available recombinant endo-β(1,3)d-glucanase known to reproducibly and reliably degrade the fission yeast cell wall β(1,3)-d-glucan. Therefore, when this enzyme was discontinued it became extremely necessary to find a new recombinant endo-β(1,3)-dglucanase with similar properties to replace Quantazyme in cell wall β(1,3)-d-glucan degradation studies [23,24,60]. Currently, some commercial enzymes described as endo-β(1,3)-d-glucanases may be useful as putative substitutes for Quantazyme; however, so far, none of them have been tested in the cell wall of S. pombe or any other fungi. Thus, the aim of this work was to identify those commercial recombinant enzymes exhibiting a specific and efficient β(1,3)-d-glucanase activity, free of any additional residual activity and able to replace Quantazyme in fission yeast cell wall analysis. For this purpose, we tested 12 commercially available recombinant endo-β(1,3)-d-glucanases and compared their activity to that of Quantazyme. Here, we show that GH16 enzyme from Pyrococcus furiosus (called PfLam16A) [61,62], was able to perform a complete and reliable cell wall β(1,3)d-glucan degradation similar to that of Quantazyme. Hence, the use of PfLam16A in future studies on the cell wall in either fission yeast or other fungal species in general appears to be quite promising.

Results and discussion
The fungal cell wall is an essential structure that provides osmotic resistance and mechanical strength to fungal cells. It must be physically robust to withstand the force of turgor pressure within the cell. On the other hand, since it is also the interface between the fungal cell and the external environment, it must be a highly dynamic structure, capable of changing its composition and architecture in response to variable environmental conditions [3,5]. Thus, to understand the mechanisms whereby fungi react to environmental conditions, it is necessary to have methods and tools to determine the cell wall composition throughout the life cycle of a cell [16].
In this work, we used the fission yeast as a model for studying cell wall composition [5], which has been described as a fast, simple and reliable tool for analyzing cell wall polysaccharides [63,64]. The protocol employed has been improved over time with only some minor modifications. Initially, this protocol allowed the quantification of three cell wall polysaccharides, α-glucan, β-glucan (mixture of β(1,3)-d-and β(1,6)-d-glucans) and galactomannoproteins, and the percentage that each of them represents in both the cell wall and the cell [16,[19][20][21]65] (Fig. 1). Later on, the protocol was improved in order to discriminate the different β-glucans and to quantify the percentage of each of the four main fungal cell wall polysaccharides: α-glucan, β(1,3)-d-glucan, β(1,6)-d-glucan and galactomannoproteins [14,22,23] ( Fig. 1). Thus, the current protocol used for fission yeast cell wall fractionation involves three consecutive steps: (1) 14 C-labelling of the cell; (2) isolation of cell wall; and (3) fractionation of the cell wall polysaccharides by using both enzymatic (Zymolyase 100T complex and recombinant β(1,3)-d-glucan-hydrolyzing enzyme called Quantazyme) and chemical (Fehling's reagent) fractionations ( Fig. 1). As mentioned above, since Quantazyme is no longer available, we sought to identify an alternative recombinant endo-β(1,3)-d-glucanase able to degrade the cell wall β(1,3)-d-glucan. Having said enzyme would not only permit degradation results similar to those achieved using Quantazyme, but would also ensure the continuity of studies on the fission yeast cell wall, as well as the cell walls of other fungi in general.
First, the endo-β(1,3)-d-glucanase activity for each enzyme was tested according to the conditions specified by the supplier. These conditions were specifically obtained through the enzymatic degradation of purified β(1,3)-d-glucan (laminarin or β(1,3)-d-glucan from barley) and β(1,3)(1,4)-glucan (lichenan), but not in the context of the fungal cell wall, where β(1,3)-d-glucan is closely intertwined with other polymers. Table 1 shows the list of recombinant enzymes tested, the percentage of cell wall degradation and the reaction conditions in which each enzyme exhibited maximum activity.
The recombinant enzymes ALam55A, BhLam81A, TmLam16A and ZgLam16A (from NZYTech) showed insufficient cell wall degradation, where the values obtained were well below from those obtained using Table 1 Cell wall degradation by specific recombinant endo-β(1,3)-d-glucanases a Reaction condition in which each enzyme exhibits the maximum % of cell wall degradation (mean ± SD from at least two independent experiments). In addition to these conditions, each enzyme was tested according to the conditions described in Additional file 3: Table S3 b One unit of Quantazyme activity is defined as the amount of enzyme required to produce a 0.001 decrease in A 800 per minute from a suspension of brewer's yeast (Saccharomyces cerevisiae) as substrate in 33.5 mM potassium phosphate monobasic buffer, pH 7.5 with KOH, 60 mM β-mercaptoethanol at 25 °C c One unit of E-LAMHV activity is defined as the amount of enzyme required to release one µmole of glucose-reducing sugar equivalents per minute from laminarin β(1,3)-d-glucan (10 mg/mL) as substrate in 100 mM sodium acetate buffer, pH 5.0 at 40 °C d One unit of E-LICACT activity is defined as the amount of enzyme required to release one µmole of glucose-reducing sugar equivalents per minute from barley β-dglucan (5 mg/mL) as substrate in 100 mM sodium phosphate buffer, pH 6.5 at 40 °C e One unit of Bglu110 activity is defined as the amount of enzyme required to release one µmole of glucose-reducing sugar equivalents per minute from lichenan β(1,3)(1,4)-d-glucan (10 mg/mL) as substrate in 100 mM sodium phosphate buffer, pH 7.0 at 75 °C  Table S1), showed excessive cell wall degradation, well above 53%, as compared to the Quantazyme control (Table 1). Despite being recombinant, these enzymes exhibited cell wall degradation that corresponded to the degradation of more than just one type of polysaccharide. Thus, these enzymes showed the signs of either containing contamination or having an activity that was stronger than that of Quantazyme. Alternatively, excessive β(1,3)d-glucan degradation could cause the release of other cell wall polysaccharides that have never been detected using Quantazyme. Finally, the recombinant 3.2.1.39 enzymes PfLam16A and CtLam81A (NZYTech, Additional file 1: Table S1 and Additional file 2: Table S2) [61,62,82] exhibited optimal percentages of cell wall degradation that were similar to the control (Table 1). However, CtLam81A presented the disadvantage that it precipitated during the reaction, generating a rare viscous reaction mixture that was difficult to process and to quantify the efficacy of the degradation. Next, in an attempt to improve the activity of low-efficient enzymes and to reduce the precipitation caused by CtLam81A, all enzymes were again tested under different reaction conditions (Additional file 3: Table S3). All combinations analyzed using different buffers, pH and incubation temperatures did not significantly alter the previous results obtained for ALam55A, BhLam81A, CtLic16A and ZgLam 16A, which were still degrading less than expected. In contrast, the recombinant enzymes E-LICACT, E-LAMHV, TmLam16A, TnLam16A and TpLam16A were highly sensitive to changes in buffer, pH and/or incubation temperature. They presented a broad range of cell wall degrading activities ranging from 8% (BhLam81A) to 73% (E-LAMHV) (Additional file 3: Table S3). This indicated that the activity of the enzymes assayed was not stable or reliable. Instead, they varied considerably when conditions other than those specified by the supplier were used. Therefore, these enzymes were not suitable for substituting Quantazyme in cell wall analysis. The stability and/or variability of the enzymes PfLam16A and CtLam81A were also analyzed, being two enzymes that had shown optimal percentages of cell wall degradation in previous experiments. While CtLam81A was extremely variable, with an activity that ranged from 14 to 81%, and still caused the reaction mixture to precipitate, PfLam16A, on the other hand, was found to be highly stable. The percentage of cell wall degradation was within the optimal range for all conditions tested, except when the reaction was carried out using decreasing amounts of enzyme or during incubation times that only allowed for partial cell wall degradation (Additional file 3: Table S3).

Reaction conditions % of cell wall degradation
Then, the cell wall degradation was also analyzed by combining two enzymes simultaneously in the same reaction. The aim was to identify combinations capable of degrading the cell wall like PfLam16A or Quantazyme ( Table 2). In all cases, a reaction condition was used in which the single enzyme, including PfLam16A, showed insufficient cell wall degradation. Under these conditions, some of the combinations still were unable to sufficiently degrade the cell wall, whereas others showed excessive cell wall degradation. The only enzyme combination that had the ability to degrade the cell wall to around 54% was that of CtLam81A and TmLam16A. Unfortunately, in this case the enzyme mixture again precipitated, making it difficult to handle the reaction.
Finally, and taking into account all previous results, recombinant PfLam16A was selected as the best enzyme. Subsequently, it was used to perform additional analyses before being selected as a substitute for Quantazyme (Table 3) [61,62]. In order to determine the optimal conditions for cell wall degradation by PfLam16A, different conditions combining various parameters, such as the buffer used, pH, temperature, incubation time and the amount of enzyme, were assessed. In the case of sodium acetate (pH 5.0), a slight increase in cell wall degradation was detected at longer incubation times. This could be due to the fact that at acidic pH the cell wall can be partially hydrolyzed, helping the enzyme to release some oligosaccharides to the medium [89]. However, at a higher pH, undesirable acidic hydrolysis was prevented ( Table 3). Most of the conditions tested were found not to be optimal, because cell wall degradation was insufficient. However, optimal cell wall degradation was achieved using the following buffers: citrate/phosphate 100 mM pH 5.5; MES 100 mM pH 5.5; sodium acetate 100 mM pH 5.0 and sodium phosphate 100 mM pH 6.0 and 6.5; and one specified by the supplier. All of these buffers, except sodium acetate pH 5.0, permitted stable and reproducible enzyme activity, with similar percentages of cell wall degradation being achieved at longer incubation times and with larger amounts of enzyme. Thus, the following conditions were established as the standard reaction (Table 3): 20 μg of PfLam16A in buffer sodium phosphate 100 mM pH 6.5 with an incubation of 20 h at 70 °C.
In order to confirm that PfLam16A and the reaction conditions established could achieve a similar percentage of cell wall degradation as that obtained using Quantazyme, separate reactions using PfLam16A and Quantazyme were carried out simultaneously ( Table 4). As expected, both enzymes resulted in reproducible and highly similar percentages of cell wall degradation; thus, it was confirmed that PfLam16A [61,62] was a definite substitute for Quantazyme for the study of cell wall β(1,3)-d-glucan degradation.
Finally, in order to show the usefulness of the proposed protocol, the complete fractionation and analysis of the cell wall polysaccharides of fission yeast was performed using recombinant endo-β(1,3)-d-glucanase PfLam16A ( Table 5). As expected, the percentages obtained for each polysaccharide, including β(1,3)-d-glucan, were in agreement with those previously described for fission yeast using Quantazyme [16,22,23].
In sum, a series of 12 commercial recombinant endoβ(1,3)-d-glucanases were analyzed, and PfLam16A was found to be the only enzyme that produced reliable and reproducible results. Moreover, PfLam16A was able to specifically and completely degrade cell wall β(1,3)-d-glucan of the fission yeast S. pombe without affecting other polysaccharides. Thus, our results show that this enzyme is the best suitable substitute for Quantazyme for the fractionation and analysis of cell wall polysaccharides from fission yeast and other fungi in general (Table 4; Fig. 3).

Strains
The Schizosaccharomyces pombe strain used in this study was the wild type 972 h − .

Growth media
The growth media used were YES (Yeast Extract with Supplements) and YES 0.5% Glc (YES with low glucose). Normal YES medium contains 30 g/L glucose, 5 g/L yeast extract, 250 mg/L adenine, 250 mg/L histidine, 250 mg/L leucine, 250 mg/L lysine and 250 mg/L uracil, sterilized by autoclaving. Alternatively, EMM (Edinburgh minimal medium) and EMM 0.5% Glc can also be used [90].

Fractionation and analysis of cell wall polysaccharides
This protocol has been adapted to quantify the three major polysaccharides of the S. pombe cell wall (α-glucans, β-glucans, and galactomannoproteins) by using new commercially available recombinant endoβ(1,3)-d-glucanases (from NZYTech, Megazyme and Prokazyme) and is based on the enzymatic and chemical fractionation of cell wall polysaccharides. Although the new protocol is essentially the same as one previously described [14,16,22,23], it now includes information describing the search for a new recombinant endo-β(1,3)-d-glucanase that is both efficient and reliable, and able to replace Quantazyme (MP Biomedicals), which is no longer commercially available. Quantazyme is the only recombinant endo-β(1,3)-d-glucanase known to date that can completely and reproducibly degrade S. pombe cell wall β(1,3)-d-glucan. Since we have shown that the new recombinant endo-β(1,3)-dglucanase PfLam16A (NZYTech) has a similar activity to that of Quantazyme, this protocol can be used for the analysis of any fungal cell wall. Briefly, the fractionation and analysis of the polymer composition of the cell wall is carried out in three main steps: (A) Fig. 3 Comparison between complete cell wall fractionations using Quantazyme or PfLam16A. Cell wall fractionation data using Quantazyme are from previous work [92]. There is no significant difference between the data obtained from cell wall fractionations using Quantazyme or PfLam16A (A) 14 C-glucose labeling of the cells 1. Prepare a stock of unlabeled wild-type cells using the following method. These cells will be used as carrier cells in Step 9: i. Incubate wild-type cells in 500 mL of YEPD at 28ºC, with shaking at 200 rpm for 48 h, until late stationary phase. ii. Collect the cells by centrifugation at 4000g for 5 min, wash the pellet with distilled water, recentrifuge, and resuspend the cell pellet in 50 mL of 1 mM EDTA, 0.02% NaN 3 (sodium azide). The cell suspension can be stored at 4 °C for up to 12 months and the final cell concentration is usually around 10 10 cells/mL. Carrier cells are added in Step 9, described below, to minimize the loss of either 14 C-labeled cells or cell walls during the different centrifugation steps.
2. Prepare cells in early log-phase in liquid medium (in the case of fission yeast, YES rich medium or EMM minimal medium) by growing, with shaking, the culture at the same temperature as that described below for the medium containing d-[U- 14 C]glucose. The medium should contain 0.5% or 1.0% of glucose (normal YES contains 3% and EMM contains 2%) in order to increase the efficiency of the subsequent incorporation of 14 C-glucose into the cell. 3. Dilute the cells in 14 mL of the same medium, calculating the appropriate dilution in order to collect the cells in early log-phase at 1.0 to 1.5 X 10 7 cells/ mL (A 600 = 0.7-1.0). 4. Transfer the 14-mL cell culture to two new flasks (7 mL each): One culture will be used to monitor cell growth (unlabeled), and 3 µCi/mL of d-[U- 14 C]glucose (Hartmann Analytic) will be added to the other. When required, the concentration of 14 C-glucose can be increased to 10 or 20 µCi/mL. 5. Incubate both cultures at the desired temperature with shaking. Use an incubation time that allows 14 C to efficiently incorporate into the cells, taking into consideration that during each cell cycle, 50% of the cell material is newly synthesized and consequently 14 C-labeled. A greater amount of labeling can be obtained using longer incubation times that allow more cell cycles to occur, or by increasing the concentration of 14 C-glucose in the medium. Use the unlabeled cell culture to monitor cell growth. 6. When the monitored unlabeled cells have reached the desired absorbance (1.0 to 1.5 × 10 7 cells/mL), transfer the labeled cells to a 10 mL centrifuge tube and collect by centrifugation (at 4000g for 10 min). 7. Carefully discard the supernatant in the appropriate radioactive container; save some supernatant so to avoid losing any cells. 8. Wash the cells twice to eliminate the residual radioactive medium with 10 mL of 1 mM EDTA and centrifuge after each wash at 4000g for 10 min. 9. Resuspend the labeled cells in 10 mL of 1 mM EDTA and add 150 µL of carrier cells (prepared according to Step 1). Centrifuge, wash with 1 mM EDTA and then spin again at 4000g for 10 min. If the carrier cells are added before eliminating the remaining radioactive medium, they can incorporate some 14  Cool the tube for 10 min at 4 °C, briefly spin the tube and mix the content to homogeneity using a vortex. 21. Take two 50-µL aliquots of cell walls and mix with 2 mL of liquid scintillation cocktail in liquid scintillation vials. Vortex and keep at 4 °C until measuring its radioactivity together with the other fractions (see Step 27 below). The radioactivity in these aliquots corresponds to the total incorporation in the cell wall fraction. 22. To inhibit the growth of undesirable aerobic microorganisms that might consume the isolated cell wall, add 10 µL of 2% NaN 3 (sodium azide) to the remaining 1.0 mL of cell wall suspension, mix well and store the cell walls at 4 °C for up to 2 weeks.

(C) Fractionation and analysis of the cell wall polysaccharides:
Once the cell walls are 14 C-labeled and isolated from the rest of cellular components, proceed to fractionate the cell wall polysaccharides as follows: 23. Cell wall degradation with Zymolyase 100 T: (a) Prepare a stock solution of Zymolyase 100 T at 5 mg/mL in 50 mM citrate/phosphate buffer, pH 5.6. The stock can be stored at -20 °C for several years. In the case of PfLam16A, 1 M sodium phosphate buffer, pH 6.5 is the optimal 10 × buffer to use. In the case of Quantazyme add 30 µL of 10 × buffer (10 × is 335 mM potassium phosphate monobasic, pH 7.5 with KOH) and 30 µL of 10 × β-mercaptoethanol (10 × is 600 mM β-mercaptoethanol). The final 1 × buffer contains 33.5 mM potassium phosphate monobasic, pH 7.5 with KOH and 60 mM β-mercaptoethanol. (c) Add from 10 to 400 units or from 5 to 300 µg of the corresponding endo-β(1,3)-d-glucanase into two of the tubes. This corresponds to a volume from 5 to 100 µL of the corresponding stock solution of recombinant endo-β(1,3)d-glucanase (prepared in the corresponding buffer as specified by the supplier). Add the same volume, from 5 to 100 µL of distilled water, into the other two tubes, which will be used as the controls (no enzymatic degradation). Table 1 lists all of the recombinant endoβ(1,3)-d-glucanases used in this work. In the case of PfLam16A, the optimal enzyme concentration is 20 µg per reaction. (d) Top up the volume in each tube to 300 µL with distilled water and mix briefly by vortexing. (e) Incubate with shaking in a thermoshaker or a roller in the conditions specified by the supplier for each enzyme. In the case of PfLam16A, the optimal condition is an incubation time of 20 h at 70 °C.
• Each tested enzyme has an optimal condition according to the specifications of the supplier (buffer, pH, temperature). • The enzymes exhibiting a good percentage of cell wall degradation (about 55%, which is like that of Quantazyme, or slightly lower) were then tested under different conditions in order to assess their stability and to determine their maximum capacity to degrade cell walls. Each polysaccharide fraction can be expressed as a percentage of the radioactivity in the cell, according to the total amount of glucose incorporated into the cell, or as a percentage of the radioactivity in the cell wall, according to the total amount of radioactivity in the cell wall (the sum of all fractions is the total amount of the cell wall, which is 100%). The first set of data is the total amount of each polysaccharide relative to the cell and the second one shows the proportion of each polysaccharide with respect to the cell wall. The analysis of these percentages in different altered yeast strains may reveal defects in the proportion with respect to either the cell, or the cell wall, or both structures.