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Fermentation Microbiology  |  Applied Microbiology

Microbiol. Biotechnol. Lett. 2023; 51(4): 457-464

Received: September 19, 2023; Revised: November 7, 2023; Accepted: November 16, 2023

A Novel Approach for Assessing the Proteolytic Potential of Filamentous Fungi on the Example of Aspergillus spp.

Anna Shestakova1, Alexander Osmolovskiy1, Viktoria Lavrenova1, Daria Surkova1,2*, Biljana Nikolić3, and Željko Savković3

1Department of Microbiology, Faculty of Biology, Lomonosov Moscow State University, 119234 Moscow, Russia
2Faculty of Biology and Biotechnology, HSE University, 101000 Moscow, Russia
3Faculty of Biology, University of Belgrade 11000 Belgrade, Serbia

Correspondence to :
Daria Surkova,

Proteolytic enzymes produced by filamentous fungi can degrade various fibrous and globular proteins along with other metabolites that may also find application in biotechnology. In this study, the effect of proteolytic enzymes of 22 Aspergillus strains on various proteins was investigated using protein-containing diagnostic media. Subsequently, a new parameter estimating secreted proteinases specificity towards fibrous or globular proteins without its advanced biochemical research - index of severity of proteolytic action (ISPA) - was suggested. This index determines mycozymes specificity in following manner: its value increases with greater affinity to fibrous proteins, decreases if there is higher affinity to globular proteins. ISPA value was the lowest (0.52) for Aspergillus domesticus, indicating the highest specificity to globular proteins, the highest one (1.26) for A. glaucus, whose proteinases best hydrolyzed fibrous proteins. However, the highest overall proteolytic potential was observed for Aspergillus melleus. The ability to produce acid, alkali and extracellular pigments was evaluated for all isolated strains as well.

Keywords: proteases of micromycetes, proteases&rsquo, specificity, filamentous fungi, Aspergillus, micromycology, fungal metabolites

Proteolytic enzymes are capable of degrading various proteins and consequently find application in various industrial branches, e.g. medicine, agriculture, food production, waste disposal, etc. Modern micromycology deals with (investigates) filamentous fungi, which are known to produce high quantities of extracellular proteases featured with great proteolytic activity. Moreover, these producers are easily cultivated and can be genetically manipulated for use in biotechnological production. Proteases produced by filamentous fungi can be active against various proteins, e.g. fibrin, collagen, keratin, hemoglobin etc [1]. Micromycetes can also produce various pigments, acids and other metabolites, which can also be used in biotechnology. Micromycetes of the genus Aspergillus are ubiquitous and often are associated with various substrates; thanks to this feature they can contaminate different raw materials and surfaces. That is why investigation of isolated strains of filamentous fungi and their metabolites is essential. Detailed characterization of growth conditions, proteases, acids, alkalis and other metabolites is extremely important: on the one hand, it helps to prevent contamination and consequent degradation of some important materials, on the other, it allows finding the proper ways of usage for the emitted substances. Taking this into account, the aim of this study was to estimate the proteolytic potential of selected strains of micromycetes of the genus Aspergillus in order to define their possible appliances in the future. In addition, production of acidity, alkality and extracellular pigments were screened as well.

Microorganisms used in this research

Aspergillus spp. isolates (22 in total) were used in this study. Among them, one strain was isolated from soil near the Poblet Monastery, Spain (A. phoenicis), six strains (A. amstelodami, A. athecius, A. caespitosus, A. glaucus, A. tamarii, A. wentii) were provided from the collection of microscopic fungi of the Moscow State University (Russian Federation) while all the rest strains (listed in Table 1) were obtained from the Mycotheca of the University of Belgrade (Serbia). The strains were maintained on Czapek agar media, containing, %(w/v): NaNO3 - 0.3, K2HPO4 - 0.1, MgSO4 - 0.05, KCl - 0.05, FeSO4 - 0.001, glucose - 3.0, agar - 2.0, at 28℃ and replated every 7 days.

Table 1 . Strains used in the study.

NoSpeciesStrain NoSample location
aAspergillus amstelodamiW/NCollection of microscopic fungi of the Moscow State University (Russian Federation)
bAspergillus atheciusW/NCollection of microscopic fungi of the Moscow State University (Russian Federation)
cAspergillus aureolatusBEOFB3320mMycotheca of the University of Belgrade (Serbia)
dAspergillus caespitosusW/NCollection of microscopic fungi of the Moscow State University (Russian Federation)
eAspergillus calidoustusBEOFB3220mMycotheca of the University of Belgrade (Serbia)
fAspergillus creberBEOFB3250mMycotheca of the University of Belgrade (Serbia)
gAspergillus domesticusBEOFB3270mMycotheca of the University of Belgrade (Serbia)
hAspergillus europaeusBEOFB382mMycotheca of the University of Belgrade (Serbia)
iAspergillus glaucusW/NCollection of microscopic fungi of the Moscow State University (Russian Federation)
kAspergillus jenseniiBEOFB3200mMycotheca of the University of Belgrade (Serbia)
mAspergillus melleusBEOFB3180mMycotheca of the University of Belgrade (Serbia)
nAspergillus penicilloidesBEOFB3190mMycotheca of the University of Belgrade (Serbia)
oAspergillus phoenicisW/NPoblet Monastery, Spain
pAspergillus proliferansBEOFB3280mMycotheca of the University of Belgrade (Serbia)
qAspergillus protuberusBEOFB3240mMycotheca of the University of Belgrade (Serbia)
rAspergillus pseudoglaucusBEOFB3170mMycotheca of the University of Belgrade (Serbia)
sAspergillus ruberBEOFB3150mMycotheca of the University of Belgrade (Serbia)
tAspergillus tabacinusBEOFB3260mMycotheca of the University of Belgrade (Serbia)
uAspergillus tamariiW/NCollection of microscopic fungi of the Moscow State University (Russian Federation)
wAspergillus tennesseensisBEOFB3310mMycotheca of the University of Belgrade (Serbia)
xAspergillus tubingensisBEOFB3300mMycotheca of the University of Belgrade (Serbia)
yAspergillus wentiiW/NCollection of microscopic fungi of the Moscow State University (Russian Federation)

W/N - without number

Screening for protease production

For protease production assessment, a previously developed technique with slight modifications was used [2]. Briefly, a strain was plated on the diagnostic media, containing different protein components indicated in Table 2. The basic media premix was of the following composition, % (w/v): peptone - 0.5, KH2PO4 - 0.05; MgSO4 - 0.025; agar - 1.5. Strains were plated in the center of the Petri dish and incubated at 28 ± 0.2℃ in the darkness. After 7 days of cultivation, the plates were contrasted by the detection agents (Table 2) and the enzymatic indices (EI) were calculated (Fig. 1). Using of trichloroacetic acid, Coomassie Brilliant Blue G250 perchloric acid solution and ammonium sulfate as detection agents for protein identification are commonly used [3, 4]. During preliminary experiments all presented agents were used, and the best result was observed for detection agents from Table 2.

Table 2 . Proteins and their detection reagent used for protease production screening.

ProteinConcentration in media, % (w/v)Detection agent
Casein1.0Trichloroacetic acid, 10%
Fibrin0.3Coomassie Brilliant Blue G250 perchloric acid solution
Fibrinogen0.5Trichloroacetic acid, 10%
Hydrolyzed collagen1.0(NH4)2SO4, 39.6%
Hemoglobin0.5Trichloroacetic acid, 10%
Keratin0.3Coomassie Brilliant Blue G250 perchloric acid solution

Figure 1.An experimental scheme used to evaluate the spectrum of fungal proteolytic activities and production of acid/ alkaline metabolites. CA medium with casein, HCA medium with hydrolyzed collagen, FA medium with fibrin, FgA medium with fibrinogen, HA medium with hemoglobin, KA medium with keratin.

For EI estimation, the diameter of the hydrolysis zone (d1) was divided by colony diameter (d2). Each experiment was carried out in triplicate, and the standard deviation was also calculated for the EI. casein is conventionally known as a simple substrate to digest, and therefore used in primary assessment of proteolytic activity in plating method [5]. However, for most applications, a high substrate specificity is essential. Furthermore, specific index of severity of proteolytic action (ISPA)was calculated for all isolates according to the formula:

ISPA =average EI value for fibrous proteinsaverage EI value for globular proteins

Average EI for fibrin, keratin, hydrolyzed collagen and fibrinogen were used for each fungal strain as EI value for fibrous protein and average EI for casein and hemoglobin were used as EI value for globular proteins.

Screening for acid and alkaline metabolites production

For assessment of produced proteases’ diversity, we also investigated acid and alkali production of the strains. For the former, plating on creatine-sucrose agar (CREA-agar) was used. The composition of the media was as follows, % (w/v): creatine - 0.3, sucrose - 3.0, KCl - 0.05, MgSO4 - 0.025, FeSO4 - 0.05, K2HPO4 - 0.013, bromocresol purple - 0.005, agar - 1.5. After 7 days of incubation at 28 ± 0.2℃ in the darkness, changing of the color was registered. Acid production resulted in changing the color of the media from purple to yellow. On the other hand, a phenol red agar (PRA) was used for monitoring both acid and alkali production. Color production depends on the pH value - in acidic conditions color turns to yellow, in alkaline conditions to red. The composition of the media was as follows, % (w/v): peptone - 1.0, glucose - 1.0, KCl - 0.05, phenol red - 0.02, agar - 2.0, pH 5.6. Cultivation conditions and results observation were the same as for CREA-agar, with the only difference that alkali production changed color of the media from reddish to fuchsia.

Screening for extracellular pigment production

To determine the extracellular pigment production, tested strains were inoculated by one colony on Czapek- Dox minimal agar and Sabouraud agar [6]. The results were observed after 7 days of incubation at 28 ± 0.2℃ in the darkness as colored zones around the colonies.

Data processing and visualization

The statistical data were processed with MS Excel. The average value of the three enzymatic indices for each medium with protein was calculated and table cells were coloured in according to the values in them.

Matplotlib for Python was used to prepare graphs and diagrams. The ISPA values were shifted along the ordinate axis and a bar chart was constructed.

Venn diagram was constructed with online-tool The total proteolytic activity was considered significant if the enzymatic index on the medium with casein was greater than 1. The remaining signs were evaluated as presence/absence. The Venn diagram shows the areas corresponding to a certain feature. The intersection of areas indicates the presence of several signs at once. The number on a particular sector indicates the number of strains that have exclusively these properties. For example, 7 letters at the intersection of the green and red regions means that 7 species break down casein and release metabolites to the CREA medium. The absence of numbers in some areas means that no studied species is characterized exclusively by these properties.

Screening for protease production

In order to reach the goal, i.e. to characterize proteolytic profile, acid and alkali metabolites production, as well as extracellular pigmentation, appropriate assays were used (Fig. 2). Proteolytic potential data is summarized in Fig. 3, showing both diversity of the produced proteolytic enzymes and the highest producers of each tested proteases. Concerning diversity of the produced enzymes among all tested Aspergillus strains, A. melleus could be denoted as the species showing the most diverse proteolytic activity, i.e. hydrolyzing six out of six tested protein substrates. A. aureolatus, A. calidoustus, A. creber, A. domesticus, A. jensenii, A. penicilloides and A. protuberus hydrolyzed five protein substrtates. Among them, A penicilloides’ proteases are not active against fibrin, A. domesticus and A. jensenii could not hydrolyze collagen and hemoglobin, respectively. Proteases of A. athecius, A. glaucus, A. caespitosus, A. ruber, A. tabacinus, A. tamarii and A. tennesseensis are active against four proteins. A. amstelodami, A europaeus, A. pseudoglaucus, A. tubingensis and A. wentii secrete proteases hydrolyzing three proteins. For A. proliferans’ proteases activity was demonstrated against two substrates, while A. phoenicis, interestingly, was not proteolytically active at all. Further on, concerning intensity of proteolytic activity on different substrates, one could note that A. domesticus showed the highest potential to hydrolyze casein (EI value 4), fibrinogen (EI 3) and hemoglobin (EI 2.5), and almost the highest potential in the case of keratin (EI 1.7, in respect to the highest 1.8 for A. ruber). The last substrate (keratin) was mostly degraded by A. penicilloides and A. ruber, while the highest potential to hydrolyze collagen (gelatin) was observed in the case of A. protuberus (EI 3.7). In addition, it is worth noting hydrolysis potential of casein for A caespitosus, A. ruber and A. protuberus (EI ≥ 3), of collagen for A. jensenii, A. europeus and A. creber (EI ≥ 2.7), of fibrinogen and hemoglobin for A. protuberus (EI 2.6 and 2.0, respectively).

Figure 2.Proteases, acid and alkali metabolites and extracellular pigment production assays. (A) CA inoculated with A. eupopaeus, transparent zone represents caseinolytic activity. (B) HCA inoculated with A. calidoustus, transparent zone representing collagenolytic activity. (C) FgA inoculated with A. creber, transparent zone represents fibrinogenolytic activity. (D) CREA inoculated with A. tamarii, yellow zone represents ability to produce acid metabolites. (E) PRA inoculated with A. creber, red zone represents ability to produce alkali metabolites. (F) PRA inoculated with A. proliferans, yellow zone represents ability to produce acid metabolites.

Figure 3.Proteolytic activity (EI) of selected Aspergillus strains. CA medium with casein, HCA medium with hydrolyzed collagen, FA medium with fibrin, FgA medium with fibrinogen, HA medium with hemoglobin, KA medium with keratin.

Total proteolytic activity (caseinolytic activity) was observed for 20 Aspergillus isolates (90.91%), collagenolytic activity - for 18 strains (81.81%), fibrinolytic activity - for 16 strains (72.73%), fibrinogenolytic activity - for 15 strains (68.18%), hemoglobinolytic activity - for 11 strains (50.0%), keratinolytic activity - for 6 strains (27.27%).

Screening for acid and alkaline metabolites production

Acid/alkaline production assay demonstrated acid secretion for thirteen strains (59.09%), alkaline secretion was observed for six strains (27.27%). Concerning acidity production, widest yellow zones on CREA-agar were recorded for A. penicilloides, A. tamarii and A. tubingensis. Widest yellow zones on PRA were recorded for A. proliferans and A. glaucus. On the other hand, red color on PRA, indicating production of alkalis, was observed in the case of A. calidoustus, A. athecius and A. protuberus strains.

Screening for extracellular pigment production

Extracellular pigment production was observed for six strains (27.27%) on Czapek-Dox minimal agar and for four strains (18.18%) on Sabouraud Agar.

Data processing and visualization

In further work we have constructed a Venn diagram (Fig. 4), in order to simultaneously analyze strains’ potential in different assays, i.e. total proteolytic activity, production of alkaline and acidic metabolites, as well as of extracellular pigments. Obtained results indicated that the most tested strains (7): A. amstelodami, A. europaeus, A. glaucus, A. penicilloides, A. ruber, A. tubingensis and A. wentii showed overlapped proteolytic and acid production activities. They are followed by 4 strains (A. jensenii, A. pseudoglaucus, A. tabacinus and A. tennesseensis) showing three tested features - proteolytic activity, acidic metabolites production and pigmentation on Czapek-Dox minimal agar. Three strains (A. calidoustus, A. domesticus and A. protuberus) are featured with potential to hydrolyze casein and to produce alkalis, while two of them (A. athecius and A. aureolatus) share simultaneous potential of proteolysis, production of alkalis and production of pigments on Sabouraud agar.

Figure 4.Five-way Venn diagram showing proteolytic activity, metabolites and pigment production of selected Aspergillus strains. CzM Czapek-Dox minimal agar, SM Sabouraud Agar, CA agar medium with casein, CREA creatine-sucrose agar, PRA Phenol-Red Agar. Letters indicate different strains of micromycetes from Table 1.

On the Venn diagram, areas corresponding to a certain feature are indicated. The intersection of areas means the presence of several characteristics at once. The letter on a particular sector indicates the strains that have exclusively these properties. For example, the letter f at the intersection of the blue, brown and red areas means that Aspergillus creber break down casein, produce pigment on Czapek-Dox minimal agar and release metabolites onto the PRA. The absence of letters in some areas means that 0 species are characterized exclusively by these properties.

Taking into account the global tendency to replace the noxious chemicals from different branches of industry with the products that could be denoted as environmentally friendly, the relevance of different enzymes' applications in biotechnology is enormous [7]. Among different used enzymes, proteases are extensively applied ones. It is well known they could be used in several biotechnological and industrial sectors, such as food industry, pharmaceutical and chemical (detergent) industries [8]. Further on, they have found applications in medicine and molecular biology, i.e. diagnosis and therapy, digestion of unwanted proteins during nucleic acid purification, recombinant antibody fragment preparation, peptide sequencing and proteolytic digestion in proteomics, etc [9]. Bearing in mind these facts, searching for a new microbial sources of potentially applicative proteolytic enzymes seems to be important.

This study characterized the proteolytic potential of selected strains of filamentous fungi of the genus Aspergillus. Aspergillus micromycetes have long been known for high activity of their extracellular proteases against a variety of proteins (collagen, fibrin, fibrinogen, keratin, hemoglobin etc.) [1012]. Extracellular metabolites play an important role in the vital activity of micromycetes, participating in attachment to the surface and nutrition. Proteolytic activity has already been defined by our investigation group for some of the selected Aspergillus strains [1, 2, 6, 12], but this study provides more systematic and complete information about proteolytic potential of tested Aspergillus isolates.

All selected strains except A. phoenicis showed some proteolytic activity. Among them, only A. proliferans was not capable of degrading casein, a feature indicating general proteolytic activity. Minor caseinolytic activity (EICA ≤ 1.2) was observed for A. glaucus, A. athecius, A.wentii and A. tubingensis, while all the rest isolates showed high potential (the EICA range 1.5-4). At the same time A. proliferans, although not showing caseinolytic potential, was characterized with high collagenolytic activity and weak fibrinolytic activity that has not been seen previously in other studies. Hemoglobinolytic activity was noticed and confirmed in another study [13], while in this investigation was shown for 15 tested strains. Hemoglobinolytic enzymes may be used in biomedicine as well as fibrinolytic and fibrinohenolytic enzymes [14] Fibrinolytic and fibrinogenolytic activity, which has not been described by other investigators, has been determined for 16 and 15 strains, respectively, in this study. Although fibrinolytic activity was generally low (EIFA ≤ 1.4), the fact that it has been detected for the first time is notable. Concerning collagenolytic and hemoglobinolytic activities, they have been observed for 18 and 11 strains, respectively. The widest yellow zones on CREA were observed for A. tubingensis; this ability to synthesize acids has been confirmed by other research [15]. Finally, the keratinolytic activity was noticed for 6 Aspergillus species (27.27%). Keratinases may be used in the leather industry, biodegradation, cosmeceuticals, etc [16, 17].

Taken together all results concerning proteolytic activity, the highest proteolytic potential was observed for A. melleus due to its ability to hydrolyze all tested substrates (the value of all enzymatic indexes exceeds 1.25). The ability to dissolve fibrin clots [18], fibrinogen [19], collagen [20], hemoglobin [21] and keratin [22] was defined and confirmed. qAlso, for A. melleus the ability to secrete acids and alkaline metabolites was not found.

Further on, concerning the ability to produce acid/ alkali metabolites, it is worth to note that the widest yellow zones on CREA were observed for A. tubingensis; this ability to synthesize acids has been confirmed by other research [15]. One of the most important applications of fungal pigments is food colorization, fabric dyeing. It may be used in the cosmetic industry for sunscreens [23]. The colorant from A. oxalicum is commonly used for dyeing of various products [24].

Due to the fact that in nature micromycetes meet a variety of proteins: globular and fibrous, it is interesting to determine the specificity of the secreted proteases to fibrous or globular proteins. In this article, we suggest a new characteristic to assess specificity of secreted proteases - index of severity of proteolytic action (ISPA). It is desired to evaluate the selectivity of secreted proteases without advanced biochemical research of the enzyme. ISPA value determines mycozymes specificity to fibrous or globular proteins. ISPA values data is summarized in Fig. 5. If ISPA≤1, mycozymes hydrolyze globular proteins better; if ISPA=1, extracellular proteases are nonspecified and hydrolyzed both globular and fibrous proteins equally effectively; if ISPA≥1, mycozymes hydrolyze fibrous proteins better.

Figure 5.ISPA values of selected Aspergillus strains.

The authors have no financial conflicts of interest to declare.

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