Journal Archive

Johnson Matthey Technol. Rev., 2023, 67, (2), 159
doi: 10.1595/205651322X16482034395036

Screening of Bioactive Compounds for Biomedical and Industrial Uses from Actinobacteria Isolated from the Parsık Cave, Turkey

Identifying novel compounds from extreme environments

  • Nahdhoit Ahamada Rachid
  • Institute of Graduate Studies in Sciences, Istanbul University, Balabanaga Mah. Sehzadebasi Cd., 34134 Vezneciler, Fatih-Istanbul, Turkey*
  • Nihal Doğruöz Güngör
  • Department of Biology, Faculty of Science, Istanbul University, 34134 Vezneciler, Istanbul, Turkey
  • *Email:

Received 3rd January 2022; Revised 7th March 2022; Accepted 24th March 2022; Online 25th March 2022

Article Synopsis

The need to avoid health issues and pollution of the environment from the use of chemicals and synthetic materials inspires scientists to search for new biological compounds beneficial to human beings. Caves, being extreme environments, might be potential sources of these compounds. Actinobacteria, one of the main groups that colonise these environments, are known to generate natural bioactive compounds. To investigate the potential uses of Parsık Cave Actinobacteria, identification of this group of isolates and the investigation of their secreted biological compounds constituted the principal aim of the present study. The identification was achieved by sequencing 16S rRNA genes of 41 selected bacteria of which 28 species were identified as Actinobacteria. Microbacterium (21%) and Pseudarthrobacter (14%) were the most identified Actinobacteria genera. Antimicrobial effects of the isolates P1 and P16 were observed against standard microorganisms like Candida albicans. The gas chromatography-mass spectrometry (GC-MS) analysis of their broth showed compounds with known antimicrobial, antioxidant or anticancer properties as well as unknown compounds. Polyketide synthase (PKS) and non-ribosomal peptide synthases (NRPS) respectively were amplified in 32.1% and 53.5% of the identified Actinobacteria while 25% were found to have both NRPS and PKS amplified. Amylase, gelatinase, cellulase, deoxyribonuclease (DNase), urease and casein hydrolysing activities were observed in the identified Actinobacteria. These results show that Actinobacteria from Parsık Cave might be good sources of industrial and biotechnological compounds. Furthermore, discovery of new bioactive compounds from these bacteria is promising due to the many unknown compounds observed in the GC-MS analysis and the high percentage of NRPS and PKS gene amplification.

1. Introduction

Identification of novel bioactive compounds is a big challenge in biotechnology and industry. The problems of drug resistance may be improved by innovation and the discovery of new antibiotic, anticancer, antiviral or other biomedical products. Furthermore, the costs, health and environmental issues associated with using chemical catalysts may be mitigated by use of biocatalysts (enzymes). Microorganisms are thought to be the main candidates to produce such compounds. New or unculturable species are highly promising for the possibility of obtaining new compounds; unexplored environments which remain generally located far away from human activities are a likely source of such novel microorganisms (13).

Caves constitute one of these interesting environments. They can be defined as natural underground openings large enough for human access. These dark chambers are considered extreme ecosystems usually characterised by a constant low temperature, high relative humidity, low oxygen and poor nutrient availability (46). Despite their extreme conditions, microorganisms are thought to play an important role in the formation of caves through the formation of cave speleothems like ‘moonmilk’ and accumulation of mineral oxides (7). In fact, caves are found to be colonised by extremophile organisms such as psychrotolerant, psychrophiles, halophiles and basophiles (8, 9).

Cave microbiology consists of studying cave microbial diversity, defining the biogenic role of this microbiota in different niches of the cave and exploring their potential uses in industrial and biotechnological fields. Both cultural-dependent and culture-independent methods are used in cave microbiological studies (7, 10, 11). Through these two strategies, bacteria appear as the most identified cave microorganisms (10, 11). Actinomycetes are high guanine and cytosine content, gram-positive bacteria which constitute a large group of cave bacteria. They are mainly isolated from moonmilk deposits and other surface formations. They also inhabit cave sediments and can colonise cave waters albeit in low proportions (1013). These filamentous bacteria are well recognised for their resistance under extreme ecological conditions due to their metabolic versatility (14).

In general, Actinobacteria are well known for their secretion of primary and secondary metabolites which are valuable bioactive compounds with biomedical (antibiotic, anticancer, antioxidant) and biotechnological (enzymes, siderophores, bioremediation, self-healing concretes) properties (15, 16). It is reported that approximatively 66% of antibacterial compounds in the world are from bacteria belonging to this phylum. 55% of those compounds are from species of Streptomyces while the remaining 11% are from other Actinobacteria (15, 17). A wide range of industrial enzymes such as amylases, proteases and cellulases have been positively screened in Actinobacteria including Nocardiopsis sp., Streptomyces pactum and Thermomonospora sp. (18).

Microbes inhabiting caves have unique metabolisms to cope with specific conditions like poor nutrient availability. The oligotrophy in caves can induce nutrient competition between bacteria and hence, development of antimicrobial compounds in these bacteria (11). Caves are also mineral-rich environments. During biomineralisation, actinomycetes species secrete extracellular enzymes that are active under extreme conditions: for example, ureolytic activity (urease) in Streptomyces species has been reported during microbial-induced calcium precipitation (19). Some bioactive compounds can be produced in small amounts and specific inducers (physical or chemical) are sometimes required for their synthesis. For this reason, screening microorganisms for microbial bioactive compounds under laboratory conditions may sometimes be complicated (1, 20).

In this context, screening for the presence of genes encoding the compounds is also required. Furthermore, the characterisation and identification of these compounds requires additional tests other than phenotypic and genotypic analyses. PKS and NRPS are groups of enzymes coded by genes to produce important secondary metabolites (21, 22). The presence of these genes in microorganisms is thought to be highly related to their biosynthetic activities. Therefore, targeting these genes is an important way to screen the bioactive potential of these bacteria.

In our study, we focused on the screening of bioactive compounds (enzymes and antimicrobial compounds) of potential Actinobacteria strains from our previous study (11). Here, a group of industrially interesting enzymes have been phenotypically screened and the antimicrobial potential of the isolates has been studied through both phenotypic and genotypic analyses. Some of the bioactive compounds have been identified through GC-MS analysis.

2. Material and Methods

2.1 Molecular Identification of Species

A total of 41 isolates were selected according to the medium used for their isolation including actinomycetes-specific media: actinomycetes isolation agar, starch casein agar, international Streptomyces project-2 medium and Reasoner’s 2A agar. DNA of these isolates was isolated using the InvitrogenTM PureLinkTM Genomic DNA Mini Kit K182001 (InvitrogenTM, USA) according to the manufacturer’s procedure. To amplify the 16s rRNA gene, the polymerase chain reaction (PCR) was run using universal primers 27F (5’-AGA GTT TGA TCC TGG CTC AG-3’) and 1492R (5’- ACG GCT ACC TTG TTA CGA CTT-3’) under these conditions: initial denaturation at 94°C for 30 s, followed by 30 cycles of 94°C for 30 s, 58°C for 30 s, and 72°C for 60 s, then a final extension step was performed at 72°C for 10 min. The resulting PCR products were run on 1% agarose gel by electrophoresis before sequencing using the Sanger DNA sequencing method.

2.2 Screening for Non-Ribosomal Peptide Synthases and Polyketide Synthase Genes in Actinobacteria from Parsık Cave

Primers coding the NRPS and PKS gene regions of bacteria were used to screen the enzyme-encoding gene clusters in the identified Actinobacteria. They consist of A7R (5′-SASGTCVCCSGTSCGGTAS-3′) / AF3 (5′-GCSTACSYSATSTACACSTCSGG-3′) for NRPS gene cluster and DegKS2R (5’-GTICCIGTICCRTGISCYTCIAC-3’) / DegKS2F (5’-GCIATGGAYCCICARCARMGIVT-3’) for PKS gene cluster (23). PCR was run following the methods proposed by Amos et al. (23) with some modification. For the A7R/AF3 primers, PCR was run under the following conditions: primary denaturation at 95°C for 5 min followed by 35 cycles (denaturation at 94°C for 35 s, annealing at 55°C for 40 s and extension at 72°C for 1 min) and final elongation at 72°C for 8 min. PCR for the PKS region was run under the following conditions: primary denaturation at 94°C for 4 min followed by 35 cycles of (denaturation at 94°C for 35 s, annealing at 55.6°C for 40 s and primary extension at 72°C for 75 s) and elongation at 72°C for 8 min. The PCR products were run in a 1.2% gel electrophoresis to visualise the bands.

2.3 Antimicrobial Production Potentiality of the Isolates

Identified Actinobacteria were tested against selected standard microorganisms constituted of pathogens or opportunistic pathogens of Gram-negative and Gram-positive bacteria and one fungi strain: Escherichia coli (ATCC® 8739TM), methicillin-resistant Staphylococcus aureus (MRSA) (ATCC® 33591TM), Pseudomonas aeruginosa (ATCC® 9027TM), Staphylococcus aureus (ATCC® 6538TM), Staphylococcus epidermidis (ATCC® 12228TM) and C. albicans (ATCC® 10231TM). Fresh culture of each isolate was used to prepare 0.5 McFarland and 100 μl of each suspension was spread onto 1/2 tryptic soy agar. The incubation was at 30°C for 48 h. 0.5 McFarland of each standard microorganism was also prepared and 100 μl of each suspension was spread to tryptic soy agar medium and incubated at room temperature for 3–5 min. After that, discs of the isolates (6 mm) were cut off from the 48 h cultures and inversely placed onto the plates containing standard microorganisms. After 24 h at 30°C, every standard Petri plate was observed for the inhibition zone around the isolate’s discs. Isolates with phenotypical antimicrobial activities were selected for characterisation of bioactive compounds.

2.4 Characterisation of Bioactive Compounds

Fresh cultures were inoculated on tryptic soy broth for 24 h. The broth was chromatographically separated using an Agilent 1260 Infinity II LC System (Agilent Technologies Inc, USA) instrument and Agilent Poroshell 120 EC-C18 Threaded Column (3.0 mm × 150 mm, 2.7 μm particle size). A gradient elution of 5 mM ammonium formate in water (A) and methanol (B) was used to construct the mobile phase system. This was as follows: 0–0.5 min, 10% B; 0.5–5 min, 70% B; 5–7 min, 95% B; 7–10 min, 95% B; 10–15 min, 10% B. Sample injection was 2 μl with a flow rate of 0.5 ml min–1 and column oven maintained at 25°C.

Mass spectroscopy was performed using an Agilent 6550 iFunnel equipped with the high‐resolution accurate mass quadrupole time of flight/mass spectroscopy sheath gas flow (nitrogen 12 l min–1) and the selected scanning range m/z was between 50 and 1800. Samples were screened using Agilent MassHunter METLIN Metabolite Personal Compound Database and Library (Metlin_AM_PCDL.cdb).

2.5 Screening of Enzymatic Activities of Parsık Cave Actinobacteria

The isolates were screened for seven enzymatic activities: amylase, lipolytic activity (Lip.A), cellulase, casein hydrolase (Cas.Hydrolase), DNase, urease, proteinase and gelatinase. Except for DNase and gelatinase activities, a fresh culture of each isolate was inoculated in media composed of minimal salt agar medium and enzyme specific substrates. 1000 ml of minimal salt agar medium was composed of 0.1 g NaCl, 1 g KH2PO4, 0.1 g MgSO4, 5 g (NH4)2SO4 and 15 g agar. For DNase and gelatinase activities, respectively, DNase test agar and gelatine were directly used. During the incubation period (3–10 days) at 30°C, the growth of isolates was checked daily and clear zones, growth or change in the colour were checked for positive or negative activities (9).

3. Results and Discussion

3.1 Identification of Isolates

The identified species in our study are presented in Table I. From the 41 selected isolates, 28 have been identified as Actinobacteria. The remaining isolates are constituted of Proteobacteria, Firmicutes and three others which were not identified in the National Center for Biotechnology Information (NCBI, Table I). As previously mentioned, the bacterial diversity of sediment and surface samples from different karstic caves has been reported with a high rate of Actinobacteria (1012). Some Actinobacteria strains like Streptomyces species are known for their role in the formation of caves and cave speleothems (14, 17). This can be achieved through their metabolic reactions in the presence of some chemicals in the cave ecosystems (14). Genera like Nocardia and Rhodococcus identified in our study have been reported with potential for degradation of hazardous chemicals and organic matter (24, 25). They may play an important role in the bioremediation of natural ecosystems and polluted environments.

Table I

Enzymatic Activities of Parsık Cave Actinobacteria

Isolates Name Similarity, % Amylase Lip.A Gelatinase Urease Cas.Hydrolase DNAase Cellulase
P1 Streptomyces exfoliatus 100 (+) (+) (–) (+) (+) (–) (+)
P2 Agromyces cerinus subsp. Cerinus 99.00 (–) (–) (–) (–) (–) (–) 0
P3 Nocardioides caeni 93.41 (–) (–) (+) (+) (–) (–) 0
P4 Propionicimonas paludicola 91.89 (–) (–) (–) (+) (–) (–) (–)
P5 Microbacterium paraoxydans 99.00 (–) (–) (–) (+) (+) (–) 0
P6 Arthrobacter humicola 96.08 (+) (–) (–) (+) (+) (+) (–)
P7 Nocardia globerula 97.99 (–) (–) (–) (+) (–) (–) (–)
P8 Microbacterium maritypicum 98.15 (–) (–) (–) (–) (–) (–) 0
P9 Microbacterium esteraromaticum 91.13 (–) (–) (–) (+) (+) (–) 0
P10 Pseudarthrobacter oxydans 98.02 (+) (–) (–) (+) (+) (–) 0
P11 Pseudarthrobacter oxydans 93.54 (–) (–) (–) (+) (+) (–) (–)
P12 Microbacterium paraoxydans 99.21 (–) (–) (–) (–) (–) (–) 0
P13 Streptomyces spororaveus 97.51 (+) (–) (–) (–) (–) (–) (–)
P14 Arthrobacter halodurans 91.00 (–) (–) (–) (+) (+) (–) 0
P15 Micrococcus antarcticus 96.09 (–) (–) (–) (–) (+) (+) (–)
P16 Micrococcus sp. TM4_2 98.15 (–) (–) (–) (+) (–) (–) 0
P17 ND ND (–) (–) (–) (+) (+) (–) (–)
P18 ND ND (–) (–) (–) (–) (–) (–) 0
P19 Pseudarthrobacter polychromogenes 94.48 (–) (–) (–) (–) (+) (–) 0
P20 Rhodococcus degradans 88.06 (–) (–) (–) (+) (–) (–) (–)
P21 Kocuria rosea 86.28 (+) (–) (–) (+) (–) (–) (+)
P22 Nocardia coeliaca 96.38 (–) (–) (+) (+) (+) (–) (–)
P23 ND ND (–) (–) (–) (–) (+) (+) 0
P24 Microbacterium paraoxydans 97.11 (+) (–) (–) (–) (+) (–) 0
P25 Oerskovia turbata NBRC 15015 93.79 (+) (+) (–) (–) (+) (–) (–)
P26 Kocuria rosea 99.10 (+) (+) (–) (+) (+) (–) (+)
P27 Kocuria rosea 99.27 (+) (–) (–) (+) (+) (–) (–)
P28 Microterricola gilva 99.31 (–) (–) (–) (–) (–) (–) 0
P29 Pseudarthrobacter siccitolerans 94.76 (–) (–) (–) (–) (–) (–) (–)
P30 Streptomyces nojiriensis 96.29 (–) (–) (–) (–) (–) (–) (–)
P31 Microbacterium paraoxydans 97.30 (–) (–) (–) (+) (–) (–) (–)
P32 Exiguobacterium undae 98.69 (–) (–) (–) (–) (+) (–) 0
P33 Paenibacillus sp. CT–7 98.62 (+) (–) (–) (–) (–) (–) 0
P34 Pseudomonas putida 98.08 (–) (–) (–) (+) (–) (–) (–)
P35 Bacillus aryabhattai B8W22 99.23 (+) (–) (–) (+) (+) (–) (–)
P36 Acinetobacter guillouiae 96.94 (–) (–) (–) (+) (–) (–) (–)
P37 Stenotrophomonas rhizophila 99.00 (–) (–) (–) (+) (+) (–) (–)
P38 Pseudomonas psychrophila 99.41 (+) (–) (–) (+) (–) (–) 0
P39 Pseudomonas sp. 99.80 (–) (–) (–) (+) (+) (+) 0
P40 Citrobacter freundii 99.00 (–) (+) (–) (+) (–) (–) 0
P41 Pseudomonas azotoformans 96.92 (–) (–) (–) (+) (+) (–) 0

0 = No growth; (+) = positive results; (–) = negative results; ND = non-determined

3.2 Screening of Antimicrobial Production Potential of the Isolates

Our study aimed to determine the bioactive potentials of Actinobacteria isolated from Parsık Cave. 53% of the isolates showed bands for NRPS gene after running their PCR products on an agarose gel electrophoresis. 32% of the identified Actinobacteria showed bands for PKS gene after running their PCR products on an agarose gel electrophoresis. Isolate P1 showed a band for both regions while neither region was amplified in P16. These genes encode enzyme groups that play an important role in the synthesis of different bioactive compounds including some with antimicrobial, antioxidant and anticancer activities (2628). These observations bring us back to the potential uses of Actinobacteria isolated from the Parsık Cave in the biotechnological and pharmaceutical industries.

However, further tests are recommended to identify the different biological activities of these isolates. One of these tests is the antimicrobial production assay. This test was done by testing the phenotypical response of the isolates against some standard bacteria and one Candida species. Only two isolates showed antimicrobial activity. These were the P1 and P16 isolates. The isolate P1, identified as Streptomyces exfoliatus, showed antimicrobial activity against the fungal strain C. albicans. Antimicrobial potential in Streptomyces species has been observed previously in different studies with Streptomyces isolated from different environments (2932). Sharma et al. showed through in vitro analyses the antifungal potential of Streptomyces exfoliates MT9 against wood rotting fungi (33). Inhibition zones were observed around isolate P16, identified as Micrococcus sp. TM4_2, when tested against S. aureus and S. epidermidis. The growth inhibition of the pathogen S. aureus and the opportunistic pathogen S. epidermidis by isolate P16 shows the antibacterial potential of this isolate and promises biomedical uses for this isolate and its extracts.

Results observed through the genotypic test, contrary to the antimicrobial tests, showed high potential bioactive compound production in our isolates. This great difference between the phenotypical and genotypical results may be explained through different hypotheses such as the laboratory conditions (culture medium or temperature) which may not be favourable to phenotypical expression in the other isolates. Besides that, these isolates may show antimicrobial activities against other standard microorganisms which were not used in our study. The presence of biological activities other than antimicrobial activity like anticancer or antioxidant activity may also explain this observation.

3.3 Gas Chromatography-Mass Spectrometry Analysis of P1 and P16

The two gene clusters observed in the isolate P1 brings us back to the high potential of bioactive compounds that can be secreted by Actinobacteria species. In addition, the antimicrobial activity phenotypically observed in the isolate P16 shows the capacity for biological compound secretion in this isolate. The GC-MS analysis of P1 and P16 isolates revealed multiple products of which some were unknown. Some of the known products revealed in this study are shown in Table II and Table III. These results show a high rate of amino acids as well as different secondary metabolites. Among the bioactive compounds observed in the isolate P1 (Table II), compounds with antimicrobial functions like azaserine, adefovir dipivoxil, valclavam and leucomycin A7/A4 have been identified. Leucomycin A7/A4 are macrolide antibiotics biosynthesised by PKS enzymes (26). They are previously known to be secreted by Streptomyces species (26, 27). The insecticidal wilfordine and the anticancer agent pectenotoxin-2 have been also revealed in our study. Neomycin A, identified in the isolate P16, has been reported with high antibacterial activity against tuberculosis agent (28). The antifungal peptide coccinin has been also identified in the isolate P16 and was reported in previous studies with leukaemia cell proliferation inhibition activity and reduced activity of human immunodeficiency virus 1 (HIV-1) reverse transcriptase (34).

GC-MS analysis of the compounds released by our isolates also revealed different amino acids. In 2017, the amino acid market worldwide was reported at US$8 billion and was projected to reach US$24.4 billion by 2020 (35). This shows the high demand for these compounds in different biotechnological and industrial fields. Some of these compounds are reported to be synthesised by a diversity of microorganisms. For example, Serratia marcescens produces L-threonine (35). Glutamate is reported to be synthesised in bacteria such as Micrococcus and Microbacterium (35). Among the amino acids identified in our study, we can enumerate L-tryptophan, L-lysine, L-phenylalanine, L-arginine and DL-pipecolic acid (Table II and Table III). Lysine, tryptophan and phenylalanine are used as food and feed supplements and in the production of pharmaceuticals and cosmetics (35). L-arginine is known for its activity in secretion of hormones like insulin, prolactin, growth hormones and in muscle mass promotion and wound healing enhancement (35). It is used as a food supplement and in pharmaceuticals and cosmetics (35). In our study, some unknown compounds were recorded from the GC-MS of the P1 and P16 isolates. This may suggest the potential for new biological compounds from Parsık Cave Actinobacteria.

Table II

Gas Chromatography and Mass Spectroscopy of the Isolate P1

Compounds Retention time, min Charge, m/z Score Formula
Valclavam 3.983 330.1661 98.83 C14H23N3O6
Azaserine 1.092 191.0768 84.80 C5H7N3O4
Adefovir Dipivoxil 3.802 519.2313 95.44 C20H32N5O8P
Apramycin 4.728 562.2693 83.59 C21H41N5O11
Maculosin 5.405 261.1235 96.12 C14H16N2O3
Posaconazole 6.298 718.3628 91.28 C37H42F2N8O4
Glycobismine A 9.844 620.2767 84.92 C37H34N2O6
Bouillonamide B 20.356 645.3439 89.11 C32H45N5O6S
Stigmatellin Y 24.354 507.2711 97.17 C29H40O6
Betaine 1.205 118.0868 96.73 C5H12NO2
Aminocaproic acid 1.724 132.1020 87.32 C6H13NO2
Salivaricin A 1.770 706.2533 63.10 C28H48O12Si4
Aconitine 2.786 646.3237 91.49 C34H47NO11
Benomyl 3.203 308.1713 94.78 C14H18N4O3
Pectenotoxin 2 4.265 881.4623 83.96 C47H70O14
Wilfordine 4.446 906.2810 85.75 C43H49NO19
Leucomycin A7 5.801 780.4154 80.22 C38H63NO14
Leucomycin A4 7.246 814.4556 78.50 C41H67NO15
Lobeline 7.370 360.1932 97.34 C22H27NO2
Jamaicamide C 8.110 491.2690 40.77 C27H39ClN2O4
Thapsigargin 8.658 668.3658 91.24 C34H50O12
Leukotriene C4-d5 9.290 631.3447 73.40 C30H42D5N3O9S
Trilobolide 9.301 523.2546 97.08 C27H38O10
Malyngamide C 10.182 473.2768 65.40 C24H38ClNO5
Avermectin A2a aglycone 10.814 639.3510 90.08 C35H52O9
Daphnoline 12.124 598.2905 92.37 C35H36N2O6
Melleolide K 12.565 452.1825 59.39 C23H27ClO6
Spinetoram 12.667 770.4805 97.90 C42H69NO10
Apigenin 14.078 271.0601 92.55 C15H10O5
Spectinomycin 18.154 333.1659 47.34 C14H24N2O7
Fumitremorgin C 19.464 397.2238 47.11 C22H25N3O3
L-Arginine 1.160 175.1197 84.63 C6H14N4O2
DL-pipecolic acid 1.036 130.0863 98.87 C6H11NO2
D-Lysine 1.047 147.1128 97.62 C6H14N2O2
L-Phenylalanine 2.741 166.0868 92.21 C9H11NO2
L-Tryptophan 4.491 205.0980 97.13 C11H12N2O2
Valclavam 3.955 328.151 98.05 C14H23N3O6
Leonurine 6.044 370.161 99.63 C14H21N3O5
Table III

Gas Chromatography and Mass Spectroscopy of the Isolate P16

Compounds Retention time, min Charge, m/z Score Formula
Azaserine 1.089 191.0770 96.75 C5H7N3O4
Dioscorine 1.224 244.1300 97.40 C13H19NO2
Flutrimazole 1.891 364.1608 87.50 C22H16F2N2
Senkirkine 3.139 366.1897 42.28 C19H27NO6
Bestatin 3.765 326.2074 85.57 C16H24N2O4
Adefovir Dipivoxil 3.856 519.2316 96.62 C20H32N5O8P
Apramycin 5.160 562.2695 94.66 C21H41N5O11
Tigecycline 5.572 603.3146 98.00 C29H39N5O8
Maculosin 5.606 261.1237 85.47 C16H16N2O3
Diprotin B 5.730 328.2235 99.00 C16H29N3O4
Posaconazole 6.295 718.3636 92.09 C37H42F2N8O4
Coccinin 13.646 551.2465 97.38 C26H40O11
Mycinamicin VI 7.717 668.3985 92.27 C35H57NO11
Chromafenozide 7.695 417.2147 82.45 C24H30N2O3
Samandarone 9.129 326.2083 96.68 C19H29NO2
Amprenavir 9.502 523.2564 86.26 C25H35N3O6S
Glycobismine A 9.841 620.2764 85.91 C37H34N2O6
Lanceotoxin B 10.078 622.3245 91.62 C32H44O11
Vindesine 10.360 776.3983 98.01 C43H55N5O7
Torvoside G 10.823 609.3971 85.88 C34H56O9
Mycinamicin III 13.680 699.4441 93.42 C36H59NO11
Lanceotoxin A 15.204 621.2912 91.39 C32H44O12
Neomycin A 17.068 323.1940 76.74 C12H26N4O6
Dithiazanine 17.937 409.1636 47.06 C23H23N2S2
Myrsinone 19.846 312.2177 82.26 C17H26O4
Bouillonamide B 20.365 645.3458 84.72 C32H45N5O6S
Stigmatellin Y 26.497 507.2727 97.22 C29H40O6
Alpha-Chlorohydrin 35.700 111.0208 47.55 C3H7CLO2
L-Arginine 1.146 175.1190 99.60 C6H14N4O2
DL-pipecolic acid 1.033 130.0865 93.04 C6H11NO2
D-Lysine 1.044 191.0770 92.46 C6H14N2O2
L-Phenylalanine 2.738 166.0863 93.68 C9H11NO2
L-Tryptophan 4.488 205.0981 90.60 C11H12N2O2
Valclavam 3.972 328.151 80.19 C14H23N3O6
Leonurine 6.050 370.161 99.44 C14H21N3O5

3.4 Parsık Cave as a House of Actinobacteria with High Diversity of Enzymes

The enzymatic activities of microorganisms indicate which organic compounds are present in the studied ecosystem. In addition, studying these activities allows us to understand the potential use of these microorganisms for human or other applications. Enzymes can be used in environmental bioremediation and in other industries such as detergents, cosmetics, leather and textiles production. Different enzymatic activities from cave bacteria have been reported in different studies. Using enzymes secreted by psychrotolerant bacteria is thought to be economically valuable to save energy in large scale applications (9). Our isolates showed different enzymatic activities (Table I).

The physiological role of amylase enzyme, which is mainly used in the textile, food, fermentation and paper industries, is to hydrolyse starch molecules into dextrins and glucose molecules (36). It is secreted by different biological sources of which the most important is the Streptomyces species from Actinobacteria (36). 29% of our isolates showed amylase activity. Different strains of these isolates showed clear zones around their colonies after incubation in starch agar plates and staining with iodide. They include S. exfoliates, Arthrobacter humicola, Kocuria rosea and Oerskovia turbata NBRC 15015 (Table I).

Protease activity was also tested in our study. Proteases are a group of enzymes directed to hydrolyse the peptide bonds of protein molecules into amino acid chains or simple peptide molecules (37). They are synthesised from different sources including plants, animals and microorganisms. They are used in different industrial and biotechnological areas including waste treatment, pharmaceuticals, detergent production, photographic and food industries (37, 38). Microbial proteases are obtained both from fungi and bacterial sources. Most of the fungi proteases are acidic while the bacterial ones are mainly active in alkaline environments (39). Proteases from Actinobacteria are reported from different strains including Streptomyces and Nocardia isolated from different ecosystems (39).

In our study, protease activity was tested through gelatin and casein hydrolysing profiles of the isolates. 5% of our isolates showed gelatinase activity while 48% showed casein hydrolase activity (Table I). The species identified as Nocardia and Nocardioides showed gelatin hydrolysing activity while those identified as Streptomyces, Nocardia, Nocardioides, Microbacterium, K. Rosea and Arthrobacter showed protease activity by hydrolysing casein molecules. These isolates might be considered candidates for the previously mentioned areas as they can produce cold-tolerant and alkaline protease enzymes in oligotrophic areas.

Among the investigated enzymatic activities in this study is the ureolytic activity. It consists of the degradation of urea molecules to generate ammonia which provides an alkaline environment at the end of the reaction (40). The urease activity of cave microorganisms has been previously demonstrated from karstic cave isolates and they are often studied for their possible significant role in cave and speleothem formation (9, 19, 40). Cave bacteria that show urease activity mostly include strains of Proteobacteria, Firmicutes and Actinobacteria (4, 9, 19). Some Streptomyces species isolated from moonmilk of the Springtails’ Cave were reported with ureolytic activity through phenotypical and genotypical analyses (19). Micrococcus luteus from Magura Cave, Bulgaria, also showed pink colour after incubation on urea-based agar plates, related to the urease secretion ability of the bacteria (9). The precipitation of calcium by bacteria is thought to take place through different mechanisms which include the urease reaction (9). The isolation of urease positive bacteria and their in vitro calcium precipitation ability were shown in previous cave microbiological studies (4).

In our study, 65% of the selected isolates showed positive results for urease activity. They include species of Streptomyces, Arthrobacter, Propionicimonas, Microbacterium and Nocardioides. These bacteria may play a significant role in construction activities like self-healing concrete production or concrete bioremediation. In addition, they may be used to remove calcium ions from wastewater. The calcium precipitation ability of these isolates should be the subject of future studies for further industrial and biotechnological applications.

Three of the identified Actinobacteria (S. exfoliatus, O. turbata NBRC 15015 and K. rosea), which represent 9.7% of our isolates, showed positive lipolytic activity in the basal agar medium supplemented with 1% Tween 80 and CaCl2·2H2O (Table I). They formed precipitation zones around their colonies that indicate the complex formed by liberated fatty acids and calcium present in the media. Different Actinobacteria isolated from the Hampoeil Cave, Iran, were also reported with esterase activity which is one of lipolytic enzymes (41). In our previous study in Parsık Cave, only strains of Proteobacteria were identified with lipolytic activity through the VITEK® test (11). In the present study, species of the same genera show different results. This suggests the lipolytic activity of the bacteria isolated from the same cave (same and different points) may be related to the substrate. Furthermore, it may be necessary to study the activities from different isolation points in order to establish the different hydrolytic activities shown by the ecosystem isolates. Lipolytic enzymes are used in industrial fields such as detergent production (11, 41). Such lipolytic enzymes isolated from cold-tolerant bacteria might be preferred in detergent production to reduce energy use during washing.

Two of our identified Actinobacteria showed a clear zone around their colonies incubated on DNA agar plates for five days (Table I). This zone was formed after flooding 1 N hydrochloric acid on the agar plate. The clear zone is caused by the reaction of DNase secreted by the isolates on the DNA (depolymerisation) present in the medium. DNase enzymes are known for different functions depending on the microbial sources: they can cause disruption of biofilms or play a role in bacterial predation, inhibition of natural transformation or the recovery of nutrients such as carbon and phosphate (42, 43). DNase activity was previously reported from Actinobacteria species isolated from other cave ecosystems (9, 43).

Cellulases are hydrolytic enzymes that hydrolyse cellulose by hydrolysing the B-1,4 glycosidic bonds into sugars (44, 45). These enzymes are important in different applications such as the degradation of cellulosic waste, bioethanol production, textile polishing and the production of detergent, food and feed (44, 45). A diversity of bacteria including Actinobacteria isolated from different ecosystems are reported with cellulase secretion ability (44, 45). In our study, three of the identified Actinobacteria (P1, P21 and P26 isolates) showed positive results at the end of a cellulase enzyme test (Table I). Their culture on carboxymethylcellulose agar plates were stained with Congo red dye, showing a yellow hallo against a red background. Interestingly, most of our isolates did not grow on the carboxymethylcellulose agar plates. This may be explained by inappropriate incubation conditions for most of the isolates. Furthermore, the medium used was not favourable for most of these oligotrophic isolates which may cause eutrophication.

The isolate P1 which was identified as S. exfoliatus exhibited the greatest enzymatic potential (Table I). As previously shown, most actinobacterial species that exhibit high bioactive compound potential belong to the genus Streptomyces (46). A strain of S. exfoliates isolated from a soil environment in Saudi Arabia has been analysed with potential lipase and protease activities (47). In addition, in our study, both PKS and NRPS cluster genes were amplified in this isolate. This result suggests the high potential for bioactive compounds of this isolate. The isolate P1 should be further investigated as a potentially industrially useful Actinobacteria from Parsık Cave.

4. Conclusion

For the identification of Actinobacteria from Parsık Cave, 41 bacterial isolates were chosen based on their isolation media. The potential bioactive compound secretion of the identified Actinobacteria was investigated and antimicrobial effects against standard bacteria and one Candida species were phenotypically observed in the isolates P1 and P16. PKS and NRPS gene clusters were amplified in the majority of the identified Actinobacteria. GC-MS analysis were run to identify bioactive compounds produced by the P1 and P16 isolates. The identified compounds include compounds with antimicrobial, anticancer and antioxidant activities. In addition, amino acids and various unknown compounds were observed. Amylase, urease, lipase, cellulase, DNase, gelatinase and casein hydrolysing enzymes were observed in different strains of the selected isolates with great enzymatic activity exhibited by isolate P1. Our results show the potential for use of Parsık Cave isolates, especially the identified Actinobacteria, in diverse biotechnological and industrial applications. Unknown compounds observed in this study suggest the potential to isolate new bioactive compounds from these sources. In our future work, we plan to investigate novel enzymatic activities, identify biological compounds from the remaining isolates, extract and purify the already identified compounds as well as those that will be identified and propose new industrial and biotechnological products.


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The author thanks Istanbul University Scientific Project Unit, Turkey (BAP Project No: FHZ-2017-26457) for the financial support.

The Authors

Nahdhoit Ahamada Rachid is a biologist from Istanbul University, Turkey. In 2021 she obtained her MSc in Microbiology from the Department of Fundamental and Industrial Microbiology, focusing on human impacts on cave microbial diversity. Currently, she is a PhD student in the same department. Her research interests include cave microbiology, bioremediation, microbial ecology and biotechnology.

Nihal Doğruöz Güngör is a Professor in the Department of Fundamental and Industrial Microbiology at Istanbul University. She obtained her doctorate at Istanbul University in 2008, focusing on microbiological corrosion of copper. Her research interests include cave microbiology, antimicrobial activities of bacteria, microbial corrosion and biotechnology.

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