A new record of Actinobacteria isolated from soil in Jerusalem and their enzymatic potential

Introduction

Actinobacteria represent one of the most diverse groups of filamentous bacteria capable of surviving in a number of ecological niches due to their bioactive potential. They are representative of terrestrial microorganisms and usually are isolated from soils. Actinobacteria have gained special importance as the most potent source of antibiotics (Kandasamy et al., 2012) and other bioactive secondary metabolites (Solecka et al., 2012). Their metabolic potential offers a strong area of research. Accordingly, the role of actinomycetes in biotechnology and medicine is well known and these industries are always looking for novelty bioactive compounds. While most of the studies on actinobacteria have focused on antibiotic production, only few reports have focused on their enzymatic potential.

Actinobacteria are considered as a promising source of a wide range of enzymes. Some of them are produced on an industrial scale, but many other remained to be harnessed. The bacteria have the ability to degrade a wide range of hydrocarbons, pesticides, and aliphatic and aromatic compounds (Sambasiva Rao et al., 2012). They perform microbial transformations of organic compounds, a field of great commercial value. Members of many genera of actinobacteria have potential for use in the bioconversion of underutilized agricultural and urban wastes into high-value chemical products (Crawford, 1988). Some actinobacteria secrete enzymes responsible for the degradation of lignocelluloses in lignin, cellulose and hemicellulase, others may secrete enzymes that can only partially achieve this breakdown (Mason et al., 2001). Here the purpose of this preliminary study was to isolate and screen new actinobacterial isolates for their ability to degrade organic compounds via the secretion of enzymes like amylase, cellulase, protease, tyrosinase, lipase, catalase, and phosphatase. Due to the high disturbance level of biodeterioration occurred in the area of Al-Aqsa mosque in Jerusalem, Israel, the soil specimens from such place may contain novel actinobacterial colonies. Meanwhile, to the author’s knowledge, no previous studies concerned organisms involved in biodeterioration in this location.

Materials and methods

Results and discussion

A total of 35 actinobacteria isolates were obtained from the two different soils collected from Northern part of Al-Aqsa mosque in Jerusalem. From Site 1, only 17 isolates were recovered in which four genera, Actinomadura, Streptomyces, Elylrosporangium and Actinopolyspora, were represented (Table 1). Site 2 was represented by more diverse genera of 18 isolates, Streptosporangium, Actinomadura, Nocardiopsis, Nocardia, Elytrosporangium and Actinopolyspora (Table 1). Genus Actinomadura was represented with the highest frequency in both sites (52.95% and 33.33% for site 1 and site 2 respectively). Despite the fact that isolation methods reveal only a minor fraction of the real existing microbes (Groth et al., 1999) we could demonstrate a great diversity among actinobacteria in the studied sites. To our knowledge (Thaer et al., 2009), this is the first study reporting identification of actinomycetes from this terrestrial environment. Meanwhile, the genera obtained are the first to be recorded in Jerusalem or in the whole country.

The differences observed among the genera of actinobacteria identified in both sites studied, may be due to the different human activities, including construction in such area. These differences may also indicate that the methods proposed and employed for actinobacteria isolation may not the suitable for site 1, and that soil pretreatment should be sought to explore the other genera inhabiting the area. Machavariani and colleagues (2011) found that preliminary treatment of soil by chemical substances like adrenaline and heteroauxin exerts a positive influence on the germination of actinomycete spores and contributes to the natural product activity of the isolated strains. Moreover, they demonstrated that soil pretreatment with different chemical substances play a role for the most complete isolation of actinomycetes that inhabit certain soil.

When screening enzyme activities, 32 of the 35 actinobacteria isolate showed a good amylolytic activity (Figure 1A) and the other three isolates (site 2), Streptosporangium sp.2, Nocardia sp.2 and Elylrosporangium sp.4, had a moderate activity (Figure 1B). However, all isolates from site 1 had a good amylase activity (Table 1). For other enzymes, although actinomycetes are well known as potent degraders of cellulose, lignin, chitin and other complex polysaccharides (El-Fiky et al., 2003; McCarthy & Broda, 1984; Prasad et al., 2012; Wilson, 1992) none of our isolates were able to produce cellulase enzyme. Our results seem to be in contrast with previous studies, and this may be explained by the unsuitable culture conditions for cellulase production such as optimal pH as reported by (Rathnan & Ambili, 2011). In their study, they showed that cellulase enzyme production by Streptomyces sp. using fruit waste as substrate was the highest at alkaline pH.

Figure 1. Enzymatic activities of different actinomycetes isolated from soil, Al-Aqsa Mosque, Jerusalem.

(A and B) showing good to moderate amylase activity respectively; (C and D) showing moderate protease activity indicated by clear zones around colonies; (E) showing good tyrosinase activity indicated by deep brown pigments and (F) showing no activity for two different actinomycete isolates.

Proteases represent one of the most important groups of enzymes and have been shown to play a role in many industrial and medical fields (Prakash et al., 2013). They can be used in detergent, food pharmaceutical, leather, waste processing industries and silk industries. Proteolytic enzymes have already been used in various forms of therapy and their use in medicine is gaining interest. Therefore, searching for new actinomycete strains still is the focus of many studies (Prakash et al., 2013). Our results revealed that only three isolates, Streptosporanium sp.1, Streptosporangium sp.2 and Actinomadura sp.10 were able to produce protease enzyme (Figure 1C and D). These three isolates were obtained from site 2. However, the site 1 and the reset of isolates from site 2 completely failed to show any protease activity. This rather low estimate of active strains may indicate that the method used for preliminary screening is not accurate (Jani et al., 2012). In addition, the low activity observed may suggest that our culture conditions are not optimal for such test (Jani et al., 2012).

The search for novel tyrosinases is still in need due to their potential in industrial applications and medical purposes. Tyrosinases have been suggested as potential tools in treating melanoma and as potential antioxidants, antiviral agents and immunogens (Popa & Bahrim, 2011). Among the isolates, only few showed tyrosine activity but all of these exhibited strong activity (Table 1). Only one isolate out of 17 (5.9%) in site 1, Elylrosporangium sp.3, showed high production of tyrosinase and the rest failed to show any activity (Figure 1E and F). Meanwhile, four isolates out of 18 (22.2%) of site 2, Streptosporangium sp.2, Kitasatosporia, Nocardia sp.2, and Actinomadura sp.15 showed different tyrosinase activity. These isolates had a high potential for enzyme production except Nocardia sp.2 which showed moderate activity.

For lipase production, all isolates from site 1 failed to show any activity. However, three isolates (16.7%) out of 18 isolates of site 2, Nocardiopsis sp.1, Nocardiopsis sp.2 and Kitasatosporia sp., were able to produce lipase in a weak to moderate manner of activity (Table 1).

Catalases are ubiquitous enzymes and have been isolated from a broad range of procaryotic and eukaryotic organisms (Zámocký et al., 2012). Actinobacteria are aerobic bacteria and would be expected to have catalase activity; however, our actinomycete isolates recovered from both studied sites showed different ranges of positive activity (Figure 2). The catalase activity was observed at pH 7 of the growing medium for all isolates. Out of the isolated actinomycetes, Kitasatosporia sp. was the most active catalase producer, followed by 42.9% of the isolates have moderate catalase activity (Table 1). However, 18 isolates (51.4%) including different genera showed weak activity. The low expressed activity may be due to shortage of manganese ions or iron concentration in the growing medium, since catalases rely on iron or manganese for their activity (Mishra & Imlay, 2012). Further studies are needed to identify the factors that weaken the scavenger process.

Figure 2. Catalase activity of different isolates.

A and B show the activity of isolates from site 1 and site 2 respectively. Evolved oxygen bubbles indicated a very strong activity and weak bubbles indicated a weak activity.

Since phosphate always exists in unavailable form for plant growth, phosphatase activity is an important mechanism of solubilizing inorganic phosphate (Sharma et al., 2013). In our study, all strains listed in Table 1, grown at room temperature (approximately 27±2^°C) on basal medium supplemented with CaHPO₄ (5 g/l) were tested for phosphatase activity at pH 7.0. Among the isolates, only Streptosporangium sp.1 and Nocardiopsis sp.2 showed phosphatase activity. The failure to detect of phosphatase activity by the remaining isolates may be due to the medium composition (Fredrikson et al., 2002). In a recent study done by Ghorbani-Nasrabadi et al., (2013), it was shown that substitution of nitrogen source in the growth medium by organic or inorganic nitrogen sources resulted in a significant reduction of phosphatase activity.

In conclusion, the need for low cost enzymes that can be applied in diverse biotechnological industries could be satisfied with the discovery of novel enzymes and metabolites. Moreover, the application of genetic engineering techniques in enzyme manufacturing is dramatically sparking the exploitation of new enzymes and the development of new enzyme properties. Actinobacteria have been proved a reservoir of important enzymes and metabolites due to their versatile genetic repertory (Prakash et al., 2013). Identification of new actinobacterial isolates in unique ecological environments could yield molecules that could become future players in green technology. Therefore our study contributes to explore new ecological sites for actinobacterial identification. Actinobacterial isolates that inhabitant the northern part of Al-Aqsa Mosque, showed a diverse population in the two sites where the soil was collected. These genera have been identified in each site for the first time. However more suitable growth conditions should be tested to explore the metabolites and enzymatic activities of these organisms.