Hydrolytic Enzymes Producing Bacterial Endophytes of Some Poaceae Plants

Although endophytes have been widely defined as microorganisms that live in the tissues of healthy plants for all or part of their life cycle, recent studies have revised this definition to include all microorganisms, including pathogens that can colonize the internal tissues of plants (Hardoim et al. 2015; Compant et al. 2021). Endophytic bacteria (EBs) have been isolated and characterized from different plant parts, including roots, stems, leaves, seeds, fruits, tubers, ovules, and nodules of various plants such as agricultural crops, meadow plants, plants grown in extreme environments, wild, and perennial plants (Afzal et al. 2019). EBs can contribute to plant health and development like Plant Growth Promoting Rhizobacteria (PGPR). In general, PGPR and EBs directly or indirectly affect the growth and development of the plant. EBs stimulate plant growth through various mechanisms such as nitrogen fixation, phytohormone production, nutrient uptake, and providing the plant with tolerance to abiotic and biotic stresses (Kandel et al. 2017). These properties make these bacteria important for various biotechnological applications in agriculture. Also, they have the potential to produce a variety of secondary metabolites like alkaloids, steroids, terpenoids, peptides, polyketones, flavonoids, quinols, and phenols with an application in agriculture, pharmaceutical and industrial biotechnology (Singh et al. 2017).

Microbial enzymes with high catalytic activities are used in many areas of the industry because they are more stable, cheaper, and can be obtained in large amounts by fermentation methods (Singh et al. 2016). Examples of industrial areas affected by discoveries of these enzymes include detergent agents, leather processing, degradation of xenobiotic compounds, food processing (bakery, meat, dairy, fruit, and vegetable products), pharmaceuticals (synthesis of pharmaceutical intermediates), biofuels (low-energy ethanol production process), and other enzyme related technologies (Singh et al. 2016). Although many bacterial isolates from various sources have been reported for the production of cellulase, protease, amylase, pectinase, lipase, asparaginase, etc., the studies involving the examination of endophytic bacteria in terms of biotechnological extracellular enzymes are relatively few (Carrim et al. 2006; Jalgaonwala and Mahajan 2011; Khan et al. 2017). Therefore, endophytic bacteria can represent a new source of enzymes with different application potentials.

In addition to entry through openings and wounds, endophytic bacteria actively colonize plant tissues using hydrolytic enzymes, such as cellulase. It was proposed that cell wall-degrading enzymes such as cellulases, xylanases, and pectinases might be responsible for plant and microbe interactions and intercellular colonization of roots (Verma et al. 2001; Kandel et al. 2017). Therefore, more knowledge on their production is also needed to understand the relationship between endophytic bacteria and plants.

The aim of this study was to examine endophyte bacteria isolated from various cultivated and wild plants of Poaceae family in Van province, Turkey, in terms of their potential to produce industrially important proteases, amylases, lipases, cellulases, xylanases, and pectinases and to perform a phylogenetic affiliation of the strains possessing relatively high enzyme activity profiles by 16S rRNA gene sequence analysis.

In this study, a total of 128 endophyte bacteria isolated from various cultivated and wild grain plants (Poaceae family) were used. For all the isolates, the EI of each enzyme activity is given in Table SI. Since endophytic bacteria offer a relatively new source of genes, enzymes, and secondary metabolites, we aimed to investigate several biotechnologically important extracellular enzymes of endophytic origin. By this purpose, endophytic bacteria isolated from Van province, Turkey, were evaluated for the presence of hydrolytic enzymes, including cellulases, xylanases, pectinases, amylases, proteases, and lipases (Fig. 1). They successfully demonstrated a variety of enzyme activities. It was revealed that lipases, proteases, amylases, cellulases, pectinases, and xylanases were produced with relative frequencies of 74.2%, 65.6% and 55.4%, 32%, 21.8%, and 7.8%, respectively (Fig. 2).

Fig. 1.

Fig. 2.

After the enzyme activity measurements were completed, 16 isolates revealing relatively high EI value for at least one enzyme tested were selected to perform a phylogenetic affiliation based on the 16S rRNA gene amplicon sequencing analysis. Also, among these selected strains, one producing none of the enzymes was selected for the identification (Table I).

The 16S rRNA gene amplicon sequencing of 16 isolates was successfully achieved. The ~ 1,500 bp 16S rRNA gene contains nine variable regions (V1–V9) in a highly conserved order. Since next-generation sequencing platforms provide an appropriate read of full-length the 16S rRNA gene intragenomic variants, they provide a better taxonomic resolution at species or strain level (Johnson et al. 2019). Illumina MiSeq method yielded full-length reading of the 16S rRNA gene amplicons for almost all strains. The lowest 16S rRNA gene reading length belongs to the strain G119Y1T with 70.1%, which nevertheless covers the V1‒V5 regions (Johnson et al. 2019) (Table II). As a result of pairwise comparisons of the 16S rRNA gene sequences on EzBioCloud server, five Paenibacillus sp. (G100Y1, G90Y2, G83S3, G80K3, G70K2), four Bacillus sp. (G119Y1T, G105S1, G113Y3, G105Y1), two Pseudomonas sp. (G88K1, G118S2T), two Curtobacterium sp. (G107Y2, G90S1), one Micrococcus sp. (G116K1T), one Streptococcus sp. (G117Y1T), one Chryseobacterium sp. (G42K2) were identified (Table II).

Except for strain G117Y1T, the 16S rRNA gene amplicon sequencing results of all strains yielded 99‒100% similarity (Table II). The 16S rRNA gene sequences alone may not be sufficient to identify a new species, but it can indicate that a new species is isolated (Tindall et al. 2010). The 94.58% similarity with G117Y1T is far below the threshold necessary to identify a new species (Stackebrandt and Goebel 1994; Stackebrandt and Ebers 2006), and, thus, this strain may represent a new species or even genus (Fig. 3). Noteworthy, strain G117Y1T gave positive results in terms of all enzymes except pectinase (Table I).

Fig. 3.

Different studies in the literature show that our identified strains belonging to seven different genera were endophytes (Verma et al. 2001; Rashid et al. 2012; Khan et al. 2017; Afzal et al. 2019). The different species of these genera produce high-potential bioactive compounds such as antimicrobials and enzymes to be used in the fields such as medicine and bioremediation, especially in agriculture (Doddamani and Ninnekar 2001; Schallmey et al. 2004; Lacava et al. 2007; Grady et al. 2016; Roy et al. 2018). Although the number of strains that we identified molecularly comprise a small cluster within all 128 isolates, they could reveal the diversity and support the literature data.

Carrim et al. (2006) presented the enzymatic activity of endophytic bacteria ranking as follows: protease (60%), amylase (60%), and lipase (40%). They did not detect cellulase and pectinase activities. Jalgaonwala and Mahajan (2011) detected 50% cellulase-positive endophytic bacteria in their study. On the other hand, our results revealed a high number of bacterial isolates with cellulase, lipase, and protease activities. Also, we have found a significant number of pectinase-positive isolates (Fig. 2). Despite the relatively limited number of studies, the percentage of endophyte bacteria with the positive scores for each of these enzymes varied due to the high species diversity.

Among the identified strains, Bacillus spp. (B. toyonensis, B. halotolerans, B. subtilis subsp. inaquosorum) except B. idriensis showed especially high cellulase activity among six tested enzymes (Tables I and II). The strain G105S, which, in contrast to other strains, did not produce the above enzymes, was closely related to B. idriensis (99.58%) (Table II). However, B. idriensis that possessed protease, cellulase, and pectinase activities, was isolated as an endophyte in the study conducted by Afzal et al. (2017). Pseudomonas spp. (P. congelans, P. orientalis) were the main xylanase producers among identified strains. In general, the number of strains demonstrating xylanase activity was relatively low. For this reason, these strains belonging to the genus Pseudomonas are valuable as xylanase enzyme producers. Xylanases produced by bacteria (Bacillus spp., Pseudomonas spp., Streptomyces spp.) are efficient in a broad pH and temperature range. Therefore, they are very useful in different industries reciprocally (Burlacu et al. 2016). Among the isolates we described, Paenibacillus spp. was observed as the most productive group of lipases and cellulases. Paenbacillus species are known to produce different hydrolytic enzymes (Sakiyama et al. 2001; El-Deeb et al. 2013). Cho et al. (2008) isolated two cellulose hydrolase genes (cel5A and cel5B) from endophytic Paenibacillus polymyxa. The strain belonging to Streptococcus, Micrococcus, Curtobacterium, and Chryseobacterium showed high activity of proteases, lipases, and xylanases. Generally, in this study, Gram-positive bacteria displayed broader hydrolytic enzyme potential than Gram-negative bacteria. Published data revealed that endophyte diversity varies according to different territories, plants, and even different plant tissues (Akinsanya et al. 2015).

Although this study was carried out in line with the biotechnological perspective, extracellular enzymes should also be evaluated and discussed in terms of the relationship between endophyte bacteria and the plant hosts. For example, different levels of cellulases and pectinases were reported to be important in endophytic diazotrophic bacteria during plant cells colonization (Verma et al. 2001). Considering that the plant pathogen bacteria also synthesize the enzymes that break down the cell wall, more information about the expression and regulation of these enzymes in both groups could be crucial to understand and distinguish between these two groups of bacteria.

In this study, a potentiality of endophytic bacteria isolated from several grain plants (Poaceae family) in Van province, Turkey, to produce biotechnologically important enzymes, was revealed for the first time. Endophyte bacteria are rich sources of enzymes and new secondary metabolites for many industries due to their high species diversity and adaptation to different environments. Therefore, investigation of these isolates not only in terms of extracellular enzymes but also in terms of specific and industrially important secondary metabolites should be among the future.