Screening of protease, cellulase, amylase and xylanase from the salt-tolerant and thermostable marine Bacillus subtilis strain SR60

Isolation of thermophilic bacterial strain

The bacterial strains were obtained from aseptically collected tissues of Siderastrea stellate Verrill, 1868 (Cnidaria, Scleractinia) colonies at Cabo Branco coral reefs, Paraiba State, Brazil (7°08’50” S; 34°47’51” W). For bacterial isolation from the anthozoan, samples were suspended in sterile saline solution, agitated until homogenization was achieved and then spread over marine agar plates (pH 8.0± 0.3) containing 5 g/l peptone; 1 g/l yeast extract; 15 g/l agar diluted in sterile marine water and incubated at 55°C until adequate growth was achieved⁶. A total of 12 bacterial isolates were obtained, which were analysed for protease, cellulase, amylase and xylanase production capacity, and only the one with the simultaneous production capacity of these enzymes was selected.

For further screening of enzymatic activity described below, two bacterial colonies, isolated using the above culturing conditions, were inoculated onto each plate. A total of three replicates were performed for each salt molarity.

Bacterial identification

In order to identify the isolate, morphophysiological and molecular data were evaluated⁷. The obtained 16S rRNA gene was sequenced by ATCGene (UFRGS, Porto Alegre, RS, Brazil) using the automated sequencer ABI-PRISM 3100 Genetic Analyzer. The SR60 isolate sequence was compared to sequences deposited in the Genbank database (NCBI). For the local alignment, the BLASTn tool (NCBI) was used. MEGA 6.0 software was used for monitoring multiple sequences and for construction of a dendrogram by the Neighbor-Joining method.

Screening of protease

The isolated bacterial strains were screened production for protease on agar medium comprising 10 g/l gelatine and 20 g/l agar in increasing concentrations of NaCl (0, 0.25, 0.50, 1.0, 1.25 and 1.5 M) pH 8.0± 0.3. The inoculated plates were incubated at 48 h at 55°C and observed for the formation of zone of hydrolysis⁸.

Screening of celullase

The ability of isolate on produce cellulose was tested a plate containing 1 g/l carboxymethylcellulose (CMC); 0.5 g/l NaNO₃; 1 g/l K₂HPO4; 0.5 g/l MgSO₄∙7H₂O; 0.001 g/l FeSO₄∙7H₂O; 1 g/l yeast extract; 15 g/l agar) in increasing molarities NaCl (0, 0.25, 0.50, 1.0, 1.25 and 1.5 M) for 48 h at 55°C on pH 8.0±0.3 and then overlaid with 0.2 g/l potassium iodide for 5 min, bacterial colonies showing clear zones were considered to be cellulase producers⁹.

Screening of amylase

Amylolytic activity of culture was screened on starch nutrient agar plates containing: 10 g/l starch; 0.05 g/l NaNO₃; 1 g/l K₂HPO₄; 0.5 g/l MgSO₄∙7H₂O; 0.001 g/l FeSO₄∙7H₂O; 1 g/l yeast extract; 15 g/l agar, in increasing molarities of NaCl (0, 0.25, 0.50, 1.0, 1.25 and 1.5 M). After incubation at 55°C pH 8.0±0.3 for 48 h, the zone of clearance was determined by flooding the plates with 0.2 g/l potassium iodide for 5 min¹⁰.

Screening of xylanase

Xylanase activity was detected using a saline medium containing: (10 g/l xylan; 0.005 g/l NaNO₃; 1 g/l K₂HPO₄; 0.5 g/l MgSO₄∙7H₂O; 0,001 g/l FeSO₄∙7H₂O; 1 g/l yeast extract; 15 g/l agar) in increasing molarities of NaCl (0, 0.25, 0.50, 1.0 and 1.5 M) on pH 8.0±0.3. After incubation at 55°C for 48 h, the plates were with 0.2 g/l potassium iodide for 5 min. The clear zones around colonies indicated qualitative xylanase activity¹¹.

Bacterial identification

The SR60 isolate was revealed to be a Gram-positive spore-forming bacillus, facultative anaerobe, catalase-positive; it was negative for indole, H₂S production and citrate utilization bacterium (Table 1). Those findings led us to consider the isolate belonging to the genus Bacillus which was posteriorly confirmed by the phylogenetic analysis which revealed that the SR60 strain formed a clade with Bacillus subtilis (Figure 1). The nucleotide sequence was deposited in GenBank under accession number MH698455.1.

Figure 1. Phylogenetic tree of isolated SR60 and other related species based on 16S rRNA sequences.

The scale bar represents 0.01 substitutions per site. GenBank accession numbers of the sequences are given in parentheses.

Screening for protease, celullase, amylase and xylanase

In differential media for the production of different extracellular enzymes, it was observed that conditions of high salinity from 0 to 1.5 M NaCl, a SR60 strain showed proteolytic, cellulolytic, aminolytic and xylanolytic activity, these productions being observed by zones of enzymatic hydrolysis (Table 2). The halo detection for protease and cellulase was observed up to the maximum salinity, 1.5 M NaCl (Figure 2 and Figure 3). Cellulolytic enzymes comprise a group of glycosidic hydrolases, including endoglucanases, exoglucanases and beta-glycosidase. In general, the production of the enzyme group is mainly observed in fungi, actinomycetes and some other bacteria. The use of fungi to produce cellulases has been practiced in the food, textile, fuel and chemical industry, but the growth period for the microorganism does not match the high demand from the industries for production. In an attempt to solve this problem bacteria present rapid growth and high enzymatic production¹². Bacterial isolates produced from different environments, such as bovine ruminants, soil and in isolation, were found to produce hydrolases^12,13. Biofuel industries that use lignocellulose as the first raw material pre-treatment process for the release of cellulose, making it more accessible to the enzymatic action. During the processing of the cellulose, various compounds containing salts are used, the enzymatic catalysis being reduced or inhibited in this halophilic environment¹⁵. The extracellular production of amylase and xylanase reached an upper NaCl concentration limit of 1.0 M and 1.25 M NaCl, respectively (Figure 4 and Figure 5); however, as a bacterial cell growth molecule at the other salt concentrations.

Figure 2. Screening of protease in crescent saline molarity.

Halos around bacterial colonies are indicative of cellulose degradation.

Figure 3. Screening of cellulase in crescent saline molarity.

Halos around bacterial colonies are indicative of cellulose degradation.

Figure 4. Screening of amylase in crescent saline molarity.

Halos around bacterial colonies are indicative of cellulose degradation.

Figure 5. Screening of xylanase in crescent saline molarity.

Halos around bacterial colonies are indicative of cellulose degradation.