Introduction 

Enzymes have very old history and they have their existence from the start of ancient civilizations where people were using microbial enzymes in baking, alcohol production, brewing, cheese making etc. However, since then the number of applications of enzymes has increased tremendously with better knowledge about production and purification of enzymes from diverse sources.¹ Microorganisms have been known to produce both intracellular and extracellular enzymes commercially.²

Enzymes are becoming alternative to chemical catalysts for better efficiency ensuring economic and ecological sustainability of the industrial processes.³ Proteases are the most dominant group of enzymes from the industry point of view, constituting about sixty percent of the total enzyme market.⁴ Proteases from microorganisms have been the most widely explored enzymes since the advent of enzymology.⁵ They account for approximately two-third share of the total commercial protease sale around the globe.⁶ These enzymes have gained attention not only due to their critical role in metabolic activities in the organisms but also due to their wide applications in industries⁷ such as food, detergent, bakery, leather, pharma, infant formulas, etc. due to their features like production ease, thermal stability and wide pH range applications.⁸

Alkaline proteases are the most frequently used industrial enzyme among different types (alkaline, neutral, and acidic) of proteases due to their comparatively high activity and greater stability at high pH.⁹ Wide range of microbes were found to produce alkaline proteases, including bacteria, yeasts, moulds and mammalian tissues⁵ and these were isolated from diverse sources as well. However, bacteria were preferred as they grow rapidly under various nutritional and physiochemical conditions, need less space, could be easily maintained and were accessible for genetic operations.² A large number of bacteria have been reported to produce alkaline proteases e.g. Bacillus, Pseudomonas, Streptomyces, Micrococcus etc. However, Bacillus sp. has been the major source for alkaline protease production, accounting for about 35% of the total microbial enzyme sales.^10,11 Since the first alkaline protease ‘Carlsberg’ from Bacillus licheniformis was utilized as a detergent additive in the 1960s, a number of other bacillus species have also been reported to produce alkaline proteases.¹²

Significant advancements in agriculture, industry, bioinformatics and biotechnology have fueled the search of extremophile microorganisms for alkaline protease production.¹³ The proteases produced by microbes present in normal environment are usually unstable under extreme conditions, even with several attempts of physicochemical treatments, protein engineering and gene-shuffling methods.⁴ Microorganisms living in extreme habitats are believed to produce proteases which are stable in those situations⁴ but limited information is available reporting such microorganisms.¹⁴

In this context, the aim of the study was to isolate potent alkaline protease producing bacterial strains from poultry farm fecal soils of Rewa, Madhya Pradesh, India. Poultry farm fecal soils are known to be highly alkaline.

Materials and Methods 

Chemicals

All the chemicals and reagents used in media formulation, screening, well diffusion assay and phenotypic characterization were obtained from HiMedia, India and Fisher Scientific, USA. HiPer Bacterial Genomic DNA extraction kit, Taq Polymerase and dNTP Mix were obtained from HiMedia, India. Primers were obtained from Eurofins Genomics, Bengaluru, India. 

Collection of Samples

Alkaline Soil samples for the isolation of potent alkaline protease producing bacteria were collected from Govt. kukkut palan kendra (poultry farm) near chirahula temple, in district headquarters of Rewa of Madhya Pradesh, India. Dried fecal soil was explored for collection which are known to have alkaline pH. The samples were collected from 2 cm depth of surface using a sterile spatula. They were tested first for high alkaline pH using pH strips and transferred in a sterile polybag. The samples were randomly collected from two sites of poultry farm: site 1 and site 2. The pH of the samples was 10.0 and 11.0 respectively.

Medium

Isolation of bacteria was carried out on CPYA (Casein, Peptone, Yeast extract, Agar) medium (pH 12.0)^{15, 16} and the same media was used also for screening of protease activity and crude enzyme production. Medium (1 L) was prepared by making three separate solutions initially. Solution A: casein (5.0 g) in 0.01 N NaOH solution (400 ml); Solutions B: peptone (5.0 g), yeast extract (2.0 g), NaCl (5.0 g), K_­2HPO₄ (1.0 g), MgSO₄.7H₂O (0.2 g), CaCl₂ (0.1 g), and Agar (20 g) in 595 ml distilled water; Solution C: 6.0 g Na₂CO₃ in 20 ml distilled water. After that, these solutions were autoclaved separately to avoid coagulation of casein due to high pressure. Solution A and Solution B were mixed and maintained to pH 12.0 aseptically by adding Solution C in a drop wise manner.

Isolation and Screening

Serial dilution agar plate technique was used to carry out isolation. 0.1 ml of the 10^-1-10^-3 diluted sample was spread evenly over the CPYA medium (pH 12.0) and incubated at 37ºC for 24 h in Incubator. Appeared colonies were purified by repeated streaking of a single isolated colonies of bacteria on fresh CPYA medium plates. Production of alkaline protease was screened by streaking of pure isolates on CPYA media plates^{17, 18} and after that giving them incubation at 37ºC temperature for 24 to 48 hours. 10% Trichloroacetic acid (w/v) solution was then poured in plates and incubated for half an hour. Zone of proteolysis was observed as transparent clear zone around the colony.

Enzyme Production and Well Diffusion Assay

Isolates producing clear zone around the colony in the range of good and excellent were selected for enzyme production under submerged conditions. Erlenmeyer flasks (150 ml) having CPYA broth (50 ml, pH 12.0) were inoculated with small amount of bacterial culture. These were incubated at 37ºC temperature for 48 h. The fermented broth was centrifuged then at 7000 g for 10 minutes. The resulting cell-free crude filtrate was used for assay.

Alkaline protease production from these isolates was examined by radial diffusion assay method. In this method wells having 7 mm diameter were created on CPYA solid media plates with the help of cork borer and 0.5 ml of crude filtrate was poured into them. These plates were incubated at 37⁰C for initially 24 hrs. than up to 48 hrs. After that TCA (Tri chloro acetic acid) solution (10 %) was added to these plates and zone of diffusion was measured as alkaline protease activity. The enzyme activity was calculated as Relative enzyme activity (REA).

REA = Diameter of zone of clearance (in mm)/ (well diameter in mm)

Phenotypic Characterization

Phenotypic characterization of isolates was determined by Bergey’s manual of bacteriology¹⁹. Differential staining was performed and observations were made under Binocular microscope.

DNA Isolation, PCR Amplification and DNA Sequencing

Total genomic DNA was isolated from the most promising alkaline protease producing Isolate, using HiPer Bacterial Genomic DNA extraction kit, followed by amplification of 16s rRNA gene with universal primers by PCR. Thus, obtained Amplified DNA was further sent to Genexplore Diagnostics and Research Centre Pvt. Ltd. Ahmadabad, Gujrat, India, for DNA sequencing.

Identification

The 16s rRNA sequence was subjected to BLAST analysis against 16s rRNA gene database in NCBI (National Centre for Biotechnology Information). A phylogenetic tree was constructed with related bacterial 16s rRNA gene sequences from NCBI using MEGA7 program by maximum likelihood option.²⁰ The Neighbor-Joining method was used to infer evolutionary history.²¹ On the basis of 16S rRNA homology and phylogenetic tree analysis, the most promising isolate was identified. 16s rRNA gene sequence of the isolate was submitted to Genbank database (https://www.ncbi.nlm.nih.gov/genbank).

Results

Isolation and Screening

A large number of isolates were isolated (Fig 1) and screened for alkaline protease production. Colonies with different characteristics appearing on CPYA medium plates were isolated and screened for casein hydrolysis on CPYA medium. A total number of 3 isolates: Isolates no. PF11, PF13, PF21 who have produced a zone of clearing in the range of excellent and good were selected for further study (Fig 2).

Figure 1: Primary screening of soil samples on CPYA medium having pH 12. Click here to View Figure

Figure 2: Zone of clearance produced by selected isolates. Click here to View Figure

Enzyme Production and Well Diffusion Assay

Crude extract of Isolate no. PF21 produced highest zone of clearance of 2 cm among all the isolates, followed by PF11, PF13 which produced 1.9 cm, 1.8 cm respectively (Fig 3). Relative enzyme activity (REA) for Isolate no. PF11, PF13, PF21 were 2.71, 2.57, 2.86 respectively (Table 1). Clearly Isolate No. PF21 had the highest enzyme activity.

Figure 3: Well diffusion assay for relative alkaline protease activity Click here to View Figure

Table 1: Relative Enzyme Activity (REA) of selected alkaline protease producing isolates.

Phenotypic Characterization

Colony morphologies of all the isolates were small, round, regular. Colony color of Isolate no. PF11, PF 13, PF21 were creamy white, off white, white respectively. In gram staining, all the isolates were long, filamentous, rod shaped and Gram positive (Fig.4, Table 2). Further phenotypic characterization of isolate no. PF 21 also revealed that it is motile and spore forming.

Figure 4: Gram stain images of selected isolates. (All the images have dimension: 640-pixel x 576-pixel) Click here to View Figure

Table 2: Phenotypic characterization of selected alkaline protease producing isolates.

DNA Isolation, PCR Amplification and DNA Sequencing

Whole genome DNA was isolated from most potent isolate PF21 and PCR amplification was obtained with 16s rRNA universal 357F forward primer having sequence (5’-3’) CTCCTACGGGAGGCAGCAG and 1391R Reverse primer having sequence (5’-3’) GACGGGCGGTGTGTRCA undergoing- 1 cycle of Initial denaturation at 95⁰C for 5 min; 35 cycle of the following steps: Denaturation at 95°C for 30 sec., Annealing at 49°C for 30 sec., Elongation at 72°C for 1:30 min; and finally 1 cycle of Final Extension at 72°C for 10 min²² (Fig. 5).

Figure 5: Electro-phoregram of isolated whole genome DNA and amplified 16s rRNA gene of Isolate no. PF21. Click here to View Figure

Identification

The molecular identification of isolate no. PF21 was done using 16S rRNA gene homology. It was found by analysis with nucleotide BLAST tool that 16S rRNA sequence of isolate no. PF21 had 95.64 % identity with the sequence of Bacillus halodurans DSM497 (Genebank Accession number: NR025446). A phylogenetic tree was constructed using MEGA7 program with closely related 16s rRNA gene sequences of other bacillus species. On the basis of both, 16S rRNA homology and phylogenetic tree analysis, the isolate no. PF21 was identified as Bacillus halodurans RSCVS-PF21. 16s rRNA sequence was submitted to Genbank database (https://www.ncbi.nlm.nih.gov/genbank) with accession no. MT279908.

Figure 6: Phylogenetic analysis of Bacillus halodurans RSCVS-PF21, on the basis of related 16s rRNA sequences. Click here to View Figure

Discussion

Less number of bacterial colonies appeared on isolation from both sample site: 1 and site: 2 of poultry farm at alkaline pH 12²³ on CPYA solid media. Poultry farm soil samples were from dried fecal matter. The pH of site: 1 and site: 2 were 10 and 11 respectively. Comparatively more dense bacterial colonies appeared from Site: 1 soil sample on serial dilution.  No fungal colony appeared even after 24 to 36 hours incubation²⁴ from both sample sites as fungus are known to be slow growing. All the colonies appeared were small, round like pin head after 24 hr incubation. Pure culture was obtained by repeated streaking. Out of bacterial colonies appeared, only some were able to hydrolyze casein protein.²⁵ Selected isolates PF11, PF13, PF21 were cultured under submerged condition to produce enzymes and all the isolate showed high relative enzyme activity on radial diffusion assay. The most potent alkaline protease producing isolate no. PF21 had highest relative enzyme activity (2.86). About 1112 bp consensus sequence was obtained after amplification of 16s rRNA gene with 357F and 1391R universal primers and DNA sequencing of amplified product. On the basis of NCBI BLAST and Phylogenetic Analysis²⁶ it was identified as Bacillus halodurans RSCVS-PF21. Closest reference bacteria Bacillus halodurans DSM497 showed 95.64 % similarity with it though it was not reported to produce alkaline protease.^27-29 Some other identical bacteria were Bacillus halodurans strain ATCC 27557 (95.13 %), Bacillus okuhidensis strain GTC 854 (95.03 %), Fermentibacillus polygoni strain IEB3 (94.02 %), Bacillus nanhaiisediminis strain NH3 (94.02 %). There were also other Bacillus halodurans strains recently reported to produce alkaline proteases.^30-33 Phenotypically also Bacillus halodurans RSCVS-PF21 was related with many other alkaline bacillus species having long, rod shaped, gram positive, white colony, motile and spore forming characteristics.¹⁹ The alkaline protease produced was extracellular in nature as was checked by well diffusion assay. Crude enzyme was stable and active at pH 12 indicating its alkaline nature.

Conclusion

The study describes alkaline protease producing Bacillus halodurans RSCVS-PF21 (Genebank: MT279908) isolated from soil of Government poultry farm near Chirahula temple of Rewa, Madhya Pradesh, India. The bacterium was able to grow at pH 12. It showed high extra cellular alkaline protease activity and the crude enzyme was stable at pH 12 indicating its alkaline nature. Phenotypically and phylogenetically it was closely related with other alkaline bacillus species but it was closest to Bacillus halodurans DSM497. Further optimization of enzyme production and physio-chemical characterization of enzyme may identify the bacterium as a potential candidate for alkaline protease industry.

Acknowledgement

This work was carried out at Centre for Biotechnology Studies, Awadhesh Pratap Singh University Rewa, Madhya Pradesh, India and was supported by facilities and financial resources available to the centre, which is gratefully acknowledged.

Declaration of Competing Interest

The authors declare no conflict of interest

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