Screening And Characterization of Amylase Producing Potential Soil Bacteria from an Historical Empire Area Kondaveedu of Andhra Pradesh, India

The enzymes in the amylase family are highly significant due to their numerous potential applications in a wide range of industries, including textile, baking, food, paper, brewing, and detergent. Two types of amylases are endoamylases (α,amylase) and exoamylases (glucoamylase). While α-amylase catalyses the random hydrolysis of α-1,4-glucosidic linkages in the interior of starch molecules to produce branched and linear oligosaccharides (dextrin, maltose, maltotriose, and glucose) with different chain lengths, glucoamylases catalyse the hydrolysis of α-1,4- and α-1,6-glucosidic linkages in starch molecules (amylase and amylopectin) from their non-reducing end to produce glucose (Sudharhsan et al., 2007; Khan and Priya, 2011).

Amylases are produced by a broad variety of organisms, ranging from prokaryotes (bacteria) to eukaryotes (plants and humans). They have been separated from a range of microorganisms, including yeast, actinomycetes, fungus, and bacteria. However, amylase, which comes from bacteria and fungus, has dominated a number of uses in biotechnology-based industries. Many bacteria can produce amylases, including B. licheniformis, Bacillus subtilis, Bacillus megaterium, Bacillus steriothermophilus, Lactobacillus sp., Proteus sp., Escherichia coli, Pseudomonas sp., Strepotmyces sp., etc. (Pokhrel et al., 2013). Microbial amylases are very beneficial due to characteristics like mass production and easy genetic manipulation (Souza PM and Magalhaes, 2010). It is well known that bacterial amylases are less costly to create, more stable, and more productive.  The genus Bacillus produces a significant number of extracellular enzymes, including amylases. Bacillus sp. is a preferred choice for the commercial production of microbial enzymes due to its short fermentation cycle, safe handling, ease of manipulation, homogeneity, excellent enzyme activity under stress, and ecologically benign properties (John and Elangovan, 2013). The production of amylase from several Bacillus species shows considerable variability since amylase synthesis depends on the medium's composition and other physical parameters. It is becoming more and more important to raise the amylase titer without increasing production expenses. The optimum basal media for amylase production is selected, the most efficient Bacillus species that generate amylase are screened, and their molecular identities are ascertained in this paper. Amylases are mostly extracellular hydrolytic enzymes that break down natural polymers such as proteins, cellulose, pectin, and starch into simpler monomers (Alaria et al., 2013). The food industry uses starch-degrading enzymes for a variety of purposes, such as the solubilization and saccharification of starch, the production of high fructose corn syrup, glucose syrup, and maltose, the reduction of turbidity to produce clarified fruit juice for a longer shelf life, and the reduction of viscosity in sugar syrups (Karnwal and Nigam, 2013). Commercial sugar syrups containing glucose, maltose, and higher oligosaccharides are made from hydrolyzed starch (Hagihara et al., 2001). These industrially important microorganisms are found among the Bacillus species due to their capacity to secrete proteins into extracellular medium, rapid growth rates that lead to short fermentation cycles, and general handling safety (Pandey et al., 2000). The hydrolysates are used in fermentation as sources of carbon and as a sweetener in a variety of processed foods and beverages. The hydrolysis of starch products, including glucose and maltose, is the consequence of controlled breakdown. Among the biotechnological applications of amylases include the food, fermentation, detergent, pharmaceutical, brewing, textile, and paper industries (Miller, 1959). To meet the growing demands of these companies, low-cost amylase manufacturing is required (Kadhiresan and Manivannan, 2006).  

The primary advantages of employing microbes are their capacity to produce amylases in large quantities and their simplicity of manipulation to produce an enzyme with desired properties (Sharma et al., 2015). These amylolytic bacteria are easy to screen and assess for amylase production in a laboratory setting (Pokhrel et al., 2013). Amylolytic microbial strains are still being isolated and characterized. In this study, several hyperactive amylase-producing bacteria were isolated and screened using soil samples from the Kondaveedu forest region in the Guntur district (Andhra Pradesh).

MATERIALS AND METHODS:

Sample collection and Isolation of bacteria:

The soil samples used to check for amylolytic bacteria came from the Kondaveedu forest tract (La: 16.2517680 and Lo: 80.2540940), Guntur district, Andhra Pradesh. Soil samples were randomly obtained from the top layer of soil using a spatula. After that, they were put in sterile plastic bags and labelled with the date and site of collection. Samples were brought to the laboratory and stored at 4?C in a freezer until they were processed. Bacteria were separated from soil samples using the serial dilution agar plate method. 1gm of each soil sample was mixed with 9ml of sterile distilled water, and the combination was serially diluted up to a ten-six-fold dilution. The nutritional agar medium (NAM) plates were incubated for two days at 37ºC after 0.1 mL of each dilution was applied using the spread plate method. 25 mg of the antifungal drug nystatin was added to the medium to prevent the growth of fungal contamination. Nutrient agar medium (g/L) with 3 beef extract, 5 peptone, 5 NaCl, 15 agar, and 1000 mL of distilled water was used to create bacterial isolate slants. The isolated bacterial slants were stored at 4?C for further investigation.

Screening of amylase producing bacteria:

To measure the amylolytic capacity of isolated bacterial colonies maintained at 4ºC, a starch agar plate (g/L) containing peptone 5, beef extract 3, soluble starch 2, agar 15, and distilled water 1000 mL was employed (Sharma et al., 2015). The inoculation plates were incubated at 37ºC for three days. After three days of incubation, the plates were flooded with starch-degrading bacteria using Gram's iodine solution (1 g of iodine crystals and 2.0 g of potassium iodide dissolved in 100 mL of distilled water, kept at room temperature). When starch and iodine reacted, a dark blue starch-iodine combination blanketed the entire agar. The positive colonies had a distinct zone of hydrolysis surrounding them when saturated with iodine solution. The negative colonies on starch agar were blue-black in colour and had no surrounding hydrolysis zone (Karnwal and Nigam, 2013).

Secondary Screening of Amylase Producing Bacterial Strains

Secondary screening was based on measuring amylase in starch broth (SA: agar, 10 g; NaCl, 5 g; beef extract, 3 g; and distilled water, 1 l at 7.0 ± 0.1 pH). A loopful of bacteria were added to 50 cc of nutritional broth in Erlenmeyer flasks. The flasks were shaken in an incubator at 150 rpm for a full day at 37°C. 100 μl (3.25 x 107 cells/ml) of the culture was introduced to starch broth media after a 24-hour growth period, and flasks were then incubated at 37°C for an additional 24 h. For the amylase test, the 3, 5-Dinitrosalicylic acid (DNSA) method was modified (Ghosek, 1987). To stop the reaction, 1ml of the 3,5-dinitrosalicylic acid reagent was added. 2ml of distilled water were also added. The mixture was promptly refrigerated with ice water after boiling for five minutes.

Morphological, Biochemical and Physiological Characterization of the strain NRI-21:

Colony Morphology Analysis provided a detailed description of the bacterial isolate by examining color, shape, size, colony type, and pigmentation (Alfred, 2007). The bacterium's biology, growth patterns, physiological characteristics, structural features, and environmental adaptations were all revealed by these measures. The bacterial isolates were first identified by morphological characterization after the bacteria were plated on NAM and incubated for 2-4 days.

The slides were created using the teasing mount method and lacto-phenol Cotton Blue (LPCB) reagent. The conidia morphology of strain NRI-21 was examined using a scanning electron microscope (SEM). After being fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) for 24 h at 4°C, the samples were postfixed in 2% aqueous osmium tetroxide for 4 h. After being dehydrated in a series of graded alcohols, they were dried to a critical point using a CPD unit. The prepared samples were mounted to the stubs using double-sided carbon conductivity tape. For three minutes, a thin layer of gold coat was applied to the samples using an automated sputter coater (Model: JEOL JFC-1600).  The samples were then scanned under a scanning electron microscope (Model: JOEL-JSM 5600) at the necessary magnifications in accordance with standard procedures (Lakshman, 2019). The isolate was biochemically characterised using conventional prototocol. Additionally tested were nitrate reduction, H2S production, and casein hydrolysis. Physiological traits like the impact of temperature (20–45°C) and pH (5–9) on strain growth were investigated. When 3% hydrogen peroxide was applied to the slide surface with a loop full of bacterial culture, the formation of gas bubbles was regarded as a catalase positive score. In the Voges-Proskauers test, Barritt reagent B is added after 1-2% of Barritt reagent A. The nitrate reduction test is carried out by adding one or two drops of sulphanilic acid and one or two drops of N, NDimethyl-1-napthylamine. Citrate utilization was assessed using Simmon's citrate agar slants; the appearance of blue color in the culture after the bacterial culture was inoculated signified a successful outcome. Oxidase, urease, gelatin liquefaction, indole production, methyl red, hydrogen sulphide test, and nitrate reductions were all analysed.

Molecular characterization of the Strain NRI-21:

The genomic DNA required for the polymerase chain reaction (PCR) was obtained from the colonies grown on NAM agar for 48 h. The isolate's whole genomic DNA was isolated using the DNA purification kit (Pure Fast® Bacterial Genomic DNA purification kit, Helini Biomolecules, India) in compliance with the manufacturer's instructions. Thirty cycles of amplification (denaturation at 94°C for sixty seconds, annealing at 55°C for sixty seconds, extension at 72°C for sixty seconds, and a further five minutes at 72°C as final extension) were performed after the initial denaturation at 94°C for three minutes. A gradient PCR (Eppendorf, Germany) with a total volume of 50μL was used for the amplification processes.  Each reaction mixture included 25 μL of master mix, 22 μL of molecular grade nuclease-free water, 1 μL of DNA, 1 μL of 10 P mol forward 16S bacteria specific primer (5'AAATGGAGGAAGGTGGGGAT-'3), and 1 μL of 10 P mol reverse 16S bacteria specific primer (5'AGGAGGTGATCCAACCGCA-'3). The separation was performed in TAE buffer with 5 μL of ethidium bromide for 40 minutes at 90 volts. Agarose gel (1%) was used to analyse the PCR product, and the fragment was purified using the Helini Pure Fast PCR clean up kit (Helini Bio molecular, India) in accordance with the manufacturer's instructions. Gel Doc was used to record the bands after they were examined under UV light. Using a 3100-Avant genetic analyzer (Applied Biosystems, USA), the dideoxy chain termination method was used to directly sequence the PCR products.

Pair wise and multiple sequence alignment:

The isolate NRI-21's gene sequence was compared to the NCBI and GenBank gene libraries for bacterial species using BLAST. Pairwise evolutionary distances were computed using MEGA-6 software. The isolate's greatest parsimony strategy was applied to the phylogenetic analysis using BLAST and CLUSTAL W. The closely related homologous isolates were found, retrieved, and their sequences compared to those of the isolated strains using CLUSTAL W, which comes with the MEGA 6 Version [29]. The strain NRI-21's 16S rRNA gene sequence was entered into the GenBank database.

Growth Pattern of the Strain NRI-21:

To evaluate the growth pattern, the strain was introduced to 250 ml flasks containing 100 ml of nutrient broth. The culture broth was incubated at 30 ± 2°C using a rotary shaker operating at 180 rpm. After the flasks were harvested every two h for up to 72 h, the strain's growth was measured using the dry weight of the mycelium. Following biomass separation, ethyl acetate was used to extract the culture filtrate, and the antibacterial activity of the crude extract was evaluated using the agar well diffusion method.

Extraction of Metabolites and Antimicrobial Assay:

The strain's antibacterial activity was evaluated using the agar well diffusion assay (Christensen and Martin, 2017). A homogenous culture suspension created by suspending a 48-hour-old culture in sterile saline was used to inoculate NA broth (seed media). After that, the mixture was cultured on a rotator shaker at 180 rpm for three days at 30 ± 2°C. The fermentation medium, NA broth, was supplemented with 10% of the seed culture. The fermentation was carried out at 30 ± 2°C and agitated at 180 rpm for three days. The filtrate's antibacterial component was extracted using the solvent extraction method. The filtrate was vigorously agitated after 1:1 ethyl acetate was added.  The residue that remained after the ethyl acetate extract was dried off in a water bath was used to measure the antimicrobial activity. Ethyl acetate itself was used as a negative control. Eighty microliters of the crude extract and eighty microliters of the negative control were added to separate wells. The agar surface was treated with a traditional antibiotic disc as a positive control. Following a 48h incubation period at 37 °C, inhibition zones (mm) were measured. The bacteria employed as test organisms include Pseudomonas aeruginosa (ATCC 9027), Proteus vulgaris (ATCC 6380), Escherichia coli (ATCC 9027), Bacillus megaterium (NCIM 2187), Xanthomonas campestris (MTCC 2286), and Staphylococcus aureus (MTCC 3160). Penicillium citrinum (MTCC 6849) and Candida albicans (MTCC 183) are examples of fungi.

RESULTS AND DISCUSSION:

Isolation and Screening of bacteria from forest Soils:

Soil samples were collected at random, mixed, and processed. After isolation using the serial dilution plate approach, the NAM plate surface was treated using the spread plate technique (Figure 1). The names of the twenty-five bacterial strains that were identified from soil samples in Kondaveedu forest habitats are NRI-1 through NRI-25. Each isolate was screened for amylase production. Interestingly, the isolates NRI-6, NRI-11, NRI-17, and NRI-21 generated amylase on SCA medium plates (Figure 2). After being sub-cultured on NAM, each of the four strains was retained for further study.

Quantitative estimation of Amylase production:

The four isolates' amylase activity was measured quantitatively in NA media. The results showed that, in comparison to the other four isolates, NRI-21 produced the most amylase (28.9 IU/ml) (Figure 3). As seen in figure 3, the NRI-6 isolate had the lowest amylase production (13.9 IU/ml). There was a statistically significant difference between the three isolates (p value > 0.05).

Figure 1. Isolation of the bacteria from Kondaveedu forest soils

Figure 2. Screening of Amylase producing bacteria

A: NRI-6, B: NRI-11, C: NRI-17, D: NRI-21

Figure 3. Estimation of amylase production by various bacterial strains

*Values are statistically analyzed and represented with ± SE values (n=3)

Morphological, Biochemical and Physiological Characterization of the strain NRI-21:

The surface of the strain's colony is spherical, white, slimy, or uneven. Rod-shaped spores were seen in the strain NRI-21 (Figure 4). It is non-motile, positive for Gram's reaction, and negative for urease, gelatin production, and casein hydrolysis. The strain demonstrated a temperature range of 30 to 40 °C for growth, with 35 °C being the optimal temperature. The pH range for growth is 5.0 to 9.0, with 7.0 being the optimal value. The strain NRI-21 showed positive results for indole, methyl red, citrate utilization (Figure 5), and catalase synthesis. Tests for hydrogen sulphide, nitrate reduction, and Voges-Proskauer all produce negative results (Table 1). Based on morphological, physiological, and biochemical characteristics as well as the Bergies manual of bacteriology, the isolate NRI-21 was tentatively identified as Bacillus.

Figure 4. Citrate utilization (A: Blank, B: Strain NRI-21)

Figure 5. Scanning Electron Microscopic photograph of the strain NRI-21

Table 1: Morphological, Biochemical and Physiological characteristics of NRI-21

Molecular Identification of the Strain NRI-21:

After the phylogenetic tree was constructed using bootstrap analysis and the neighbor-joining method, the strain NRI-21 was identified as Bacillus rugosus (Figure 6). The incomplete 16S rRNA sequences of the bacterium were added to the GenBank database with accession number PX498601. The partial sequence was aligned and compared with each 16S rRNA gene sequence in the GenBank database using the multi-sequence advanced BLAST comparison tool. The 16S rRNA gene sequence was aligned for phylogenetic analysis using the CLUSTAL W tool from the MEGA 6 Version. The phylogenetic tree depicted in Figure 6 was produced using the maximum parsimony method and MEGA software Version 6.

Figure 6. Maximum Parsimony tree based on partial 16s rRNA gene sequence showing relationship between strain NRI-21 and related members of the genus Bacillus

Growth pattern and antimicrobial activity of the strain NRI-21:

Bacillus rugosus NRI-21's growth curve and antimicrobial profile were regularly examined for up to 72 h in batch culture. On the eighth hour, the strain entered the log phase, which lasted for 44 h before the stationary phase, which lasted for 48 h (Figure 7). Table 2 and Figure 8 show the antimicrobial spectrum of the strain that was cultivated on NA broth for 48 h. Secondary metabolites from the 44-hour-old culture shown potent antibacterial activity against Candida albicans, P. aeruginosa, E. coli, and P. vulgaris. Bacillus sp. strain FAS1, which was isolated from soil, shown the strongest antibacterial activity against Staphylococcus aureus, Klebsiella pneumonia, and Aspergillus niger (Moshafi et al., 2011).  Bacillus subtilis RLID 12.1 showed a broad spectrum of antibacterial efficacy against gram-positive bacteria (S. aureous, S. pyogenes), gram-negative bacteria (E. coli, P. aeruginosa), and fungi (Candida albicans, Candida glabrata), according to Ramachandran et al. (2014). The antibacterial ability of extracellular metabolites was demonstrated by a zone of inhibition that measured 12 mm against S. aureus, 11 mm against E. coli and E. faecalis, and 13 mm against Candida albicans (Sujitha et al., 2024). Escherichia coli, Klebsiella pneumoniae, Proteus vulgaris, Salmonella typhi, Serretia marcescens, and Staphylococcus aureus were the five test organisms against which the ethyl acetate extract of Bacillus flexus AVSC4 cultured in prepared media had strong antibacterial activity. This implies that under optimal culture conditions, AVSC4 generated potentially bioactive secondary metabolites.

Statistical Analysis:

* Microsoft Excel XP 2007 was used to calculate the mean ± standard deviation of the mean of three replicates.After statistical analysis, it is determined that the results are significant at the 5% level.

Figure 7. Growth pattern of the strain Bacillus rugosus NRI-21

Table 2. Antimicrobial activity of secondary metabolites produced by the strain NRI-21

Figure 8. Antimicrobial activity of cultures crude extract against Pseudomonas aeruginosa

1. NRI-21; B. NRI-11; C. NRI-17