Isolation and Biochemical Characterization of an Acidophilic, Detergent-Stable Amylase-producing strain of Providencia rettgeri from the soil of Patnitop region, J&K

 

Afreen Anwar, Daljeet Singh Dhanjal, Reena Singh, Chirag Chopra*

School of Bioengineering and Biosciences, Lovely Professional University, Phagwara, Punjab, India.

*Corresponding Author E-mail: chirag.18298@lpu.co.in

 

ABSTRACT:

Amylases are almost ubiquitous and their vital role in biotechnological industries and process such as starch saccharification, detergent, food, and textile industry have prompted the isolation of microbes from different niches. Thus, our current study focusses on the isolation of amylase producing bacteria from Patnitop region. After isolation, the bacterial strain was characterised, during which the highest activity of the enzyme was found to be at 40oC and pH 2.0. The metal ion Mg2+ induced amylase activity whereas detergents like SDS, CTAB could not inhibit the enzyme. The bacterial strain showed significant stability under acidic conditions. On performing 16S rRNA sequencing, the amylase producing bacteria were identified to be Providencia rettgeri.

 

KEYWORDS: Amylase, Characterization, Jammu and Kashmir, Metagenomics, Providencia rettgeri.

 

 


INTRODUCTION:

In Patnitop, a tourist spot in Udhampur area situated in Jammu and Kashmir, the micro organisms adapt to the intense cold conditions because of cold weather around the year. This area is an unexplored reservoir of resources, from wheremicrobes can be isolated, which synthesise the enzymes of industrial importance. Enzymesare defined as biocatalysts which accelerate the chemical reaction at a specific rate to complete the metabolic processes inside the cell1. Enzyme functionality is not limited to metabolic processes, but it plays a vital role in the inter-conversion of macromolecules. Enzymes are also involved in proliferation, repair and maintenance of the cell2. Henceforth, microbial enzymes must be explored as they consume fewer resources, have a high yield, do not involve in any socio-political issue and can be genetically engineered3. Thus, for their commercial value with desirable traits and low cost, the enzyme isolation and characterisation has become the primary focus of the research.

 

Amylase, a hydrolytic enzyme which hydrolyses the α-1,4 and α-1,6 glycosidic linkage to glucose, is of great industrial importance4. Amylases (specially α-amylase) have various applications in industries such asbakery, carbonated drinks, wine, dairy, pharmaceutical, textile, leather, paper, and food processing industries. Animals, microbes and plants synthesise amylase enzyme and among these,microbes are of commercial choice5,6. Amylases have also been reported to show anti-oxidant and anti-fungal properties7. As the microbes require less space and less time for proliferation, their genes can be easily manipulated8. Several microbes have been reported which synthesise the extracellular amylasessuch as Clostridium thermosulfurogenes9, Thermococcus profundus10, Halomonas meridiana11, Bacillus licheniformis12, Aureobasidium pullulans13, Bacillus subtilis14,15,16, Bacillus megaterium17, Thermotoga neapolitana18, Bacillus amyloliquefaciens19, Bacillus cereus20, Lactobacillus plantarum21, Pseudomonas stutzeri22, Comomonas kerstersii23, Pseudomonas aeruginosa15), Aspergillus niger24, Penicillium notatum25, Aspergillus oryzae26, marine actinomycetes27, Streptomyces sp.28,29.

 

The present work was carried-out to isolate and characterise the novel amylase producing cryptic bacterial strain form the Patnitop, J & K. Assessment of the effect of varied cultural conditions and optimisation of these conditions was done to attain high enzyme productivity as well as enzyme activity from the isolated strain. Identification of the isolated strain was done by biochemical tests and 16S rRNA sequencing.

 

MATERIAL AND METHODS:

Soil Collection:

The sample of soil sediment was taken from the hilly area of Patnitop Region (33.0903 N - Latitude and 75.3264 E - Longitude) in J&K, India. The sample was collected using a clean tool and stored in as terile zip-lock bag in an ice-box. After that, the sample wastransported to the lab and kept at 4C for future use.

 

Bacterial Culture Isolation and Primary Screening:

Isolation of bacterial culture was done by inoculating 100mg of both soil samples collected from the different sites in 2ml of Peptone water (2gm of Peptone in 100ml of Distilled Water) and serial diluted to 10-3-fold. After, serial diluting to100 and 1000-fold, diluted samples were spread (in duplicate) on enrichment medium (comprising of 2g Agar, 500mg Starch, 0.25g KH2PO4, 1.43g K2HPO4, 1.96g Tryptone and 0.5g NaCl per 100mLvolume of medium). The flask was then kept in a bacteriological incubator for overnight at 37C. On observing these plates, six distinct colonies were marked based on their texture and morphology. The pure cultures were isolated by repeated sub-culturing on LB agar plates. Primary screening of amylase producers was done by staining the plates with iodine and checking for the zone of hydrolysis.

 

Secondary Screening of the Pure Cultures:

The secondary screening was done to check for enzyme localisation. Amylase positive colonies were cultured and the growth media was separated by centrifugation. The cell pellet was lysed using bead-beating lysis and cell-free extract (CFE) was prepared. Both CFE and media were checked for amylase activity using the standard DNS assay31. Enzyme activity was calculated by comparing the absorbance values to a glucose standard curve. Unit definition of amylase implied the concentration of enzyme that releases 1 micromole of glucose equivalent per-minute at standard experimental conditions30. All experiments were conducted in triplicates.

 

Biochemical Characterization of Amylase:

Variation of Enzyme Activity with Temperature:

The temperature-optima was determined by estimating the enzyme activity at temperatures ranging from 20-90C. The enzyme extract (comprising 300l of Phosphate Buffer, 500l of 1% Starch and 200l of enzyme) was incubated at varied temperatures ranging from 20-90C for 30 minutes. The activity was assessed using the standard DNSAmethod.

The dependence of enzyme stability on temperature was assessed by pre-incubating the mixture of 300l of Phosphate Buffer and 200l of thee nzyme at various temperatures for 60 minutes (ranging from 20-90C). 500uL of starch substrate was added the tubes, and the reaction mix was kept at 40oC for 30 minutes. Enzyme activity was estimated using the standard DNSA method.

 

Variation of Enzyme Activity with pH Change:

The optimum pH for enzyme activity was found by evaluating the enzyme activity upon varying pH by the reaction mixture (comprising 300l of Buffer, 500l of 1% starch and 200l of enzyme) by employing the following buffers, each of concentration 0.1MKCl (pH 1 and 2), Sodium Acetate buffer (pH 3.0 to 6.0) and Tris-HCl (pH 7.0 to 9.0). The reaction was performed at 40oC for 30 minutes. The enzyme activity was assessed by DNSAmethod.

 

The enzyme stability with varying H was assessed by pre-incubating the mixture of 300l of the buffer of different pH and 200l of then zyme for 60 minutes. Next, 1% of starch was added to the mix, and the reaction was incubated at 40oC for 30 minutes. The enzyme stability was assessed by the standard DNSAmethod32, using the unit definition and the glucose standard curve.

 

Optimum Ionic Cofactor for the Enzyme:

The optimum metal ions for the enzyme was assessed by performing the amylase assay with or without 2mM or 5mM metal ions. The following metal ions were used Ca2+, Mg2+, Co2+, Zn2+, Fe2+, Mn2+, and Cu2+and Hg2+ to assess the effect of metals ions on the amylase activity. All the reactions were thencarried out at 40oC for 30 minutes and the enzyme activity was quantified by the standard DNSA method31,32,33.

 

Study on Enzyme Inhibition

Amylase inhibition was studied by incubating the reaction mix (300l of Phosphate buffer, 500l of substrate and 200l of enzyme) in presence or absence of 2uL or 5uL of various inhibitor stock solutions. The following stock solutions of inhibitors were used 30% H2O2, 10% SDS, 10% Triton X100, 10% Tween20, 8M Urea, 10% CTAB, 6M Guanidine hydrochloride, 100mM PMSF to evaluate the inhibition of the amylase by the inhibitors. All the reactions were carried at 40oC for 30 minutes and amylase activity was quantified by the standard DNSA method31,34,35.

 

Screening of Bacterial Strains by Biochemical Tests:

The Gram Staining, Endospore Staining and Biochemical Tests named Catalase, Urease, Indole, Oxidase and Mannitol Agar test were performed according to the protocol of Cappucino and Shermann (2008)36.

 

Molecular-Based Characterization:

16S ribosomal RNA genotyping was performed at a third-party company Yaazh Xenomics. The 16S sequence that was obtained was analysed using the nucleotide BLASTn tool of NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi). BLAST results were analysed for identity, query coverage as well as the accession number of the subject with which the query shared similarity.

 

RESULTS AND DISCUSSION:

Bacterial Culture Isolation and Primary Screening:

The isolation of the bacteria was done by inoculating the enrichment media with the soil sample for overnight. Six distinct colonies were selected, and pure cultures were isolated. Primary screening was performed on starch agar plates followed by staining with iodine. Upon staining, three colonies (2, 4 and 6) showed the clear hydrolysis zone as illustrated in Figure. 1.

 

 

Fig. 1: Starch Agar plates showing zone of hydrolysis by amylase producing bacteria

 

Secondary Screening of the Pure Cultures:

Amylase producing cultures were named PTamy002, PTamy004 and PTamy006. The secondary screening was performed by checking enzyme localisation through the standard DNS assay. Culture media and CFE were incubated in a reaction mix with or without 1% starch and reaction buffer. Enzyme activity was represented in IU/mL. It was found that the enzyme was liberated out of the cell as the activity was found in the culture medium.

 

Biochemical Characterization of Amylase:

Variation of Enzyme Activity with Temperature:

The variation in enzyme activity with temperature was studied by using the standard amylase assay at varying temperatures, followed by quantification of glucose released. It was observed that the enzyme showed the highest activity of 114IU/mL at 40oC (Figure. 2a). The activity was significant until the temperature of 70oC as more than 50% of optimum activity was retained. The stability of the enzyme was determined by incubating the enzyme at varying temperatures followed by amylase assay at optimum temperature. It was found that the enzyme was the most stable at 40oC (Figure. 2b). At this temperature, the maximum amylase activity of 0.054001μg/ml was observed.Most mesophilic bacteria synthesise amylase at 37-60C32. The result of optimised temperature and stability showeda positive correlation with the amylase producing bacterial species, which showed optimum activity in the range of 40-45C37,38.


 

 

Fig 2: Biochemical Characterization of Amylase; a) Temperature Optimum, b) Effect of Temperature on Amylase Stability, c) pH Optimum, d) Effect of pH on Amylase Stability, e) Effect of Metal ions on Amylase Activity, f) Effect of Inhibitors on Amylase Activity.

 

 


Table 1: Morphological and Biochemical Testing of Amylase-producing Bacteria

Sample

Gram Staining

Endospore Staining

Catalase

Urease

Indole

Oxidase

Mannitol Salt Agar

PTAmy001

+

-

+

+

-

+

-

 


 

Variation of Enzyme Activity with pH Change:

The impact of pH on enzyme activity was studied by carrying out amylase assay in buffers of varying pH, followed by estimation of glucose production. It was found that the enzyme showed the highest activity (157.08 IU/mL) at a low pH of 2.0, which remained stable till pH 4.0 as it retained more than 50% of optimal activity (Figure. 2c). This meant that the amylase PTamy002 was acidophilic. The stability was also impacted upon by variations in pH. It was observed that the amylase showed maximum stability at pH 2.0 (Figure. 2d). Most amylase producing bacteria are found to be active in the pH range of 6-738. However, Providencia rettgeri showed stability in the alkaline pH (i.e. 8), which showed a similar result with thermostable Bacillus sp.39.

 

Optimum Ionic Cofactor for the Enzyme:

Amylase assay was performed in presence or absence of metal ions (2mM or 5mM). Each reaction was followed by estimation of glucose production. It was found that the most favourable ion for our amylase was Mg2+ at a concentration of 5mM yielding the enzyme activity 318.72 IU/mL, which was more than 200% of the control (no metal ions). Whereas magnesium and calcium ions increased the enzyme activity, it was found that cobalt, zinc, iron (III), Cu (II), and Hg (II) inhibited the enzyme activity (Figure. 2e). According to previous reports, Mg2+ ion was the best inducer for the microbial growth as well as to aide in amylase synthesis. Various reports have favoured the Mg2+ion, as the best inducer of amylase activity as seen in Bacillus sp.40,41.

 

Study on Enzyme Inhibition:

Inhibition of enzyme by hydrogen peroxide, sodium dodecyl sulphate, Triton X100, Tween20, Urea, CTAB, Guanidine hydrochloride and phenylmethanesulphonyl fluoride was studied. It was found that Triton X-100 at a concentration of 0.02% increased the amylase activity by 24%. Also, 0.02% of CTAB increased amylase activity by 80%. SDS, Tween-20, 0.05% CTAB and 0.05% Triton X-100 did not affect the enzyme activity. Whereas H2O2, 8M urea and 6M guanidine hydrochloride reduced the activity of the enzyme (Fig. 2f). Thus, it could be concluded that the amylase PTamy002 was detergent stable. Antony et al. (2014) have also reported that the amylase showed stable activity in the presence of detergents SDS as well as CTAB. Previous reports have stated Bacillus sp. Calp12-7 to be SDS-stable, and Klebsiella SSTA2 was stable in presence of detergents SDS, CTAB and Tween 8042,43.

 

Characterisation of Bacterial Strain for Gram Staining, Endospore Staining and Biochemical Tests:

The select biochemical tests and staining were conducted per the standard protocols. The result obtained for the bacterial strain for gram staining, endospore staining and the biochemical testis depicted inTable.1.

 

Molecular-based strain identification:

Strain identification by PCR amplification of 16S rRNA sequencing was carried out through Yaazh Xenomics, Chennai, India.

 

The sequence was analysed using the BLASTn tool of NCBI. The sequence was aligned to the 16S rRNA database of Bacteria and Archaea. The results showed similarity with the 16S rRNA gene sequence of Providencia rettgeristrain SU18-12. The sequence showed 86.77% identity to the Providencia rettgeri16S rRNA gene with a query coverage of 94%. The organism has been reported previously to be amylase positive44.

 

CONCLUSION:

The amylase producing bacteria was identified as Providencia rettgeri. This bacterial strain showed optimum activity of amylaseata temperature of 40oC and pH2.0. The enzyme activity was stable till a temperature of 70oC and pH 4.0. The metal ion, Mg2+ was found to be the best inducer of the amylase activity. H2O2, urea and guanidine hydrochloride inhibited the amylase. This study shows that these gram-positive, spherical-shaped, non-endospores-producing bacteria acquire the ability to produce the amylase enzyme, which can have significant applications in the field of biotechnology and it assures to serve the industrial purpose. The biochemical tests showed the strain to be positive for catalase, urease and oxidase and tested negative for endospore staining, indole production and Mannitol salt agar test. In future, the bacterial strain can be engineered to achieve functionality under harsh conditions of temperature, to produce a potent enzyme with abroad spectrum of industrial applications. An example of future application of amylase is in production of alcohol by fermentation45.

 

ACKNOWLEDGEMENT:

The authors thank the senior administration of Lovely Professional University for providing support for completion of the project.

 

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Received on 20.01.2020 Modified on 11.03.2020

Accepted on 17.04.2020 RJPT All right reserved

Research J. Pharm. and Tech. 2020; 13(12):5958-5962.

DOI: 10.5958/0974-360X.2020.01040.9