Academia Journal of Agricultural Research 5(7): 162-168, July 2017 DOI: 10.15413/ajar.2017.0119 ISSN: 2315-7739 ©2017 Academia Publishing

Research Paper

Bacillus licheniformis from Hot spring of Rampur, Himachal Pradesh, India: Potential producer of thermostable α-amylase

Accepted 12th April, 2017 ABSTRACT Strains of Bacillus licheniformis isolated from the soil of hot water spring in Rampur district of Himachal Pradesh India were screened for the production of the thermostable α-amylase. Studies were carried out to observe the optimum temperature and pH for the production of α-amylase which was 70 and 8.0°C respectively. Various production parameters for B. licheniformis RA31 with accession no. KT989882 were optimized as carbon source soluble starch (1.5% w/v), nitrogen source tryptone and gelatin (0.5% w/v), metal ion MgCl2 (0.2% w/v), incubation time 72 h, incubation temperature 70°C, inoculum age 24 h and inoculum size of 0.4% v/v. Key words: Bacillus licheniformis, amylase, starch, thermophilic, alkaliphilic, tryptone.

INTRODUCTION The glycolytic pathway and the citric acid cycle stand as prototype for energy producing pathways in cells. These pathways vary from organism to organism but the basic enzymes of both glycolysis and the citric acid cycle seem to be conserved in the vast majority of organisms (Schnarrenberger and Martin, 2002).

Glycolysis is responsible for the breakdown of sugars, in particular, glucose which is a major source of energy for many organisms. Glucose is found in nature in large quantities, primarily in a polymeric state as cellulose, starch and glycogen. Cellulose and starch are produced predominantly in higher plants and glycogen is the main sugar storage polymer in animals.

Starch is formed from glucose units linked linearly by α(1→4) glycosidic bonds and branched off to another chain by α(1→6) glycosidic bonds. Starch is broken down by amylase which is produced from plants, animals and micro-organisms (Kaneko et al., 2005; Mohamed et al., 2011; Aygan et al., 2014). The production from plant and animal sources is limited for several reasons but amylase from microbes can be produced in high concentrations and

abundant quantity for the necessary industrial market requirements.

Amylases (α-amylase, β-amylase and γ-amylase) are among the most important enzymes in present-day biotechnology. The amylase family of enzymes is of great significance due to its wide area of potential application. Amylase is used in processed food industry for solubilization and saccharification of starch, alcohol fermentation in brewing, preparation of digestive aids, production of cakes, fruit juices and for converting starch into fructose, glucose and high maltose syrups (Nielsen and Borchert, 2000). They are also used to slow down the staling of baked products, reduction of viscosity of sugar syrups and haze formation in juices (Kent, 2003).

Amylases in detergent industry, especially, the liquid detergents are the second type of enzymes used in the formulation of enzymatic detergent (Gupta et al., 2003). In textile industry, amylases are used in the desizing process to remove starch from fabrics that acts as strengthening agents to prevent breaking of the warp thread during the weaving process (Ahlawat and Singhania, 2009).

Sharma H., Singh J., Batra N. and Jagtar Singh* Department of Biotechnology, Panjab University, Chandigarh, India. *Corresponding author. E-mail: jagtar@rediffmail.com. Tel: 91-172-2534085, +91-9876775160.

Academia Journal of Agricultural Research; Sharma et al. 163

In paper industry, coating treatment makes the surface

of the paper to appear smooth and strong, thus, improving the writing quality of the paper. Amylase was derived from fungi, yeast, bacteria and actinomycetes; however, enzymes from Aspergillus species and Bacillus sp. due to their industrial applications have dominated the industry.

Thermostable α-amylases are generally used as it decreases the contamination risk and saves energy by reducing the reaction time. Solubility of many reaction components such as polymeric substrates is significantly improved at higher temperature and hydrolysis carried out at higher temperatures also minimizes polymerization of D-glucose to iso-maltose (Pandey et al., 2000). Alkaline characteristics of amylase isolated from alkalophilic organisms are important features in the detergent industry (Van Der Maarel et al., 2002).

For the thermal stabilities of enzymes to be utilized in various fermentation processes, extreme thermophilic bacteria such as Rhodothermus marinus and mesophilic bacteria such as Bacillus megaterium, B. macerans and B. coagulans are generally selected and utilized (Saroja et al., 2000; Gimbi and Kitabatake, 2002). Most thermostable α-amylase utilized in the industry is produced from Bacillus licheniformis (Hmidet et al., 2010). Highly thermostable α-amylases are also obtained in hyperthermophilic and thermophilic Archaea such as Pyrococcus furiosus, Thermococcus hydrothermalis, T. profundus, Sulfolobus acidocaldarius and S. solfataricus (Goyal et al., 2005; Hernandez et al., 2006).

Due to the earlier mentioned advantages of thermostable enzyme and their operational stability and long shelf life, the bacterial diversity of hot water spring in Rampur district of Himachal Pradesh, India was explored. Among all the isolates, one with the best amylolitic activity was selected for the optimization of the media components and other process parameters. MATERIALS AND METHODS Sample collection, screening and isolation of bacterial isolates Samples were collected aseptically from various parts of hot water spring in Rampur district of Himachal Pradesh, India in sterile sample containers. Temperature and pH of sample site was noted. Samples were further processed in the laboratory within 24 h. Serial dilutions of the soil and water samples were spread onto Luria Bertani (LB) agar plates for separation of bacterial isolates after incubation of plates at 50°C for 24 h.

The isolates were incubated at 50°C for 24 h on starch agar plate and thereafter, plates were flooded with Lugol’s reagent (mixture of iodine-potassium iodide) to test for enzyme hydrolysis which was indicated by formation of clear zones around the colonies. Every colony that showed a clear zone was selected as an amylolytic isolate.

Identification of strains by morphological and biochemical methods

Out of forty two colonies, twenty three were amylase positive. Among these, eight were observed for morphological and biochemical characters (Aneja, 2003) and identified through molecular 16S rRNA methods (Jiang et al., 2006).

Identification of strains by Molecular methods (DNA extraction, 16S rRNA gene amplification, sequences and phylogenetic analysis) The pure cultures of the amylolytic bacterial strains were inoculated separately in 20 ml of LB and grown at 50°C for 24 h. Isolation of total genomic DNA from the cultures was then carried out (Sambrook et al., 1989). Molecular genetics identification of the isolates was done by amplification of 16S rRNA gene with bacterial universal primers 27F (5-AGAGTTTGATCCTGGCTCAG-3) and 1492R (5- CGGCTACCTTGTTACGACTT-3) (Frank et al., 2008).

PCR was carried out in a thermal cycler with total volume of 25 and 2 µl, respectively diluted genomic DNA with initial denaturation at 94°C for 180 s, followed by 35 cycles of 94°C for 60 s, 50°C for 45 s and 72°C for 105 s with a final temperature of 72°C extension for 5 min. Amplified product was then analyzed directly by electrophoresis on (1.5% w/v) agarose gel. Gel extraction of the PCR amplified DNA was done using QIAquick Gel Extraction Kit (QIAGEN).

PCR product after purification was used in sequencing reaction. Sequencing was performed using an automated sequencer ABI 3130 by BioServe Biotechnologies (India) Pvt. Ltd. Data from the dendrogram was mined using the BioEdit software and contigs obtained used for finding its homologs from NCBI.

Enzyme assay

One ml reaction mixture containing soluble starch (1% w/v) and 100 µl of the enzyme samples were incubated at 50°C for 10 min. 50 µl of each reaction mixture was used for the estimation of glucose by DNS method (Behal et al., 2006). The amount of reducing sugars liberated during the assay was estimated by measuring the color development at 540 nm by UV-VIS spectrophotometer. One unit of enzyme activity was defined as the amount of enzyme which releases 1 µmol of reducing group per ml per minute with standard enzyme assay.

Optimization of process parameters for enzyme production

Effect of temperature on α-amylase production The optimum temperature for the enzyme production was

Academia Journal of Agricultural Research; Sharma et al. 164 evaluated by growing culture at different temperatures ranging from 50 to 100°C.

Effect of pH on α-amylase production The effect of pH on amylase production was assessed by culturing the bacterium in media with different pH ranging from 4 to 10 and incubated at 70°C with 150 rpm. Enzyme activity was assessed as earlier described.

Optimization of media components for production of enzyme from selected isolated RA31 Basal media containing per liter: (soluble starch 10 g, MgSO4.7H2O 0.5 g and K2HPO4 0.16 g) was used. Crude enzyme obtained was assayed as per the protocol. The enzyme production was optimized by studying cultural and environmental variables like carbon, nitrogen, metal ions and additives in the basal media.

Optimization of carbon source The bacterial culture was grown in a basal media containing different carbon sources (glucose, fructose, xylose, sucrose and mannitol etc) in place of soluble starch (1% w/v).

Effect of organic and inorganic nitrogen source on production of amylase Basal media with different organic nitrogen sources like peptone, biopeptone, skim milk, gelatin, yeast extract, tryptone and beef extract (0.5% w/v) were used individually and in different combinations of tryptone and gelatin to study their effects on the production of enzyme. Inorganic nitrogen sources (ammonium sulphate, ammonium chloride, ammonium oxalate, urea and L-glutamic acid) were also checked for their utilization by isolate.

Effect of metals on production of enzyme Effect of metals on production of enzyme was assessed by inoculating the production medium with sulfates and chlorides of metal ions (Al3+, Ba2+, Ca2+, Fe2+, Fe3+, K+, Mg2+, Mn2+, Na+, Zn2+, Cu2+ and Ni2+). The enzyme activity was measured at regular intervals of incubation at 50°C for 96 h.

Inoculum age and inoculum concentration Production media was inoculated with 12, 24, 36 and 48 h,

respectively for old bacterial culture to optimize the inoculum age. Various concentrations ranging from 100 to 1000 µl of 24 h grown inoculum were incubated at 70°C and 150 rpm to optimize the inoculum concentration. The enzyme assay was carried out as earlier described after every 12 h. Effect of inhibitors on production of enzyme Media containing common inhibitors like SDS, EDTA, tween 20 and Triton X-100 (1% w/v) were used to study their effects on the production of enzyme and assay was performed as per the protocol (Figure 2). RESULTS Among the twenty-three (23) amylase positive isolates, eight (8) isolates with amylase activity were screened for further analysis. Morphologically, all were bacilli and gram positive and biochemically positive for amylase, catalase, oxidase and negative for indole and hydrogen sulphide test. They were identified as B. licheniformis by 16S rRNA gene sequencing. All the isolates were enzymatically active in a temperature range of 50 to 100°C and pH range of 7 to 10 but maximum activity was observed at 70°C and pH 8 respectively. Isolate RA31 showed maximum activity among these screened isolates. Figure 1 shows the maximum growth and enzyme activity in LB starch in 78 h and basal media in 72 h.

Table 1 shows the effect of different carbon sources on enzyme production where maximum enzyme activity was observed in soluble starch. Among the different concentrations of soluble starch (0.5, 1.0, 1.5, 2.0 and 2.5% w/v, respectively) maximum activity was observed at 1.5% w/v soluble starch at 72 h and activity further decreased with the increase in starch concentration.

Effect of different nitrogen sources individually in basal media showed increase in relative enzyme activity in the presence of tryptone and gelatin. Table 2 shows various combinations of basal media and nitrogen sources used. Combination of tryptone and gelatin (0.5% w/v) enhanced growth and enzyme activity. Inorganic nitrogen sources showed no significant increase in enzyme activity. 24 h old inoculum in 0.4% (v/v) concentration was optimized as the inoculum age and inoculum size respectively. Table 3 shows the effect of metal ions where Zn2+ > Cu2+ >Mn2+ were the potent inhibitors. Chloride derivatives of (Al3+ , Fe2+, Ca2+ and K+) reduced the enzyme activity to 90% sulphates of (Al3+, Na+ and Mg2+) and (Ba2+, Ca2+ and Ni+) reduced the activity to 80 and 70%, respectively. Chloride derivatives of (Na+ and Ba2+) and sulphates of (Fe2+ and K+) reduced the activity by 60 and 50% respectively when compared with the chloride derivative of Mg2+

.

Among the tested inhibitors and chelators, tween 20 and

Academia Journal of Agricultural Research; Sharma et al. 165

0

20

40

60

80

100

120

24 36 48 60 72 84 96 120

Re

lati

ve

am

yla

se a

ctiv

ity

an

d

ba

cte

ria

l g

row

th (

%)

Time (h)

Growth in BM

Growth in LBM

Activity in BM

Activity in LBM

Figure 1: Comparative growth of Bacillus licheniformis RA 31 and amylase activity in Luria broth media (LBM) and Basal media (BM).

Figure 2: Effect of inhibitors on the production of α-amylase from Bacillus licheniformis RA31. LB (luria broth), BT1 (Basal media and 0.5% tryptone), BT1G1 (Basal media, 0.5% tryptone, 0.5% gelatin), BT1G2 (Basal media, 0.5% tryptone, 1.0% gelatin), BT1G3 (Basal media, 0.5% tryptone, 1.5% gelatin), BT1G4 (Basal media, 0.5% tryptone, 2.0% gelatin), BT1G5 (Basal media, 0.5% tryptone, 2.5% gelatin), BSG2 (Basal media, sodium chloride, 1.0% gelatin), BSG2T1 (Basal media, sodium chloride 1.0% gelatin, 0.5% tryptone), BSG2T2 (Basal media, sodium chloride 1.0% gelatin, 1.0% tryptone), BSG2T3 (Basal media, sodium chloride1.0% gelatin, 1.5% tryptone), BSG2T4 (Basal media, sodium chloride 1.0% gelatin, 2.0% tryptone), BSG2T5 (Basal media, sodium chloride1.0 % gelatin, 2.5% tryptone).

Triton X-100 reduced the activity to 50% whereas EDTA> SDS> β mercaptoethanol further reduced the activity in the mentioned order.

DISCUSSION Most of the amylase producing bacteria reported is

Academia Journal of Agricultural Research; Sharma et al. 166

Table 1: Effect of different carbon sources on the production of α-amylase from Bacillus licheniformis RA31.

Enzyme activity U/ml

Carbon 72 h

Carbon 72 h

D Fructose 2.7 Sucrose 2.3

D Glucose 4.5 D Cellobiose 1.4

D Mannose 4.1 Lactose* 5.9

D Galactose 2.3 D Melibiose 4.1

Inositol 8.6 D Maltose* 2.7

Mannitol 0.9 Salicin 0

D Sorbitol 0.9 Aesculin 2.3

D Xylose 2.3 D Raffinose$ 2.7

D Arabinose 7.2 D Trehalose+ 1.8

D Ribose 5.9 Glycerol 1.4

Xylitol 0.5 Glycogen 23.1

Dulcitol 0 Starch 28.7

L Rhamnose* 1.8 (*) Monohydrate; (+) Dihydrate; ($ ) Pentahydrate

Table 2: Effect of different nitrogen sources on the production of α-amylase from Bacillus licheniformis RA31.

Relative enzyme activity (%)

Nitrogen 72 h

Nitrogen 72 h

LB 30.4 BT1 101.4

BSG2 62.3 BT1G1 100

BSG2T1 16 BT1G2 26.0

BSG2T2 21.7 BT1G3 18.8

BSG2T3 15.9 BT1G4 18.8

BSG2T4 14.5 BT1G5 17.3

BSG2T5 17.4

Table 3: Effect of different metal ions on the production of α-amylase from Bacillus licheniformis RA31.

Enzyme activity U/ml

Metal ions 0.005%

Selected metal ions with varied concentration

Metal 72 h

Metal 72 h Metal Concentration (%) †72 h

*Al3+ 25.8 †Al3+ 27.1

Al3+

0.005 27.6

*Ba2+ 25.3 †Ba2+ 16.3 0.01 12.2

*Ca2+ 25.8 †Ca2+ 26.2 0.02 12.7

*Fe3+ 26.7 †Fe3+ 27.1 0.05 2.7

*Fe2+ 20.3 †Fe2+ 5.9

Ca2+

0.005 25.8

*K+ 19.4 †K+ 20.3 0.01 8.5

*Mn2+ 12.2 †Mn2+ 12.2 0.02 10.8

*Mg2+ 26.7 †Mg2+ 27.6 0.05 12.6

*Na+ 26.2 †Na+ 20.8

Mg2+

0.005 27.29

*Zn2+ 0.9 †Zn2+ 0 0.01 28.00

*Cu2+ 3.2 †Cu2+ 0 0.02 28.95

*Ni+ 23.1 †Ni+ 26.7 0.05 6.32

*Representing metal sulphates and † as metal chlorides.

Academia Journal of Agricultural Research; Sharma et al. 167 acidophilic but B. licheniformis RA31 produced α-amylase at a high temperature of 70°C and that also in alkaline conditions, thereby, making this enzyme distinct from the reported literature. Based on enzyme activity at optimum temperature and pH, isolate RA31 was assorted for study. RA31 identified as B. licheniformis was assigned the accession no. KT989882. Optimum temperature of 70°C and pH 8.0 for the α-amylase production was same as described by (Bozic et al., 2011; Kiran and Chandra, 2008) respectively. With the use of basal media, the peak of growth and enzyme activity shifted to 72 from 78 h, respectively indicating the utilization of basal media components were more effective.

Starch is a very common carbon source in Bacillus species but tryptone and gelatin utilizing bacteria were reported in the researches of Zhang et al. (2015), Chakraborty et al. (2011), Rao and Satyanarayana (2007) and Malhotra et al. (2000) from hot water springs, thus, indicating B. licheniformis RA31 as a potential thermophile. Hiroshi et al. (2001) in their study observed a reduction in the presence of chloride derivatives (Al3+, Fe2+, Ca2+ and K+). Inhibition of activity by β mercaptoethanol indicated that histidine and disulphide bonds are essential for the activity (Kikani and Singh, 2011; Shafiei et al., 2010). Conclusion

Amylases are industrial important enzymes. Ample of plants, animals and microbial sources were explored for the production of the amylases but only few strains of fungi especially Aspergillus sp. and thermophilic Bacillius strains were able to meet the criteria for the commercial production. To use the rest of the microbes for the industrial purpose there is a need to study the structural and functional relationships of these unexplored microbes which would further intend to discover new enzymes with different properties.

AKNOWLEDGEMENT

The financial support provided by University Grants Commission, New Delhi, in the form of Basic Scientific Research Fellowship (No.F.7-148/2007 BSR) is highly acknowledged. REFERENCES Ahlawat S, Dhiman SS, Battan B, Mandhan RP, Sharma J (2009). Pectinase

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Cite this article as: Sharma H, Singh J, Batra N, Singh J (2017). Bacillus licheniformis from Hot spring of Rampur, Himachal Pradesh, India: Potential producer of thermostable α-amylase. Acad. J. Agric. Res. 5(7): 162-168. Submit your manuscript at http://www.academiapublishing.org/journals/ajar