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Partial characterization and optimization of alkaline amaylase production by Bacillus circulans from Lonar Lake

Tambekar D H, Raut N S and V R Dhundale

PG Dept. of Microbiology SGB Amravati University, Amravati 444602 (M.S.) India

diliptambekar@rediffmail.com

ABSTRACT

In India the Lonar Crater Lake, popularly called as the Lonar Soda Lake is situated in the Buldhana District of the Maharashtra State, which is occupied by saline water and harbors various unidentified, unique haloalkaliphilic bacterial species which produces thermo-alkaliphilic enzyme. The present study deals with isolation, production and partial characterization of amylase producing bacterial culture isolated from the alkaline Lonar Lake. Total thirty seven bacterial cultures were isolated by using different enrichment media. Out of thirty seven, six bacterial strains were selected on the basis of their amylolytic activity and then studied their phenotypic and biochemical characters. The strain CW2 (2) was selected for 16S rDNA sequencing and production, partial characterizations of amylase on the basis of their maximum amylolytic activity. Phylogenetic analysis based on 16S rDNA gene sequences indicated that strain CW2 (2) was affiliated to phylum Firmicutes with genera Bacillus. Newly isolated Bacillus circulans CW2 (2) strain was found to produce alkaline amylase at optimum pH 10 and optimum temperature was found at 50°C. The best substrate concentration 1250 µg/ mL and enzyme concentration 3500 µg/ mL was observed. The present studies indicated that the isolated Bacillus circulans CW2 (2) have ability to produced amylase which can be exploited for biotechnological potential.

Key words: Lonar lake, haloalkaliphiles, Bacillus, amylase.

INTRODUCTION The alkaline Lonar crater is situated in Buldhana district of Maharashtra state (Latitude 19º 58’, Longitude 76º 36’). Based on geological studies, it is postulated that the lake originated as a meteorite impact crater around 50-60 thousand years ago (Fredriksson et al., 1973). The uniqueness of Lonar lake is its salinity and alkalinity with pH 9.5- 10.5 (Joshi et al., 2007). Lonar Lake occupied by saline water which was formed by meteoritic impact on basaltic rock and it has been well known as an inland saline lake with a considerable amount of sodium carbonate and chloride (Kanekar et al., 2000). Extracellular enzyme like amylase, lipase, protease and celluloses producing bacterial strains were isolated from water and sediment samples of alkaline Lonar Lake (Joshi et al., 2007). Microbial enzyme could be usable resources in the pharmaceutical since the enzymes prepared with suitable properties with the advent of new knowledge in biotechnology, the spectrum of enzyme application has widened in many other fields, such as clinical, medicinal, and analytical chemistries, as well as their widespread

relevance in starch saccharification (Shanmughapriya, 2009). The enzyme has better resistance to alkali and some other denaturing chemicals in the reaction mixture. It is also thermostable organism growing in naturally alkaline habitats may how analyses with special characteristics (Tambekar and Tambekar 2012). Therefore, attempt was made to isolate new species of bacillus which can produce good quality of amylase useful in the detergent and textile industry. Alkaline amylase producing bacteria are of great importance in detergent and textile industry due to their high thermostability and pH stability and most important industrial enzymes, accounting for about 60% of total enzyme market (Kanekar et al., 2000; Borsodi et al., 2005). Very less study has been done on amylase from Bacilli of Lonar Lake which can withstand high temperature as well as high pH and has wide application in different industries. As there is large demand of amylase, isolation and production of amylase enzyme is most important to fulfill this demand (Srinivasan et al., 2009).

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Tambekar et al.,

The present study aims to deals with the isolation, purification, partial characterization of amylase producing bacteria isolated from the alkaline Lonar Lake. MATERIALS AND METHODS Collection Lonar lake water and sediment sample Total four sediment and eight, water samples were collected in year 2012 from alkaline Lonar Lake. Water samples collected in sterilized plastic bottles and sediments in sterilized plastic bags were transferred to laboratory for isolation and identification of bacteria followed by their screening for amylolytic activity. Isolation of Alkaliphiles The 1.0 g of soil sample was transferred to 100 mL sterilized distilled water in 250 mL conical flask and agitated (200 rpm) at 37°C for 15 min in shaker. The suspension was then diluted to 10-7 dilutions. One mL of each diluted sample was lawn into Petri Plates containing nutrient agar medium (pH 10) and inoculated at 37°C for 24 h. Screening of bacterial alkaliphiles Individual bacterial colonies were screened for amylolytic activities on Starch agar medium (Starch 1.0, Peptone 5.0, Yeast Extract 1.5, Beef extract 1.5, Sodium Chloride 5.0, Agar 20.0, pH 10). The pH of the medium was adjusted to pH 10 with 1N NaOH before and after sterilization. The inoculated plates were incubated at 37°C for 48 h, floods the iodine solution into the plate. The halo zone was observed for amylolytic activity of isolates. Identification of bacterial isolates The bacterial identifications based on morphological, cultural and biochemical characteristics. The isolates were also tested for their growth at different temperatures and pH and NaCl concentration (Joshi et al., 2007). These isolates were identified in accordance with Bergey's Manual of Systematic Bacteriology (Sneath, 1986). The identified strains were maintained on nutrient agar slants having pH 10 at 4.0°C. The selected strains were then analyzed by 16S rRNA at NCCS, Pune and BLAST identification was made. Phylogenetic Analysis The 16S rDNA sequences were analyzed via RDPII using SEQUENCE-MATCH (Version 2.7) to identify the most closely related database sequences. Multiple Sequence Alignment of approximately 900 bp sequence was done by using Clustal X version 1.8. The phylogenetic tree was constructed from evolutionary distances using the neighbor-joining

method of Mega 4 program package (Kumar et al., 2004). Preparation of crude enzyme extracts The 100 mL Starch nutrient medium (Starch 1gm, Peptone 5.0, Yeast Extract 1.5, Beef extract 1.5, Sodium Chloride 5.0, pH 10). The sterile broth was inoculated with culture and incubated for 48h at 37°C in incubator. After 48 h incubation, centrifuged the broth at 5000 rpm for 15 min. The supernatant served as crude enzyme source. Optimization of crude amylase enzyme The standard graph of maltose was prepared by adding different concentration of standard maltose (1 mg/mL) into a series of test tubes and added 2mL DNS solution incubate all tubes in boiling water bath for 5 min after incubation add 1mL NaK tartarate to stop the reaction. Estimation of Amylase Estimation of amylase was carried out with 2.5 mL of starch in a test tube; 2.5 ml of PO4 butter, 1mL of NaCl and 1 mL of enzyme source was added and incubated for 15 min at 370C. After incubation 1 mL of DNS+NaoH solutions was added and incubates in boiling water bath for 5 min after incubation 1 mL of Nak+ tartarate was added to stop the reaction. Characterization of Amylase: Effect of pH on alkaline amylase activity The effect of pH on alkaline amylase was determined by assaying the enzyme activity at different pH values ranging from 6.0 to 12 using the PO4 buffer systems with concentration of buffer was 0.2 M. Effect of temperature on alkaline amylase activity The effect of temperature on alkaline amylase activity was determined by incubating the reaction mixture (pH 10) for 15 min at different temperature ranging from 30°C to 80°C. Effect of substrate on alkaline amylase activity The effect of substrate concentration on alkaline amylase activity was determined by incubating the reaction mixture for 15 minutes with different substrate concentration, ranging from 0.5 mg / mL to 4 mg/mL. Effect of enzyme on alkaline amylase activity The effect of enzyme concentration on alkaline amylase activity was determined by incubating the reaction mixture (pH 10) for 15 minutes at different enzyme concentration ranging from 0.5mL to 4mL. The activity of the amylase was then measured as per assay procedure.

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RESULTS AND DISCUSSION In the present study a total of 37 bacterial cultures were isolated from water and sediment sample of Lonar Lake, maintained on slant of nutrient agar (pH 10). Then these cultures were inoculated on alkaline starch agar at pH 10 for studying their amylolytic activity. Out of thirty seven, twenty one

showed amylolytic activity. Out of twenty one, six bacterial culture were found prominent amylase producer. The strain CW2(2) select for further characterizations, screening was done on the basis of maximum amylolytic activity and subjected to phenotypic, biochemical characteristics and 16S rDNA sequencing.

Table 1. Biochemical characteristic of Bacillus circulans CW2(2) isolated from Lonar Lake

Morphology

Gram character +

Growth at pH

pH 7 +

Utilization of

Glucose +

Shape of Bacteria Rod pH 8 + Arabinose +

Arrangements of Cell

Chain pH 9 + Mannitol +

Motile + pH 10 + Xylose -

Spore

Spore bearing + pH 12 + Lactose -

Position Central Growth at NaCl

1% NaCl + Trehalose +

Shape Cylindrical 7% NaCl + Sucrose +

Swollen Sporangia -

Biochemical Characters

Catalase + Cellobiose -

Culture characters

Pigment White Oxidase - Galactose -

Shape Irregular Indol - Maltose -

Elevation Umbonate MR - Fructose +

Edge Undulate VP + Salicin -

Growth at temperature

45º C + Citrate + Hydrolysis

of

Starch +

50º C + Urease - lipid +

55º C - Nitrate + Casein +

Fig 1: Phylogenetic tree based on a comparison of the 16S ribosomal DNA sequences of Lonar lake isolates and some of their closest phylogenetic relatives. The tree was created by the neighbor-joining method. The numbers on the tree indicates the percentages of bootstrap sampling

Bacillus circulans strain B230 GU9046...

Bacillus sp. KZ AalM Mm2 GU726177

Bacillus sp. KZ AalF Mm4 GU726174

Bacillaceae bacterium KVD-1982-07 DQ4...

Bacterium L194WBS.88 EU935293

Bacillus sp. R-33179 AM910332

Bacillus sp. R-30632 AM910246

Bacillus circulans NBRC AB271747

Bacillus sp. C1(2010b) HM171927

Bacillus circulans N3 AB215100

Bacillus sp. M3T4B4 GQ246730

Bacterium L194WBS.184 EU935241

Bacillus circulans RIGLD BC1 HQ315829

Bacillus circulans ATCC 4513 NCDO1775

Bacillus circulans D1 EU116046

Bacillus circulans CW22 JQ319541

64

0.005

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Tambekar et al.,

The morphologically CW2 (2) was single cell arrangement with rod shaped, Gram positive. Cells were highly motile by flagella and produce spores that were ellipsoidal and lie at central position in swollen sporangium with capsulated. Culturally the strain CW2(2) were white pigmented and characteristically very small 1 mm in diameter, shape was toruloied and elevation was effuse with undulate edges along with arboresent internal structure. The CW2 (2) strain were able to grow at pH 7 - 12 and 0.5 - 7% NaCl concentrations. The strain CW2(2) produced the catalase, oxidase and not produced indole and positive for the methyl red and Voges Proskauer test, citrate was utilized. Nitrate was not reduced to nitrite and unable to hydrolyze the urea. The glucose, mannitol, arabinose, sucrose, cellobiose, raffinose, galactose, salicin, sorbitol were utilized but maltose, fructose, xylose and trehalose were not utilize by CW2 (2) strain. Bootstrap analysis was used to evaluate phylogenetic tree stability according to a consensus tree from the neighbor-joining based on 1,000 replicates for each. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain CW2(2) was affiliated to phylum Firmicutes with genera Bacillus (Fig 1). The highest similarity values with the sequences of obligately alkaliphilic and alkalitolerant, aerobic endospore forming bacteria. Lonar lake strain CW2(2) was showed high similarity to B. circulans D1 EU116046. The stipulations prevailing in the industrial relevance in which enzymes are used rather extreme, particularly with regards to temperature

and pH. Therefore, there is a continuing attention to become better the stability of enzymes and to encounter the fulfillment set by particular applications. In this respect, thermostable enzymes have been proposed to be industrially relevant (Tambekar and Tambekar, 2012; Prakash and Jaiswal, 2010). Members of diverse genera have been reported to produce different types of enzymes. So far not many amylases with optimum activity under alkaline conditions have been studied from the various soda lakes (Martins et al., 2001; Hashim et al., 2004). Alkaline amylase production was maximum at pH 9-10.5. Maximum amylase production was recorded after 48 h of incubation at 37°C. In the effect of substrate concentration on enzyme activity of amylase, the Michalies Menten constant (KM) and Maximum velocity (VMax) was found to be 1250 µg/ml and 400 µg/ml (fig 3) by Line weaver-Burk plot. The optimum enzyme concentration required for maximum activity of amylase 3500 µg/ml (fig 5). The optimum pH and temperature required for maximum activity of amylase was 10 (Fig 6) and 50ºC respectively (Fig 7). The Bacillus circulans species from Lonar Lake produce alkaline amylase and maximum growth at pH 7-12. The isolated bacterial Bacillus circulans strain produces the amylase enzyme which has theomorphic, alkaliphilic and has potential to produce good quality amylase which can use in the industry. The Bacillus circulans species were most efficient for amylase producing at pH 10 incubated at 370C for 48 h.

0

2

4

6

8

10

12

14

16

18

20

CW

2(2

)

D5

B8

OC

W3(1

)

B5

AW

3(2

)

BS

1(1

)

DW

2(3

)

DW

2(5

)

OB

W3(2

)

BW

4(3

)

CS

4(1

)

DW

1(1

)

A1

CS

1(1

)

DW

4(1

)

A2

BW

2(1

)

AW

4(3

)

BW

3(2

)

B4

Zon

e st

arc

h h

yd

roly

sis

(mm

)

Bacterial Strains

Fig 2: Zone of amylolytic activity of bacterial strains

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The amylase produced from this species was highly efficient at thigh temperature, and tolerate the other environmental conditions. Lonar Lake bacterial strains were promising source of the extracellular enzyme producer and exhibiting

alkaline thermostability making it valuable tool in industrial enzyme production. Amylase enzyme has importance in various industries like detergent industry, textile, paper industries etc (Horikoshii et al., 2006; Tambekar and Dhundale, 2012).

Series 1

f(x)=3.4754*x+2.6059; R²=0.9855

-9 -8 -7 -6 -5 -4 -3 -2 -1 1 2 3 4 5 6 7 8 9

-8

-6

-4

-2

2

4

6

8

x

y

1 / Vmax = 1 / 0.4

= 2.5

1 / - Km = 1 / - 0.80

Km = 1.25

X - Axis 2 cm = 0.5 mg / mL

Y - Axis 1 cm = 1 mg / mL

-1 / [S] 1 / [S] --->

Fig 3: Line weaver-Burk plot

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0 1 2 3 4 5

Con

ce

ntr

atio

n o

f M

alto

se

Substrate concentration

Fig 4: Effect of substrate concentration on Amylase

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

0 2 4 6

Co

nce

ntr

atio

n o

f M

alto

se

Enzyme Concentration

Fig 5: Effect of enzyme concentration on Amylase

0

0.1

0.2

0.3

0.4

0.5

0.6

0 1 2 3 4 5 6 7 8 9 10 11 12 13

Co

nce

ntr

atio

n o

f M

alto

se

pH

Fig 6: Effect of pH on Amylase

0

0.2

0.4

0.6

0.8

1

1.2

0 20 40 60 80 100

Con

ce

ntr

atio

n o

f m

alto

se

Temperature

Fig 7: Effect of temperature on Amylase

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Tambekar et al.,

In the present investigation, we have determined the optimum parameters for maximum production of alkaline amylase by the newly isolated thermophilic bacterium Bacillus circulans. The current study was carried out with the purpose of defining amylolytic enzymes in haloalkaliphilic bacterial strain isolates, which are adapted to live at extreme saline and alkaline environments. Following the discovering of the amylolytic

enzymes, they were further characterized to determine their tolerance with pH, temperature substrate concentration and enzyme concentration. The amylolytic enzymes from our halophilic isolates are also active under high temperature 50°C and pH 10. The abilities of Bacillus circulans CW2(2) to produce extracellular amylase suggest that they might be of potential value for biotechnological exploitation.

LITERATURE CITED Borsodi AK, Micsinai A, Rusznyak A, Vladar P, Kovacs G, Toth EM, Marialigeti K, 2005. Diversity of alkaliphilic and alkali tolerant bacteria cultivated from decomposing reed rhizomes in Hungarian Soda Lake. Microb ecol., 50: 9-18. Fredriksson K, Dube A, Milton DJ and Balasundaram MS, 1973. Lonar Lake, India: An impact crater in basalt. Sci., 180 (4088): 862-864, doi: 10.1126/Science.180.4088.862. Hashim SO, Delgado O, Kaul RH, Mula FJ and Mattiasson B, 2004. Starch hydrolysing Bacillus halodurans isolates from a Kenyan soda lake. Biotechnol Lett., 26: 823–828. Horikoshii K, Diurak M, Kojima M, Kanai M and Inoue A, 2006. Isolation and characterization of a feather-degrading enzyme from Bacillus pseudofirmus FA30-01. Extremophiles, 10:229–235. Joshi AA, Kanekar PP, Kelkar AS, Shouche YS, Wani AA, Borgave SB and Sarnaik SS, 2008. Cultivable bacterial diversity of alkaline Lonar Lake, India. Microb Ecol., 55(2): 163- 172. Kanekar PP, Nilegaonkar SS, Sarnaik SS and Kelkar AS, 2000. Optimization of protease activity of alkaliphilic bacteria isolated from an alkaline lake in India. Bioresource Technol., 85(1): 87- 93. Kumar S, Tamura K and Nei M, 2004. MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform., 5:150–163. doi:10.1093/bib/5.2.150. Martins RF, Davids W, Abu Al-Soud W, Levander F, Radstrom P and Hatti-Kaul R, 2001. Starch hydrolyzing bacteria from Ethiopian soda lakes. Extremophiles, 5: 135–144. Prakash O and Jaiswal N, 2010. Amylase: An Ideal Representative of Thermostable Enzymes. Appl Biochem Biotechnol., 160: 2401-2414. DOI 10.1007/s12010-009-8735-8734. Shanmughapriya S, Kiran GS, Selvin J, Gandhimathi R, Baskar TB, Manilal A and Sujith S, 2009. Optimization, Production, and Partial characterization of an alkaliphilic amylase produced by sponge associated marine bacterium Halobacterium salinarum. MMD047. Biotechnol Bioproc Engi., 14: 67-75, DOI/ 10.1007/s12257-008-0060-1. Sneath PHA, 1986. Endospore forming Gram Positive Rods and Cocci. In: Murray RGE, Brenner DJ, Bryant MP (eds) Bergey’s manual of systematic bacteriology, 1st edn. Williams and Wilkins, Baltimore, Md, 1104–1207. Srinivasan TR, Das Soumen, Bal Krishnan V, Philip R and Kannan N, 2009. Isolation and characterization of thermostable protease producing bacteria from tannery industry effluent. Rec Res Sci Technol., 1(2): 63-66. Tambekar DH and Dhundale VR, 2012. Studies on the physiological and cultural diversity of bacilli characterized from Lonar Lake (MS) India. Biosci Disco., 3(1):34-39. Tambekar DH and Tambekar SD, 2012. Partial characterization and optimization of alkaline protease production of Bacillus pseudofirmus from Lonar Lake. Int J Adv Pharm Biol Sci., 2(1): 130-138.

How to Cite this Article: Tambekar D H, Raut N S and V R Dhundale, 2013. Partial characterization and optimization of alkaline

amaylase production by Bacillus circulans from Lonar Lake. Biosci. Disc., 4(2):202-207.