a temperature and salt-tolerant l-glutaminase from gangotri region of uttarakhand himalaya: enzyme...

A Temperature and Salt-Tolerant L-Glutaminasefrom Gangotri Region of Uttarakhand Himalaya:Enzyme Purification and Characterization

Lokendra Kumar & Balvinder Singh &

Dilip Kumar Adhikari & Joydeep Mukherjee &

Debashish Ghosh

Received: 24 April 2011 /Accepted: 19 January 2012 /Published online: 26 February 2012# Springer Science+Business Media, LLC 2012

Abstract Purification and characterization of halotolerant, thermostable alkaline L-glutaminase from a Bacillus sp. LKG-01 (MTCC 10401), isolated from Gangotri region ofUttarakhand Himalaya, is being reported in this paper. Enzyme has been purified 49-fold fromcell-free extract with 25% recovery (specific activity 584.2 U/mg protein) by (NH4)2SO4

precipitation followed by anion exchange chromatography and gel filtration. Enzyme has amolecular weight of 66 kDa. L-Glutaminase is most active at pH 11.0 and stable in the pH range8.0–11.0. Temperature optimum is 70 °C and is completely stable after 3 h pre-incubation at50 °C. Enzyme reflects more enhanced activity with 1–20% (w/v) NaCl, which is furtherreduced to 80% when NaCl concentration was increased up to 25%. L-Glutaminase is almostactive with K+, Zn2+, and Ni2+ ions and Km and Vmax values of 240 μM and 277.77±1.1 U/mgproteins, respectively. Higher specific activity, purification fold, better halo-tolerance, andthermostability would make this enzyme more attractive for food fermentation with respect toother soil microbe derived L-glutaminase reported so far.

Keywords L-Glutaminase . NaCl . Thermostability . Alkaline pH

Appl Biochem Biotechnol (2012) 166:1723–1735DOI 10.1007/s12010-012-9576-0

L. Kumar : B. SinghDepartment of Biochemistry, Sardar Bhagwan Singh PG Institute of Biomedical Sciences and Research,Balawala, Dehradun 248161, India

L. KumarDepartment of Biochemistry, Dolphin PG Institute of Biomedical and Natural Science,Dehradun 248007, India

D. K. Adhikari :D. Ghosh (*)Biotechnology Area, Indian Institute of Petroleum (CSIR), Dehradun 248005, Indiae-mail: [email protected]

J. MukherjeeSchool of Environmental Studies, Jadavpur University, Kolkata 700032, India

Introduction

L-Glutaminase has inculcated significant buzz in food industry as a potential flavor-modulating agent, imparting a savory flavor as it increases food’s glutamic acid content. Itfinds application in food fermentation by hydrolyzing L-glutamine to produce highly savoryamino acid L-glutamic acid imparting a unique taste called umami and thereby regarded as akey enzyme that controls the delicious taste of fermented foods [1, 2]. Still, there is plenty ofroom for research on glutaminases including the isolation of salt- and thermotolerantenzymes, which would significantly enhance their applications in the food industry [3].

Therefore, salt-tolerant glutaminase may play potentially significant roles in food fer-mentation process that require high-salt environments. The two isozymes of Micrococcusglutaminase (I and II) were found to be highly salt tolerant. Glutaminase I was stable andexhibited about 1.3-folds higher activity in the presence of 8–16% NaCl than in the absenceof NaCl, while glutaminase II was not stabilized and activated under the same condition.These properties of glutaminase I except for its optimal pH (pH 8) render it suitable for usein food fermentation [4]. Halophiles are being adopted extensively in food industries as feedadditives for their biotechnological potential in different fields, mainly as sources ofextracellular enzymes or compatible solutes [5–7]. In this paper, we are presenting purifi-cation and characterization of a halostable and thermotolerant bacterial (Bacillus sp. LKG-01, MTCC 10401) L-glutaminase from Himalayan soil. Very few reports of microbialenzymes have been found to be isolated from different areas of Himalayas [8–10]. This isthe first report of such enzyme bioprospected from this region with the best of ourknowledge based on various published peer-reviewed literature.

Experimental Methodologies

Microorganism: Culture Conditions and Identification

LKG-01 was maintained by routine culture in TFY medium supplied with 1.5% L-glutamine(composition in grams per liter: yeast extract 3, tryptone 5, NaCl 5, glucose 10, pH 7.2–7.4) at37 °C and 120 rpm for 30 h. Gram characteristics have been studied by Gram staining.Taxonomic characterization was done based on nucleotide sequence of 16S rDNA gene.Extraction and purification of DNA were carried out by GenElute™ Bacterial genomic DNAkit (Sigma Aldrich, USA) following manufacturer’s instructions. Forward primer 5′-AGAGTTT-GATCCTGGCTCAG-3′ and reverse primer 5′-ACGGCTACCTTGTTACGACTT-3′ were usedfor PCR amplification followed by amplicon purification with HiPurA™ PCR product purifica-tion kit (Himedia, India). Sequenced amplicon data were analyzed in Bioedit and then alignedwith submitted sequences available in NCBI database using program ClustalW.

L-Glutaminase Assay

Supernatant of LKG-01 culture broth was used as crude enzyme solution. L-Glutaminasewas assayed by direct nesslerization according to Imada et al. [11]. Reaction mixture recipecontained 1 ml 1% L-glutamine in citrate phosphate buffer (pH 7.0) and 1 ml crude enzyme,which was cooked at 30 °C for 1 h and then terminated with addition of 0.5 ml 1.5 Mtrichloroacetic acid. Liberated ammonia was determined using 0.5 ml Nessler’s reagent andquantified spectrophotometrically at λ480 nm. Enzyme and substrate blanks were used ascontrols. Ammonia concentration of reaction was determined in terms of standard quantity

1724 Appl Biochem Biotechnol (2012) 166:1723–1735

(NH4)2SO4 solution. Protein was measured by method described by Lowry et al. [12] usingbovine serum albumin as standard. During chromatographic purification steps, proteinconcentration was measured as a function of its absorbance at λ280 nm. One unit of

L-glutaminase was defined as amount of enzyme that liberates 1 μmol of NH3 under optimalassay conditions. Specific activity of L-glutaminase was expressed as activity of enzymeunits per milligram protein released.

Enzyme Purification

Protein was precipitated from 2 L of filtered medium with addition of (NH4)2SO4 up to 30%saturation. Precipitate was resuspended in 20 mM Tris–chloride buffer (pH 8.5) and dia-lyzed. Concentrated protein solution was loaded in a DEAE-cellulose (Himedia, India)column (0.7×2.5 cm, Himedia, India) at a flow rate of 0.2 ml/min pre-equilibrated with20 mM Tris–chloride buffer and eluted fractions (1 ml each) were collected. Column waswashed with same buffer until λ280 of effluent became zero. Bound proteins were then elutedwith a concentration gradient of sodium chloride (0–0.5 M NaCl) in same buffer and activefractions were collected. Protein concentration and enzyme activity of each fraction weredetermined. Active fractions were pooled, concentrated, and reloaded to pre-equilibratedSephadex G 100 and Sephadex G 50 (Fluka chemicals, Switzerland) columns (2.25×35 cm),respectively, and eluted with same buffer. Fractions of 2 ml each were collected at a flow rateof 5 ml h−1. Active fractions were pooled and purity was checked by sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS–PAGE) on a discontinuous gel containing5% stacking and 7.5% resolving gel of pH 6.8 and 8.8, respectively, by method of Laemmliet al. [13]. Detection was done by Coomassie brilliant blue R-250 staining method.

Enzyme Characterization

Molecular Weight Determination by SDS–PAGE

Molecular weight was estimated by comparing relative mobility of proteins of different molecularsize compared to standard molecular weight marker (97, 66, 45, 31, and 14 kDa) in SDS–PAGE.

Effect of pH and Temperature on Activity and Stability of Purified Enzyme

pH dependence of L-glutaminase was determined over a wide pH range of 5.0–12.0. Activityof purified L-glutaminase was measured at different pH values. pH of reaction mixture wasadjusted using 100 mM of one of the following buffers: citrate buffer (pH 5.0–6.5), Tris–chloride buffer (pH 7.0–9.0), glycine–NaOH buffer (pH 9.5–11.0), and Na2HPO4–NaOHbuffer (pH 11.5–12.0). Enzyme activity was determined by assay method as describedearlier. In order to determine pH stability, purified enzyme was dissolved in all above-mentioned buffers. Pre-incubation was performed in each buffer at 50 °C temperatures for3 h. Residual enzyme activity was measured for each aliquot at an interval of 30 min. Eachvalue is a mean from triplicate tests.

To study the effect of temperature on activity of purified enzyme, reaction mixture wasincubated at different temperatures ranging from 30 to 90 °C (instead of 30 °C of standardassay method) for 30 min and relative activity was determined with respect to maximumexhibited activity as 100%. To evaluate thermal stability, purified enzyme solution wasincubated at temperatures of 30–90 °C for up to 3 h. Residual enzyme activity was recordedat 30-min intervals during this 3-h period as described earlier.

Appl Biochem Biotechnol (2012) 166:1723–1735 1725

Effect of NaCl on Activity and Stability of L-Glutaminase

To study the effect of NaCl on enzyme activity, purified enzyme was dissolved in 100 mMglycine–NaOH buffer (pH 11.0) containing NaCl of different concentrations ranging from0–25% (w/v). Enzyme activity was measured with 1% substrate solution at 70 °C withcooking for 30 min. Enzyme stability was determined at various salinity levels; purifiedenzyme with 100 mM glycine–NaOH buffer (pH 11.0) was appended with 0–25% salinityand cooked for 4 h at 70 °C. Residual enzymatic activity was determined with aliquotscollected at every 30-min intervals by same procedurementioned earlier. Values are percentagesof observed maximum activity of enzyme. Each value is a mean from triplicate tests.

Kinetic Determinations

Initial reaction rate for glutamine hydrolysis was calculated by estimating glutaminaseactivity at different L-glutamine concentrations of 70 to 7,000 μM; Michaelis constant(Km) and rate of reaction (Vmax) were determined according to Lineweaver and Burk [14].

Substrate Specificity and Metal Ion Effects of the Purified L-Glutaminase

Relative activity of enzyme was measured on a broad range of substrates by standardnesslerization. All substrates were used at 10 mM concentrations in 0.1 M Tris–Cl buffer(pH 8.5). Those substrates showing no activity were reevaluated with 100-min cooking. Onehundred percent relative activity was based upon the amount of glutamine converted intoglutamic acid and ammonia. Effect of various metal ions was investigated on the activity ofpurified L-glutaminase as shown in Table 3. Enzyme solutions were incubated at 70 °C for1 h in 50 mM glycine–NaOH buffer (pH 11.0) with Ca2+, Mn2+, Fe3+, K+, Co2+, Mg2+, Zn2+,Ba2+, and Ni2+ (final concentration of 5 mM). Residual activity was measured with L-glutamine as a substrate. Enzyme treated without any metal salt was considered as control.

Results

Characteristics of the Microorganism

Morphological and physiological characteristics of isolate LKG-01 were observed as Gram-positive rod (Fig. 1), catalase positive, indole test positive, and positive to fermentation testfor glucose and xylose, which can grow in TFY medium supplied with 1.5% L-glutamine[OD at λ60000.674 (1:20 dilution), after 30 h]; 1,393 bp of 16S rDNA gene was sequencedand submitted to NCBI GenBank (accession no. GU247967). Based on nucleotide sequenceof 16S rDNA gene, isolate LKG-01 was classified as a Bacillus sp. Taxonomic position hasbeen shown in the phylogenetic tree (Fig. 2). Strain Bacillus sp. LKG-01 has been depositedin “Microbial Type Culture Collection,” Institute of Microbial Technology (CSIR), Chandigarh(India) with accession no. MTCC 10401.

Enzyme Purification and Molecular Weight

Purification results of LKG-01 L-glutaminase are summarized in Table 1. Approximately 49-fold purification of crude enzyme was achieved with a recovery of approximately 24.9%,specific activity of finally purified enzyme being about 584.2 U mg−1 proteins. Purified


enzyme migrated as a single band in SDS–PAGE under reducing conditions, suggesting that

L-glutaminase was purified up to homogeneity. Apparent molecular weight was revealed bySDS–PAGE was about 66,000 Da (Fig. 3).

Effect of pH and Temperature on Activity and Stability of L-Glutaminase

Purified L-glutaminase remained active over a broad pH range, with maximum at pH 11.0(Fig. 4). More than 80% of maximum observed enzyme activity was reached betweenpH 8.0 and 11.0 and beyond this range it declined. At pH 9.0, 10.0, or 11.0, this L-glutaminase retained more than 80% activity for 3 h; however, at pH 10.0, it was reduced

Fig. 2 Phylogenetic dendrogram, indicating the position of the Bacillus sp. LKG-01

Fig. 1 SEM image of Bacillussp. LKG-01


to only 24% within 2 h (Fig. 5). Optimum temperature of purified L-glutaminase was 70 °Cand beyond that enzyme activity gradually ceased. Enzyme was highly stable over atemperature range of 50–70 °C (Fig. 6). Enzyme retained 100% activity at 50 °C temperatureand 20% of activity at 80 °C up to 3 h. At 70 °C, more than 50% of enzyme activity wasevaluated for same span of time (Fig. 7).

Effect of NaCl on Activity and Stability of L-Glutaminase

This L-glutaminase enzyme exhibited appreciable activity in presence of up to 25% (w/v)NaCl with an optimal concentration of 1% (Fig. 8). Enzyme also remained 100% active inabsence of NaCl but started losing activity as NaCl concentration was increased from 20% to25%. At 25% NaCl concentration, enzyme retained 60% of its activity within 4 h (Fig. 9).

Table 1 Summary of purification of L-glutaminase from Bacillus sp. LKG-01

Purification step Total activity(U)

Protein(mg)

Specific activity(U/mg)

Purificationfold

Recovery(%)

Crude extraction 10,690 897 11.91 1.0 100

30% (NH4)2SO4 fraction 6,870 313.41 21.92 1.84 64.2

DEAE ion exchange 4,470 31.64 141.25 11.86 41.8

Sephadex G-100 4,050 12.06 335.8 28.20 37.8

Sephadex G-50 2,670 4.57 584.2 49.05 24.9

14 KD

31 KD

45 KD

66 KD

97 KD

1 2 3 4 5Fig. 3 SDS–PAGE of thepurified LKG-01


Kinetic Determinations

Kinetic parameters Km and Vmax of enzyme were determined from Lineweaver–Burk doublereciprocal plots of L-glutaminase activity at 70 °C using various concentrations of L-glutaminase as substrate (Fig. 10). Km and Vmax values of L-glutaminase were 240 μMand 277.77±1.1 U mg1 proteins, respectively.

Substrate Specificity and Metal Ion Effects of the Purified L-Glutaminase

Beside L-glutamine, which was hydrolyzed with the highest specific activity, L-glutamine–L-asparagine was also hydrolyzed, although with a lower affinity (Table 2). L-Glutaminase ofBacillus sp. LKG-01 turned out to possess a weak affinity toward D-glutamine; however, D-asparagine did not serve as substrate. Effect of various metal ions was investigated on

4 5 6 7 8 9 10 11 12 13

20

40

60

80

100

% R

elat

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acti

vity

pH

Fig. 4 Effect of pH on theactivity of L-glutaminase fromLKG-01

0 30 60 90 120 150 1800

20

40

60

80

100

% R

elat

ive

acti

vity

Time (min)

pH 5 pH 6 pH 7 pH 8 pH 9 pH 10 pH 11 pH 12

Fig. 5 Effect of pH on activityand stability of LKG-01L-glutaminase


activity of purified L-glutaminase as shown in Table 3. Enzyme exhibited appreciable activity inpresence of K+, Zn2+, and Ni2+, and almost 80% activity was retained with all tested metal ions.

Discussions

Very few appreciable halotolerant, thermostable alkaline L-glutaminase from Bacillus sp. hasbeen bioprospected [1]. This paper describes such a protein able to deaminate L-glutamineappending all these criteria. LKG-01 L-glutaminase was purified from culture supernatant inthree steps by (NH4)2SO4 precipitation, DEAE ion exchange chromatography, and Sephadexgel filtration. Purified enzyme showed high recovery with comparison to enzyme reportedby Klein et al. [15] and higher specific activity (584.2 U mg−1 protein). LKG-01 L-

20 30 40 50 60 70 80 90 10040

50

60

70

80

90

100

110

% R

elat

ive

acti

vity

Temperature (0C)

Fig. 6 Effect of temperature onactivity of LKG-01 L-glutaminase

0 30 60 90 120 150 18010

20

30

40

50

60

70

80

90

100

110

% R

elat

ive

acti

vity

Time (min)

50 0C 60 0C 70 0C 80 0C

Fig. 7 Effect of temperature onthe activity and stability ofLKG-01 L-glutaminase


glutaminase has a molecular weight of approximately 66,000 Da, and as for catalyticproperties of LKG-01 L-glutaminase, Km for L-glutamine has been found to be 240 μM.

LKG-01 L-glutaminase was optimally active 60–70 °C. At 80 °C, LKG-01 displayed ahalf-life of 60 min, and no significant reduction in activity was observed at 60–70 °C andenzyme was active in a broad range temperature (30–90 °C). Jeong-Min et al. [16] inves-tigated thermostability of L-glutaminase from Lactobacillus sp. KCTC3594 had a half-life of30 min at 60 °C and optimal temperature 40 °C. L-Glutaminase from Bacillus sp. LKG-01was found to be salt-tolerant as relative activity displayed 109% with 20% (w/v) NaCl and70% in presence of 25% NaCl. In a previous study of halostability of L-glutaminase fromLactobacillus reuteri, KCTC3594 was found up to 5% [17] and it has been reported thatwith increase in salt concentration, enzyme activity decreased in case of Micrococcus luteusK-3 [18]. In case the of glutaminase of Lactobacillus rhamnosus, relative activity was 1.8times higher in presence of 2.5% NaCl which was also its highest activity [1], compared to

0 5 10 15 20 250

50

100

150

200

250

300

Rel

ativ

e A

ctiv

ity

(%)

Concentration of NaCl (w/v %)

Fig. 8 Effect of NaCl (percentweight/volume) on LKG-01L-glutaminase

0 50 100 150 200 250

60

70

80

90

100

resi

dual

act

ivit

y (%

)

time in min

5% NaCl 10% NaCl 15% NaCl 20% NaCl 25% NaCl

Fig. 9 Effect of NaCl (percent)on the activity and stability ofLKG-01 L-glutaminase


Bacillus sp. LKG-01 with 2.2-fold activity in presence of 2.5% NaCl. Comparative featureof various characteristics of L-glutaminase from Bacillus sp. LKG-01 with recent reportedarticles is described in Table 4.

Since food fermentation is generally done in presence of considerable salinity and attemperatures above 40 °C [19], LKG-01 L-glutaminase could be a potential candidate.Present work strongly suggests that L-glutaminase is useful to increase the amount ofglutamic acid released from enzymatically hydrolyzed proteins in presence of salt and/orat elevated temperatures.

Hydrolytic activity of LKG-01 enzyme displayed high levels of activity under neutral toalkaline conditions with no activity below pH 5. The highest level of activity was measuredat pH 11. Interestingly, enzyme was extremely stable, with little loss of activity after

Table 2 Substrate specificity ofLKG-01 L-glutaminase Substrate Specific activity

(U/mg)Residual activity(%)

L-Glutamine (control) 584.2 100

D-Glutamine 105.1 18

L-Asparagine 11.6 02

D-Asparagine 46.7 08

L-Glutamine–L-asparagine 525.7 90

L-Asparagine–D-asparagine 140.2 24

D-Glutamine–L-asparagine 280.4 48

L-Aspartic acid 0 0

D-Aspartic acid 0 0

L-Glutamic acid 0 0

D-Glutamic acid 0 0

L-Isoasparagine 0 0

N-Acetyl–L-asparagine 29.2 5.1

0.0 0.2 0.4 0.6 0.8 1.0

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

Y = 0.1264 + 0.0036XK

m = 35.11 g/L

Vmax

= 277.8 U/mg

[1/V

]

[1/S]

Fig. 10 Line weaver–Bark plotof LKG-01 L-glutaminase for thekinetic analysis of the reactionrate, at a series of concentrationof substrate L-glutamine


incubation for 3 h under alkaline conditions up to pH 11. pH stability profile of LKG-01depicts that enzyme retained almost good activity at broad range pH 6.0–11.0 up to 3 h atroom temperature compared to Lactobacillus sp. KCTC3594. LKG-01 displayed alkaliphilicproperties that have been previously detected only in enzymes isolated from Bacilluspasteurii [15]. Glutaminase activity was detected at a pH range from 5.0 to 11.0 whichwas broader than 7.5 to 9.0 for cyanobacterium Anabaena sp. PCC7120 [20], or 6.0 to 9.0for L. rhamnosus [1]. LKG-01 L-glutaminase can be used to increase amount of glutamicacid by hydrolysis of glutamine in stressful conditions like elevated temperature.

In conclusion, results of present study recommend the usage of Bacillus sp. LKG-01 as asource of halotolerant, thermostable L-glutaminase activity in higher alkaline condition oftenrequired in food fermentation processes.

Table 3 Effect of metal ions onLKG-01 L-glutaminase activity Metal ions Specific activity (U/mg protein) % Relative activity

Control 584.2 100

Ca2+ 519.9 89

Mn2+ 461.5 79

Fe3+ 426.4 73

K+ 590.0 101

Co2+ 479.0 82

Mg2+ 514.0 88

Zn2+ 560.8 96

Ba2+ 519.9 89

Ni2+ 554.9 95

Table 4 Comparative feature regarding various characteristics of L-glutaminase from Bacillus sp. LKG-01

Strain Optimum Residualactivity(%)

NaClconcentrationlimit (%/M)

Reference

Temperature(°C)

pH

Bacillus sp. LKG-01 70 11 108 20% Present work

Lactobacillus reuteriKCTC3594

40 7.5 50 15% Jeong-Min et al. [16]

Lactobacillus reuteriKCTC3594

40 7.5 ≤100 5% Jeong et al. [17]

Stenotrophomonas maltophiliaNYW-81

60 9.0 ≤100 10% Wakayama et al. [19]

Aspergillus oryzae RIB40 30 8.0–9.0 40 3M Masuo et al. [21]

Micrococcus luteus K-3 – – 300 3M Yoshimune et al. [18]

Lactobacillus rhamnosus 50 7.0 90 15% Weingand-Ziade et al.[1]

Cryptococcus nodaensis sp. 70 8.0 85 18% Sato et al. [22]

Bacillus pasteurii 37 9.0 100 – Marcus et al. [23]

Bacillus subtilis RSP-GLU 41 8.0 100 – Thadikamala andReddy [24]


Acknowledgments Financial support provided by UCOST Dehradun (UCS&T/R&D/HSS-10/07-08/1855/2) is sincerely acknowledged. Facilities provided by Dr. M O Garg, Director, Indian Institute of Petroleum(CSIR) and Dr. AK Sharma, Director, SBSPGI are sincerely acknowledged.

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a temperature and salt-tolerant l-glutaminase from gangotri region of uttarakhand himalaya: enzyme...

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