ISOLATION AND …sciencejournal.in/data/documents/Trends-Biotech-Res.-Volume-5-1-3... · ISOLATION...

www.sciencejournal.in

Volume 5, Issue 1 (2016) ISSN 2320–0421(Print); ISSN 2320–043X(Online) © 2016 DAMA International. All rights reserved. 9

ISOLATION AND IDENTIFICATION OF Α-AMYLASE PRODUCING PSYCHROTROPHIC

ENTEROBACTER SP. NACASA2

Umesh U. Jadhav1, Sachin B.

Chavan

2,

Vishal V. Dawkar

3, Ashok D. Chougale

4 and Manohar V. Padul

5*

1Department of Power Mechanical Engineering, National Tsing Hua University, No. 101, Sec.2, Kuang Fu Rd., 30013,

Hsinchu, Taiwan ROC. 2P.G. Department of Biochemistry, N.A.C. & S. College, Ahmednagar-414 001, Affiliated to University of Pune, India.

3Plant Molecular Biology Unit, Division of Biochemical Sciences, National Chemical Laboratory, Pune, India.

4Mass Spectrometry

and Proteomics group Organic Chemistry Division, National Chemical Laboratory, Pune, India

5Department of Biochemistry, Dr. Babasaheb Ambedkar Marathwada University, Aurangabad-431004, India

*(Corresponding author Email: [email protected])

ABSTRACT

This study reported the isolation and characterization of cold adapted Enterobacter sp. NACASA2. The ability of

this bacterium, isolated from garden soil, to biosynthesize α-amylase was investigated. Maximum α-amylase

production was obtained at 24 h of incubation (56.13 U/mg protein/min). The optimal temperature and pH were 35

°C and 7.0, respectively, for growth and enzyme production. Enterobacter sp. NACASA2 showed maximum

amylase production with starch and tryptone. The bacterium withstands 0.8% NaCl concentration in medium. The

enzyme production was slightly inhibited by 10 mM, Zn2+

(19.11%) and Cu2+

(32.56%) while strongly inhibited by

Hg2+

(100%).

KEY WORDS: α-amylase, Cold adapted bacterium, Salt tolerance.

INTRODUCTION

A diverse range of microbes have been discovered in cold environments and include representatives of the bacteria,

eucarya and archaea (Cavicchioli and Thomas, 2000). Their cold-active or cold adaptive enzymes have received a great

deal of attention because they are essential in some fundamental scientific study areas and have the ability to withstand

certain industrial reaction conditions (Deming 1998). The high flexibility of cold-active enzymes enables increased

complementarities between the active site and substrate, at a low-energy cost, resulting in high specific activity at low

temperatures (Cavicchioli et al 2002). Likewise, the high activity of cold- adaptive enzymes at low and moderate

temperatures offers potential economic benefits (Gerday 2000). Isolation of industrially important cold-adapted

enzymes like α-amylase, cellulase, β-galactosidase, lipase, proteases, etc has been done previously Cavicchioli et al

2002.

α-amylase (EC 3.2.1.1) are glycoside hydrolases which cleave the α-1,4-glycosidic linkages in starch and maltodextrins

with an endo mechanism, i.e., in a random fashion within the polysaccharide molecule (Ballschmiter et al 2006).

Enzymatic hydrolysis of starch by α-amylase yielding a mixture of maltose and glucose has wide applications in

industry. It is used in brewing and fermentation industries for the conversion of starch to fermentable sugars (Farid et al

2002), in the textile industry for designing textiles and in the laundry industry in a mixture with protease and lipase to

launder clothes (Upadek and Kottwitz 1997; Ito et al 1998), in the paper industry for sizing (Maarel et al 2002), and in

the food industry for preparation of sweet syrups (Rao and Satyanarayana 2003).

Recent microbial studies of the deep sea have led to significant new discoveries of unusual microbial diversity, new

species and cold-adaptive enzymes (Feller and Gerday 2003). Cold adapted α-amylase was isolated and purified from

Nocardiopsis strain that was isolated from deep sea sediments (Zhang and Zeng 2008). In the present research we

isolated Enterobacter sp. NACASA2, which was able to produce α-amylase at low temperature. This work is in an

incipient stage and further studies may exploit the potential of Enterobacter sp. NACASA2 for pilot and large scale

applications.

MATERIALS AND METHODS

Screening, isolation of microorganism and culture conditions

Garden soil collected from botanical garden of N.A.C. & S. College, Ahmednagar, India was used for screening of α-

amylase producing bacterial strains. One gram soil was added to 100 ml Bushnell and Hass medium. The composition

mailto:[email protected]



of medium was magnesium sulphate 0.20, calcium chloride 0.02, monopotassium phosphate 1.0, dipotassium

phosphate 1.0, ammonium nitrate 1.0, ferric chloride 0.05 and starch 2.0 g/l. The flasks were incubated at 15 ◦C under

shaking (120 rpm) as well as static condition. The culture showing α-amylase production at static condition was

acclimatized by transferring 5 ml aliquots from the flask to the fresh starch-containing medium with various

concentration of starch increased from 0.2 to 1%. The starch-containing agar plates were inoculated with 0.1 ml

suspension from these flasks. The isolated colonies were transferred to the starch-containing Bushnell and Hass

medium and selected on the basis of high α-amylase activity. Out of these isolates one showing high α-amylase

production under static condition was identified as NACASA2 and used for further study. The pure culture was

maintained on starch-containing agar slants at 40C.

16S rDNA sequencing

16S rDNA sequencing of isolated bacteria was carried out at “Molecular diagnostic center”, Pune, India. The

nucleotide sequence analysis of the sequence was done at Blast-n site at NCBI server

(http://www.ncbi.nlm.nih.gov/BLAST). The sequence was refined manually after crosschecking with the raw data to

remove ambiguities and submitted to the NCBI, the accession number is HM179100. The evolutionary history was

inferred using the Neighbor-Joining method (Saitou and Nei 1987). The optimal tree with the sum of branch length =

1.53397188 is shown (Fig. 1). The percentage of replicate trees in which the associated taxa clustered together in the

bootstrap test (500 replicates) is shown next to the branches (Felsenstein 1985). The evolutionary distances are

computed using the Maximum Composite Likelihood method(Tamura et al 2004) and are in the units of the number of

base substitutions per site. All positions containing gaps and missing data were eliminated from the dataset (Complete

deletion option). There were a total of 204 positions in the final dataset. Phylogenetic analyses were conducted in

MEGA4(Tamura et al 2007).

Assay for α-amylase enzyme activity

Amylase activity was determined by detecting the amount of reducing sugars liberated. The reaction mixture (1 ml)

contained 0.25 ml of 1% soluble starch, 0.25 ml of 0.4 M Tris-HCl buffer (8.0) and 0.5 ml of enzyme (obtained from

Enterobacter sp. NACASA2). The reaction was terminated by addition of 2 ml of 3, 5-dinitrosalycylic acid reagent

after incubation for 30 min (Sengupta 2000). The protein concentration was measured with bovine serum albumin as a

standard (Lowry, 1951).

Effect of incubation temperature and pH on α-amylase production

Temperature and pH for maximum α-amylase production was quantified. Variable temperature (0 to 45 0C) and pH

range (1 to 9) were employed for production of α-amylase. The initial pH of Bushnell and Hass medium was adjusted

using concentrated HCl/NaOH. For the study of effect of temperature the flask were incubated at various temperatures.

Optimization of carbon sources

Different carbon sources (xylose, lactose, maltose, and sucrose) were used at a concentration of 1.0% (w/v). These

were used to replace starch in standard growth medium. The flasks were incubated at 15 °C.

Optimization of nitrogen sources

Different nitrogen sources (ammonium sulphate, peptone, ammonium per sulphate, beef extract, malt extract, and

tryptone) at a concentration of 1.0% (w/v) were used. The flasks were incubated at 15 °C.

Effect of various concentration of sodium chloride on amylase production

The effect of various concentrations of sodium chloride on α-amylase production was studied. Various concentrations

of sodium chloride (0.2, 0.4, 0.6, 0.8 and 1.0%) were added in culture medium. The flasks with various concentrations

of sodium chloride were inoculated with Enterobacter sp. NACASA2. After incubation at 15 0C for 24 h the amylase

production was measured and compared with control (without sodium chloride).

Effect of various metal ions on amylase production

The effect of metal ions on α-amylase production was measured. Various metal ions were added in culture medium at a

concentration of 10 and 100 mM respectively. Metals used were Zn2+

, Cu2+

and Hg2+

. The flasks with metal ions were

inoculated with Enterobacter sp. NACASA2. After incubation at 15 0C for 24 h the amylase production was measured

and compared with control (without metal).

http://www.ncbi.nlm.nih.gov/BLAST



Statistical analysis

Data were analyzed by one-way analysis of variance (ANOVA) with Tukey-Kramer multiple comparisons. Readings

were considered significant when **P was <0.01, and ***P<0.001. Graphpad software was used for this analysis.

RESULTS AND DISCUSSION

Isolation and identification of α-amylase producing bacteria

Several isolates were obtained during enrichment process. Potential isolate was selected and used for further studies on

the basis of rapid and extensive growth on starch and designated as NACASA2. Typical cultural morphologies of

isolate on starch agar were tiny, circular, pale yellow colored and entire. Cellular characteristics revealed Gram-

negative motile rods (Table I).

Table 1. Colony characteristics of Enterobacter sp. NACASA2

Various biochemical tests were performed for the identification and characterization of bacteria. The isolate was

catalase, citrate positive, and indole, methyl red negative. We also checked the ability of this isolate to utilize various

sugars. Starch was hydrolyzed and most sugars were utilized (data not shown).

Figure 1. Phylogenetic tree of the Enterobacter sp. NACASA2 and related organisms were aligned based on 16S

rDNA sequences (neighbor-joining tree).

SIZE SHAPE COLOR MARGIN OPACITY ELEVATION CONSISTENCY GRAM’S

NATURE

MOTILITY

1MM CIRCULAR PALE

YELLOW

ENTIRE OPAQUE RAISED SMOOTH GRAM

NEGATIVE

MOTILE



The 16S rDNA amplicon of the isolate was used to determine the sequence of the nucleotides. The sequence was

submitted to NCBI. The accession number for the sequence is HM179100. A BLASTn search was undertaken to find

out the similarity of the sequence with the most similar 16S rDNA sequences of the GenBank database. The sequence

of 16S rDNA of the strain was aligned to related sequences retrieved from the database to construct a phylogenetic tree

(Fig. 1). Comparison with 16S rDNA sequences available in the GenBank database revealed that NACSA2 is closely

related to species belonging to the genus Enterobacter. Therefore the name to this strain is designated as Enterobacter

sp. NACASA2. There is a report in literature on α-amylase production by Enterobacter cloacae IIT-BT08 (Kumar and

Das 2000).

Effect of incubation time on amylase production

The enzyme production was initiated within 12 h and further increased in exponential phase. Maximum amylase

production was obtained at 24 h of incubation (56.13 U/mg protein/min) (Fig. 2). Maximum production of amylase was

obtained after 24 h in Bacillus cereus (Anto et al 2006) and Bacillus subtillis (Asgher et al 2007). Production of

amylase at comparatively earlier stages of fermentation suggested the usefulness of the strain for enzyme production.

Figure 2. Effect of incubation time on amylase production.

Effects of temperature and pH on amylase production

Temperature has been identified as a factor controlling secretion of extra cellular enzymes and their activity in

psychrophilic bacteria (Barbaro et al., 2001). The production of α-amylase by Enterobacter sp. NACASA2 was

measured at various temperatures at pH 7.0. The Enterobacter sp NACASA2 showed production of enzyme between

15 to 45 0C and the optimum temperature for production was at 35

0C (Figure 3).

This is in agreement with the previous observations (Zhang and Zeng 2008). Enterobacter sp. NACASA2 was able to

produce significant amount of amylase enzyme at 15 0C. Psychrotroph is essentially a mesophilic microorganism that

can also grow under cold conditions below 15 0C (Takeshi et al 2001). The cold adaptation of this microorganism is

likely to be due to, at least in part, its ability to produce cold-active enzymes which exhibit higher catalytic activities at

low temperatures than its mesophilic and thermophilic counterpart’s do (Margesin and Shinner 1994). Recently, much

attention has been made to the application of psychrotrophs, and their cold-active enzymes in biotechnology (Gerday et

al 2000). For example, cold-active hydrolases such as proteinases, amylases, cellulases and lipases from

microorganisms have been used as additives in laundry detergent (Margesin and Shinner, 1994).

0

10

20

30

40

50

60

0 12 24 36 48

Time (h)

Enzy

me

acti

vit

y (

U/m

g p

rote

in/m

in) Enzyme

activity



Figure 3. Effect of temperature and pH on amylase production.

The enzyme is very sensitive to pH. Therefore the selection of pH is very essential for the production of α-amylase

(McMahon et al 1999). The effect of initial pH of the medium was studied (Fig. 3). Most of the Bacillus amylase has

optimum pH values of from 5.0-8.5 (Burhan 2007; Liu and Yan 2008) and an acidic amylase from Bacillus

acidocaldarius has an optimal pH of 3.5 (Mutuso 1986). Alkaline amylases reported previously had optimum pH

values of 9.0 to 11.0 (Burhan et al 2003). However, we found Enterobacter sp. NACASA2 has an optimal pH value of

7.0 for amylase production. Also Enterobacter sp. NACASA2 showed broad pH range of 1.0 to 9.0. Similar broad pH

range of 3-8 for α-amylase production was also reported (Gupta et al 2008).

Effects of carbon and nitrogen source on amylase production

Carbon sources have been shown to have different effects on amylase production (Farez-Vidal et al 1992). The

influence of the carbon source on α-amylase production by Enterobacter sp. NACASA2 was investigated using

different carbon sources as shown in Fig. 4. Enterobacter sp. NACASA2 showed maximum amylase production with

starch. Sucrose also supported the amylase production. Xylose showed lowest amylase production. Our results are in

good agreement with previous findings who obtained greater production with starch as the sole carbon source (Fang

and Demain 1989). Also similar observations were reported by other researchers (Gupta et al 2008; Ezeji 2005). In

contrast to this the enzyme production by B. subtilis was higher with glucose than with starch (Jamuna and

Ramakrishna 1992). Rhamnose is reported as the best carbon source for the production of enzyme (Jamuna and

Ramakrishna 1992).

Effect of the nitrogen source on the α-amylase productivity from Enterobacter sp. NACASA2 was studied using

various nitrogen sources having starch (1%) as the carbon and energy source (Fig. 5).

Tryptone was found to be the best nitrogen source for the production of enzyme. After tryptone, malt extract showed

good amylase production. Enterobacter sp. NACASA2 showed least amylase production in the presence of ammonium

sulphate as compared to other nitrogen source. Several workers reported various nitrogen sources as stimulators for

enzyme production (Gupta et al 2008; Ezeji 2005; Jana and Pati 2007).

0

10

20

30

40

50

60

70

15 25 35 45

1 3 5 7 9

Temperature/pH

Enzy

me

acti

vit

y (

U/m

g p

rote

in/m

in)

for

pH

0

20

40

60

80

100

120

U/m

g p

rote

in/m

in)

for

tem

per

ature

pH

Temperature

http://www.ncbi.nlm.nih.gov/pubmed?term=




Effect of salt concentration on the production of amylase

The salinity was found to be a significant factor in the production of amylase. The Enterobacter sp. NACASA2 showed

amylase production in a medium supplemented with NaCl. The amylase production was optimum in the medium

supplemented with 0.8% NaCl (Figure 6).

Figure 4. Effect of various carbon sources on amylase production.

Figure 5. Effect of various nitrogen sources on amylase production

0

20

40

60

80

100

120

Xylose Lactose Maltosr Sucrose Starch

Carbon source

Enzy

me

acti

vit

y (

U/m

g p

rote

in/m

in)

Enzyme activity

0

5

10

15

20

25

30

35

40

Ammonium

sulphate

Peptone Ammonium

per sulphate

Beef extract Malt extract Tryptone

Nitrogen source

En

zy

me a

cti

vit

y (

U/m

g p

rote

in/m

in)

Enzyme activity



Figure 6. Effect of various concentrations of NaCl on amylase production.

The amylase activity was approximately 75% higher than that of the control. Similar results were reported by others

(Shanmughapriya et al 2009).

Effect of metal ions on the production of amylase

The production of amylase by Enterobacter sp. NACASA2 was assayed in the presence of 10 and 100 mM of various

metal ions. As shown in Table II the enzyme production was strongly inhibited by Hg2+

.

Table 2. Effect of metal ions on amylase production

Metals Concentration (mM) Percent inhibition (%)

Control 0 0

Zn2+

10 19.11

100 90.44

Cu2+

10 32.56

100 95.87

Hg2+

10 100

The enzyme production was slightly inhibited by 10 mM Cu2+

, and Zn2+

. The effect of Zn2+

also varied between

amylases. For instance, it had a potent inhibitory effect on the amylases from Bacillus SMIA2 and Bacillus A3-15

(Burhan 2007; Carvalho et al 2008); on the other hand, slight inhibition in the activity of the enzyme observed in

Bacillus cohnii US147 and Bacillus subtilis JS- 2004 (Asgher et al 2007; Raoudha et al 2009). It had no effect at all on

the enzyme of anaerobic archeon strain HJ21 and Aspergillus kowashii (Wang et al 2008). Copper strongly inhibited

the enzymatic activity from Bacillus subtilis JS-2004, Bacillus sp. Strain SMIA2 and Bacillus sp. strain KSM-1378

(Asgher et al 2007; Raoudha et al 2009).

REFERENCES

Anto H., Trivedi U. and Patel K. (2006). Α amylase production by Bacillus cereus MTCC 1305 using solid state

fermentation. Food Technol. Biotechnol. 44: 241-245.

Asgher M., Asad M., Rahman S. and Legge R. (2007). A thermostable α-amylase from a moderately thermophilic

Bacillus subtilis strain for starch processing. J. Food. Eng. 79: 950-955.

Ballschmiter M., Futterer O. and Liebl W. (2006). Identification and characterization of a novel intracellular alkaline

α-amylase from the hyperthermophilic bacterium Thermotoga maritima MSB8. Appl. Environ. Microbiol. 72: 2206-

2211.

Barbaro S., Trevors J. and Inniss W. (2001). Effects of low temperature, cold shock, and various carbon sources on

esterase and lipase activities and exopolysaccharide production by a psychotropic Acinetobacter sp. Can. J. Microbiol.

47: 194-205.

0

100

200

300

400

500

600

700

0 0.2 0.4 0.6 0.8 1

Concentration of sodium chloride (%)

Enzy

me

activ

ity (U

/mg

prot

ein/

min

)Amylase enzymeactivity



Burhan A. (2007). Highly thermostable, thermophilic, alkaline, SDS and chelating resistant amylase from thermophilic

Bacillus isolate A3-15. Biores. Technol. 99: 3071-3076.

Burhan A., Nisa U., Gkhan C., Omar C., Ashabil A. and Osama G. (2003). Enzymatic properties of a novel

thermostable, alkaline and chelator resistant amylase from an alkalophilic Bacillus sp. Isolate ANT-6. Process

Biochem. 38: 1397-1403.

Carvalho R., Côrrea T., Silva J., Mansur L. and Martins M. (2008). Properties of an amylase from thermophilic

Bacillus sp. Braz. J. Microbiol. 39: 102-107.

Cavicchioli R. and Thomas T. (2000). Extremophiles In: Encylopedia of Microbiology, edn 2. Edited by Lederberg,

J., Alexander, M., Bloom, B., Hopwood, D., Hull, R., Iglewski, B., Laskin, A., Oliver, S., Schaechter, M. Summers, W.

San Diego: Academic Press Inc. 317-337.

Cavicchioli R., Siddiqui K., Andrews D. and Sowers K. (2002). Low-temperature extremophiles and their

applications. Curr. Opinion Biotechnol. 13: 253-261.

Deming J. (1998). Deep ocean environmental biotechnology. Curr. Opinion Biotechnol. 9: 283-287.

Ezeji T., Wolf A. and Bahl H. (2005). Isolation, characterization, and identification of Geobacillus

thermodenitrificans HRO10, α-amylase and α-glucosidase producing thermophile. Can. J. Microbiol. 51: 685-693.

Fang A. and Demain A. (1989). A new chemicaly-defined medium for RAC-certified strains of B. subtilis. Appl.

Microbiol. Biotechnol., 30, 144-147.

Farez-Vidal M., Fernandez-Vivas A. and Arias J. (1992). Production of α-amylase by Myxococcus cora~loides D. J.

Appl. Bacteriol., 73, 148-176.

Farid M., El-Enshasy H. and El-Deen A. (2002). Alcohol production from starch by mixed cultures of Aspergillus

awamori and immobilized Saccharomyces cerevisiae at different agitation speeds. J. Basic. Microbiol. 42:162-171.

Feller G. and Gerday C. (2003). Psychrophilic enzymes: hot topics in cold adaptation. Nature Rev. Microbiol. 1: 200-

208.

Felsenstein J. (1985). Confidence limits on phylogenies: An approach using the bootstrap. Evolution. 39: 783-791.

Gerday C., Aittaleb M., Bentahir M., Chessa J., Claverie P., Collins T., D'Amico S., Dumont J., Garsoux, G.,

Georlette D., Hoyoux A., Lonhienne T., Meuwis M. and Feller G. (2002). Cold adapted enzymes: from

fundamentals to biotechnology. Trends Biotechnol. 18: 103-107.

Gupta A., Gupta V., Modi D. and Yadava L. (2008). Production and Characterization of α-amylase from Aspergillus

niger. Biotechnol. 7: 551-556.

Ito S., Kobayashi T., Ara K., Ozaki K., Kawai S. and Hatada Y. (1998). Alkaline detergent enzymes from

alkaliphiles: enzymatic properties, genetics, and structures. Extremophiles. 2: 185-190.

Jamuna R. and Ramakrishna S. (1992). Continuous synthesis of thermostable α-amylase by Bacillus cells

immobilized in calcium alginate. Enz. Microb. Technol. 14: 36-41.

Jana M. and Pati B. (2007). Thermostable, salt-tolerant-amylase from Bacillus sp. MD 124. J. Basic Microbiol. 37:

323-326.

Kumar N. and Das D. (2000). Production and purification of α-amylase from hydrogen producing Enterobacter

cloacae IIT-BT08. Biopro. Eng. 23: 205-208.

Liu X. and Yan X. (2008). A novel raw starch digesting α-amylase from a newly isolated Bacillus sp. YX-1,

purification and characterization. Biores. Technol. 99: 4315-4320.

Lowry O., Rosebrough N., Farr A. and Randall R. (1951). Protein measurement with the Folin phenol reagent. J.

Biol.Chem. 193:265-275.

Maarel V., M., Van-der-Veen B., Uitdehaag J., Leemhuis H. and Dijkhuizen L. (2002). Properties and applications

of starch-converting enzymes of the α-amylase family. J Biotechnol. 94: 137-155.

Margesin R. and Shinner F. (1994). Properties of cold-adapted microorganisms and their potential role in

biotechnology. J. Biotechnol. 33: 1-14.

McMahon H., Kelly C. and Fogarty W. (1999). High maltose producing amylolytic system of a Streptomyces sp.

Biotechnol. Lett. 21: 23-26.

Mutuso K. (1986). A Bacillus acidocaldarius α-amylase that is highly stable to heat under acidic conditions. Agricul.

Biol. Chem. 50: 23-31.

Rao U. and Satyanarayana T. (2003). Statistical optimization of a high maltose-forming, hyperthermostable and

Ca2+

-independent α-amylase production by an extreme thermophile Geobacillus thermoleovorans using response

surface methodology. J. Appl. Microbiol. 95: 712-718.

Raoudha E., Sameh M., Ezedin B., Samir B. and Semia E. (2009). New thermostable amylase from Bacillus cohnii

US147 with broad pH applicability. Appl. Biochem. Biotechnol. 157: 50-60.




http://scialert.net/asci/author.php?author=A.&last=Gupta

http://scialert.net/asci/author.php?author=V.K.&last=Gupta

http://scialert.net/asci/author.php?author=D.R.&last=Modi

http://scialert.net/asci/author.php?author=L.P.&last=Yadava

http://www3.interscience.wiley.com/journal/5007687/home

http://www3.interscience.wiley.com/journal/114053052/issue



Saitou N. and Nei M. (1987). The neighbor-joining method: A new method for reconstructing phylogenetic trees. Mol.

Biol. Evolution. 4: 406-425.

Sengupta S., Jana M., Sengupta D. and Naskar A. (2000). A note on the estimation of microbial glycosidase

activities by dinitrosalicylic acid reagent. Appl. Microbiol. Biotechnol. 53: 732-735.

Shanmughapriya S., Kiran G., Selvin J. Gandhimathi R., Baskar T., Manilal A. and Sujith S. (2009). Optimization, production, and partial characterization of an alkalophilic amylase produced by sponge associated marine

bacterium Halobacterium salinarum MMD047. Biotechnol. Biopro. Eng.14: 67-75.

Takeshi S., Toru K., Tatsou N. and Nobuyoshi E. (2001). Cold-active lipolytic activity of psychotropic

Acinetobacter sp. Strain No. 6. J. Biosci. Bioeng. 92: 144-148.

Tamura K., Dudley J., Nei M. and Kumar S. (2007). MEGA4: Molecular evolutionary genetics analysis (MEGA)

software version 4.0. Mol. Biol. Evolution. 24: 1596-1599.

Tamura K., Nei M. and Kumar S. (2004).Prospects for inferring very large phylogenies by using the neighbor-

joining method. Proc. Natl. Acad. Sci. (USA). 101: 11030-11035.

Upadek H. and Kottwitz B. (1997). Application of amylases in detergents. In: Ee, J.H., O. Misset and E.J. Baas

(eds.). Enzymes in detergency van. 203-212. Marcel Dekker, Inc, New York.

Wang S., Lu Z., Lu M., Qin S., Liu H., Deng X., Lin Q. and Cheng J. (2008). Identification of archeon-producing

hyperthermophilic α-amylase and characterization of the α-amylase. Appl. Microbiol. Biotechnol. 80: 605-614.

Zhang J. and Zeng R. (2008). Purification and characterization of a Cold-Adapted α-amylase produced by

Nocardiopsis sp. 7326 isolated from Prydz bay, Antarctic. Marine Biotechnol. 10: 75-82.

ISOLATION AND …sciencejournal.in/data/documents/Trends-Biotech-Res.-Volume-5-1-3... · ISOLATION...

Documents

Transcript of ISOLATION AND …sciencejournal.in/data/documents/Trends-Biotech-Res.-Volume-5-1-3... · ISOLATION...