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Research Plan

On

Halophilic Enzymes

and

Their Biotechnological Applications

Submitted by Under the Supervisionof

Sumit Kumar Dr. S. K. Khare

(2007CYZ8227)

DEPARTMENT OF CHEMISTRY

INDIAN INSTITUTE OF TECHNOLOGY, DELHI



Research Plan

Submitted by: Sumit Kumar

(2007CYZ8227)

Registered Topic: Halophilic Enzyme and

Their Biotechnological

Applications

Ph.D. Supervisor: Dr. S. K. Khare

Course Work: Completed 12 Credits

CGPA: 9.5/10



Introduction

Hydrolases, especially proteases, lipases and amylases are the largest sellingindustrial enzymes. These are widely used in detergent formulations and

peptide/esters/oligosaccharide synthesis. Their demand is predicted to increase by

4-5 folds in coming years. In question, however, is the stability of these enzymesin extreme conditions, such as high salt, surfactants and organic solvents.

Enzymes in halophiles and haloalkaliphiles have evolved to function in highly

saline and alkaline conditions. These are best suited to function under suchconditions.

A systematic investigation to screen the halophilic diversity that exists in the vast

saline habitats of our country is needed for obtaining novel enzyme preparations.

It is proposed to work on above lines with following overall objectives:

1. Studies on microbial diversity of different saline habitat of India with respect to

their morphological, biochemical and enzymatic profile.

2. Screening of lipolytic activity in selected halophilic isolates and selection of

potential lipase producers.

3. Production, purification and characterization of potential lipases especially with

respect to their catalysis in high salt/ solvent medium.

4. Identification of halophilic lipase genes, cloning, overexpression, structure andfunction analysis.

5. Lipase application in high salt/ solvent system.



Review of literature

Halophiles

Halophiles have been studied from saline soils, mud and salt piles in hypersalineenvironment of California, Nevada, Great salt lake and Utah, USA; Soda lake in

Kenya; Japanese sea; inland and marine saltern of France, Canada, Germany, UK

and many other countries. Among these Halococcus and Halobacterium are prominently found halophiles. These have been extensively studied for structural

and genetic diversity and some of the species, such as, Halobacterium halobium,

Halomonas halophiles and Halocella cellulytica are well characterized (Anton et

al., 2000, Vreeland et al., 2000)

Little is known about the enzymes from these halophiles. Amongst, few reports

that have been published in this area, a cellulase complex have been purified from Halocella cellulytica which grows at 2.6 M NaCl. The half life of enzyme was

found to be 68 minutes at 50°C. A serine protease from Archbacterium strain 172

P1 is also purified and characterized. The purification to homogeneity in this case

was achieved by ammonium sulfate precipitation followed by chromatography on butyl Toyopearl 650-C. The enzyme had a Molecular weight of 44,000-46,000. It

had pH optima at 10.7, temperature optima 75-80°C and reported to be stable at

high NaCl concentration.

The biotechnological applications of halophiles and their enzymes are lessexplored. The understanding of enzymes from halophilic and haloalkaliphilic

organisms is still hazy. Their uses in hypersaline waste treatment, enhanced oil

recovery and peptide synthesis are predicted to be promising. A cellulase from a

halophilic bacterium has been successfully used for degradation of cellulose inhypersaline waste at US Waste Isolation Pilot Plant (Rodriguez-Valera, 1992). In

another interesting application, an extracellular protease from Halobacterium

halobium was used for peptide synthesis in aqueous organic system (Kim andDordick, 1997). The esterification activity of enzyme is found to be 80 fold higher

in DMSO medium. This unique behavior of halophilic enzymes in organic media

may have advantage in organic synthesis.

Structure and function analysis of halophilic enzyme gives insight in proteinfolding under a combination of extreme conditions (Favilla et al., 1997). Some of

the enzymes from halophilic and haloalkaliphilic organisms are extremely

resistant to chemical denaturation, a feature which would attract several novelapplications besides providing clues on the protein stabilities. Investigations on

these aspects would be quite interesting to pursue.



Lipases

Lipases (EC 3.1.1.3) catalyse both the hydrolysis and esterification reactions (Fig.1). These reactions usually proceed with high regio- and/or enantioselectivity. Thereasons for the enormous biotechnological potential of lipases include the facts

that they are (1) stable in organic solvents, (2) do not require cofactors, (3)

possess broad substrate specificity and (4) exhibit a high enantioselectivity(Jaeger and Reetz, 1998)

Figure 1

Their ability to accept wide range of substrate (lipids, sugar, alcohol and esters)

and to maintain activity and selectivity in organic solvents have enabled their

wide use as biocatalysts in food, detergent, pharmaceutical, leather, textile,cosmetic and paper industries (Houde et al ., 2004; Salameh and Wiegel, 2007).

Though a large number of microbes secrete lipase, only few are of industrialimportance. The production of lipases is greatly influenced by nutritional and

physico-chemical factors (Jaeger et al ., 1994; Kim et al ., 1996). For enhancement

of yields, process optimization is carried out by “one-at-a-time approach” or byResponse Surface Methodology (RSM).

Lipases have been purified and characterized from diverse microbial sources.However, no single technique or generic protocol can be defined to be the best.

The purification strategy varies from source to source.

Cell free supernatant obtained by filtration or centrifugation of fermentation brothcan be concentrated by means of ultrafiltration (Castro-Ochoa et al., 2005) or

precipitated using ammonium sulphate, acetone or ethanol (Ogino et al., 2000;Karadzic et al., 2006) or extracted with organic solvents (Hiol et al., 1999). The

precipitation step concentrates the enzyme and often purifies to about two to three

fold (Aires-Barros et al ., 1994). Such partially purified lipase preparations are aptfor use in detergent formulations.



In general, single chromatographic step does not purify lipase to required level.

Combination of chromatographic steps works better. Ion-exchange

chromatography is the most common chromatographic method. The mostfrequently employed ion-exchangers are the diethylaminoethyl (DEAE) and

carboxymethyl (CM) cellulose (Gupta et al ., 2004).

Anion-exchange chromatography on Amberlite IRA 410 for B. licheniformis

MTCC 6824 resulted in 33.5 fold purification (Chakraborty and Raj, 2008). C.

antarctica lipase B has been purified by cation-exchange chromatography to 2.4fold (Trodler et al., 2008). P. aeruginosa lipase has been purified using anion

exchanger Q-Sepharose to 12.7 fold (Singh and Banerjee, 2007). DEAE-

Sepharose ion exchange chromatography was successful in purification of B.

cereus C71 lipase to 65 fold purification (Shaoxin et al ., 2007).

Lipases having large hydrophobic surfaces may be better purified by hydrophobic

interaction chromatography (Queiroz et al ., 2001). The use of hydrophobic

interaction chromatography has increased in the past few years. Various HICmatrices have been employed for the purification of microbial lipases e.g. octyl-

sepharose CL-4B for Pseudomonas sp. strain ATCC 21808 (Kordel et al ., 1991)and Bacillus sp. H257 (Imamura and Kitaura, 2000); Fractogel TSK-isobutyl 650

for P. aeruginosa YS-7 (Shabtai and Daya-Mishne, 1992); Butyl-Sepharose 4

Fast Flow for Rhizopus chinensis (Sun and Xu, 2008) and Butyl-toyopearl for P.

aeruginosa (Karadzic et al ., 2006).

Different affinity matrices/ supports have been used to purify lipases from various

microbial sources. Some of them are ConA-Sepharose for Pseudomonas sp. strainS5 lipase (Rahman et al ., 2005); oleic acid affinity column for Rhizopus delemar

lipase (Haas et al ., 1992) and hydroxyapitite for P. simplicissimum lipase (Sztajer

et al ., 1992) leading to 30, 10.3 and 56 fold purification respectively.

Cloning of lipase gene from Galactomyces geotrichum Y05 into pPIC9K and

overexpression in Pichia pastoris GS115 has been reported by Yan et al . (2007).Overexpression led to 10.4-fold higher production over the wild type strain. The

genes of organic solvent-tolerant LST-03 lipase (Lip9) and lipase-specific foldase

(Lif9) have been cloned and expressed in E. coli (pET system). The

overexpression of the lipase gene (lip9) was achieved upon using T7 promoter and deleting the signal peptide of the lipase. Under these conditions, the

overexpressed lipase accumulated in the form of inclusion bodies (Ogino et al .,

2007). Yet another lipase gene from Pseudomonas fluorescens JCM5963 has beencloned, sequenced and overexpressed as an N-terminus His-tag fusion protein in

E. coli. The recombinant lipase (rPFL) was purified to homogeneity by Ni-NTA

affinity chromatography and Sephacryl S-200 gel filtration chromatography(Zhang et al., 2009).

Properties of Lipases: Properties of various microbial lipases are summarized in

Table 1.



Table. 1 Properties of some microbial lipases.

Source Mol wt, pH, pH, temperature Substrate specificity Comments Reference

temperature stability

optima

Acinetobacter

calcoaceticus LP009

23 kDa, pH

7, 50°C

Stable at pH 4-8,

temperatures lower than 45°C

n.s.

Enzyme inactivated with

EDTA, enzyme stabilityenhanced with Triton X-100,

Tween-80 or Tween-20

Pratuamgdejkul

andDharmsthiti,

2000

Acinetobacter sp. RAG-1

33 kDa, pH9.0, 55°C

Active attemperatures up to

70°C

Hydrolyzes wide rangeof p NP esters, but

preference for medium

length acyl chains (C6,C8)

Lipase stabilized by Ca2+,strongly inhibited by EDTA,

Hg2+ and Cu2+, retains 75%

activity after exposure toorganic solvents

Snellman et al .,2002

B.

licheniformisMTCC 6824

74.8 kDa,

pH 8.0,45oC

half-life of 82 and 48

min at

K m 29 mM, V max 0.64

mM/mg/min

Activated by Ca2+ and Mg2+,

inhibited by Co2+, Cu2+, Zn2+,Fe2+, EDTA, PMSF, no

hydrysis of triacylglycerolswith more double bonds

Chakraborty

and Raj, 2008



B. pumilus B26

(recombinantlipase)

n.s., pH 8.5,35°C

n.s. Hydrolyzes various longtriacylglycerols, C14-

C18 and triolein (C18:1)

Exhibits Ca2+ independentthermostability and catalytic

activity

Kim et al .,2002

Burkholderia

sp. lipase

30 kDa, pH

11.0.

Stable at pH 6 -12 High rate of hydrolysis

towards linseed oil, neemoil, mustard oil and almondoil, preference for long

chain (>C12)triacylglycerides

Stable in organic solvents,

activated in presence of CaCl2, MgCl2, BaCl2, stableto bleaches and proteases

Rathi et al .,

2001

P.

aeruginosa

LP 602

n.s., pH 8,

55°C

90% residual activity

at pH 8 after 5 h;

50% residual activityat 55°C after 2 h

High activity towards

melted butter, castor,

coconut oil

Insensitive towards EDTA

Dharmsthiti

and

Kuhasuntisuk,1998

P.

aeruginosa

59.4 kDa,

pI 5.5

stable at pH 7-9 K m 50 mM, V max 27.1

mmol/ min/ mg

Active in EDTA, Tween-80,

and β -Mercaptoethanol,sodium dodecyl sulphate and

dithiothio-threitol inhibitedthe activity

Singh and

Banerjee, 2007

P.aeruginosa

LST-03

27.1 kDa, pH 6.0,

37oC

stable at pH 5-8 andand below 40°C

Prefers tricaproin(C6),ethyl octanoate (C8) and

coconut oil amongtriacylglycerols, fatty

acid methyl esters andnatural oils, random

positional specificity

n.s.

Ogino et al .,2000



against triolein, high

solvent stability

P.

fluorescens

AK 102

33 kDa,pH 8.0-10.0,55°C, pI4.0

pH 4-10, stablebelow 50 ºC for 1h.

Broad specificity Enzyme stable in anionicsurfactants

Kojima et al.,1994

P.

fluorescens

NS2W

n.s., pH9.0, 55°C

Stable over pH 3-

11; stable at 0°C

with more than70% residualactivity for at least2 h

n.s. n.s.

Kulkarni andGadre, 2002

P. luteola n.s., 55°C Half-life of 116 minat 65°C

Preference formedium chainsaturated andunsaturated fattyacids

Inhibited by Sn and Zn Litthauer et

al., 2002

P.

mendocina

3121-1

62 kDa,pH 7.2-9.5, 50-65°C

Different fordifferentsubstrates

Hydrolyzes pNPbutyrate, Tween-80,olive oil

pH and temperaturekinetics, effect of variousmetal ions and EDTAdepended on the natureof the substrate.

Surinenaiteet al.,2002

Pseudomon

as sp. strain

S5

60 kDa, pH9.0, 45°C

stable at 45°C and pH6-9

highest activity in the presence of palm oil andtriolein among natural oilsand synthetic triglyceride,

Ca2+ and Mg2+ stimulated activ-ity, EDTA had no effect, highsolvent stability

Rahman et al .,2005



random positional specifi-city

Serratia

marcescens

52 kDa,pH 8.0-9.0, 37°C

n.s.Michelis-Mentenconstant 1.35 mM ontributyrin

n.s.

Abdou, 2003



Halophilic Lipase

Isolation and characterization of salt stable lipase from halophilic sources haveattracted considerable interest in recent years. Availability of such enzymes would

facilitate industrial processes that require activity at high salt concentrations.

Moderately halophilic bacteria adapted to live in a wide range of saltconcentrations (3–15% NaCl) constitute an interesting group of micro-organisms

that could be used as a source of such salt-adapted enzymes (Ventosa et al.,

1998).

It is quite possible that the structural features of halophilic enzymes that impart

stability at high salt concentrations will also confer stability in organic solvents

and at high temperatures (Adams et al., 1995).

Therefore, it seems promising to screen halophiles for lipases having novel/ new

biochemical properties.

In a recent study, Sanchez- Porro et al. (2003) isolated hydrolase-producing

moderately halophilic and halotolerant eubacteria from Spanish salterns. Only23% of the 892 strains produced extracellular lipolytic activity.

In the course of screening programme a novel, moderately halophilic bacterium(strain SM19T) that displays novel lipolytic activity has been isolated and

characterized. Strain SM19T is a Gram-negative rod that grows optimally in

culture media containing 7.5% NaCl. This is classified in the genus Marinobacter

with proposed name Marinobacter lipolyticus sp. nov.(Martıń et al., 2003).

An extremely halophilic isolate, Salicola strain IC10, showing lipase and proteaseactivities has been marked for potential biotechnological applications. The

optimum growth conditions for this strain were 15-20% (w/v) NaCl, pH 8.0, and

370C. Its lipase showed highest activity against p-nitrophenyl-butyrate (deLourdes Moreno et al., 2009).

In the study related to archaea, total 118 halophilic strains were screened for

lipolytic activity. Eighteen were found positive on rhodamine agar plates. Highestlipase activities were found at pH 8, temperature 45-650C and NaCl 3.5-4 M.

These results indicate the presence of salt-dependent and thermostable lipases in

halophilic archaeal groups (Ozcan et al., 2009).

Fifty strains of moderately halophilic bacteria were isolated from various saltyenvironments in Iran. A strain designated SA-2 was shown to be the best

producer of extracellular lipase. Biochemical and physiological characterization

along with 16S rDNA sequence analysis placed SA-2 in the genus Salinivibrio

(Amoozegar et al., 2008).



Lipolytic activity has also been reported and characterized from an extremelyhalophilic archaeon, Natronococcus sp. (Boutaiba et al., 2006). Recently,

lipolytic enzymes have also been reported in B. halodurans strain originating

from a Kenyan alkaline soda lake (Vargas et al., 2004).

Thus variety of potential lipase producers has been studied in different part of

world. In Indian context the proposed study is likely to be a useful initiative.



Methods

● Sample collection and purification of halophiles

Samples were collected from different saline habitats along the western coast of

India in sterile plastic containers. pH and other physical characteristics of thesample were recorded at the time of sample collection. Halophilic bacteria were

isolated by salt enrichment♦. Pure colonies were obtained by repeated streaking

on nutrient agar plates containing 10%/ 20% NaCl (w/v).

● Screening of enzyme activity

Lipase producers among the isolated halophilic strains were screened ontributyrin/ Rhodamine B agar plates. Potential isolates showing good zone of

hydrolysis were cultured in broth and enzyme activity was reconfirmed. Potential

enzyme producers have been selected for further studies.

• Lipase assay

Lipase activity was determined by the method of Kilcawley et al . [2002] using p-nitrophenyl palmitate as substrate. The amount of liberated p-nitrophenol ( p NP)

will be recorded at 400 nm.

Maintenance and preservation

The pure cultures were preserved on the CMB (Complete Medium Broth)* agar

media containing (10% w/v NaCl and adjusted to pH 8-10) and stored at 4°C.Subculturing was done at monthly interval.

♦ (10-20% NaCl in EM1 media containing (%): olive oil 2; MgSO4.7H2O 0.04.; MgCl2.6H2O

0.07; CaCl2.2H2O 0.05; KH2PO4 0.03; K 2HPO4 0.03; (NH4)2SO4 0.05; 0.01% of trace elements

solution containing 0.026 B, 0.05 Cu, 0.05 Mn, 0.006 Mo and 0.07 Zn).

* Complex Medium Broth (CMB) (g/l): glucose, 10; peptone, 5; Yeast Extract, 5; KH2PO4, 5.

Identification of Isolates by 16S rRNA gene Sequencing



Genomic DNA was isolated by PrepManTM Ultra sample preparation kit (Applied

Biosystems Inc., CA, and USA). The purity of DNA was checked by comparing

260/280 nm, 260/230 nm absorbance ratios and electrophorectic mobility onagarose gels. The 16S rDNA sequence was obtained by using MicroSeq ® fullgene

16S rDNA sequencing kit (Applied Biosystems Inc., CA, USA) and bacterial

identification was done by using MicroSeq®

3130 microbial identification system.

Identification of Isolates by 16S rRNA gene Bioinformatic analysis

These sequences were compared with gene sequences available at “ribosomalDNA database” (http://rdp.cme.msu.edu/) and the identity of the isolates was

established.

● Transmission Electron Microscopy (TEM)

The cells of log phase, grown in presence of salt (NaCl) were harvested bycentrifugation at 10,000 x g for 10 min at 4oC. The pellet was washed with 0.1 M

phosphate buffer (pH 7.4). The washed cells were fixed in modified Karnovsky’sfluid and processed as per the procedure of David et al . (1973). Micrographs wererecorded at TEM facility of IIT Delhi.

Optimization of culture conditions for growth and enzyme production

Nitrogen and carbon source/ concentrations, salt, dO2, pH requirements and other

relevant parameters will be optimized using response surface methodology. Scale-

up will be carried out in 3.5 L bioreactor.

● Purification of halophilic lipase

Halophilic isolates rich in lipase activity will be harvested at active growth stage

and crude extract will be prepared in appropriate buffer. Firstly ammonium sulfate

precipitation will be carried out. The ammonium sulfate fraction containing

desired enzyme activity would be subjected to combination of ion exchange/molecular sieving chromatography for further purification. Recent techniques of

affinity precipitation/ HIC will also be attempted to develop a single step

purification procedure. The homogeneity of enzyme preparation will bedetermined by SDS-PAGE. Protein content at each stage will be estimated by

Bradford method (Bradford, 1976).

Characterization of Halophilic lipase



Km of lipase will be determined using p-nitrophenyl palmitate as substrate.

Temperature and pH optima will also be determined towards same substrate.

Substrate specificity of enzyme will be determined using range of substrates andestimating corresponding activity. Enzyme kinetics and stability will also be

monitored in presence of different salt concentrations.

Molecular weight of the enzyme will be determined by SDS-PAGE and gel

exclusion chromatography.

Behavior of lipase in aqueous-organic solvent/ salt concentrations/ surfactants anddetergent systems with respect to activity and stability will be studied by

following standard protocols (Gupta et al., 2005).

Application of halophilic lipase

Lipase will be used in organic solvent/ low water system for fatty acid ester

synthesis. The reaction conditions will be optimized for maximum product yields.Enzymes stable in presence of surfactant and detergents will be studied for cleansing efficiency in detergent formulations.

Gene Cloning and Overexpression

The identity of the lipase encoding genes would be determined. N-terminal

sequencing of the purified protein will be obtained and matched with homologous

sequences in database. The gene sequence of matching protein will be used for designing appropriate primers. PCR amplified product of the test DNA (based on

above primers) will be cloned and sequenced.The lipase genes would also be cloned in inducible expression vectors (e.g. pET

system from clontech) with ‘N’ or ‘C’ terminal tags for easy purification of therecombinant lipase, which will be characterized subsequently.

● Structural and functional analysis

Bioinformatic analysis will be done to predict the three-dimensional structure of

halophilic lipase and to study the possible reasons for salt, temperature and

organic solvent stability of the protein.



Work done so far

Sample Collection

Various saline and hyper saline samples (water and soil) were collected from sea of

Calicut (Kerala), saltern near old Goa (Goa), Nagoa beach (Diu, Gujarat), Somanath

(Gujarat) and Triveni sangam (Gujarat).

Isolation of Halophilic bacteria by salt enrichment

Halophilic bacteria were isolated by salt enrichment (10-20% NaCl). The isolates were

subjected to repeat streaking on nutrient agar plates containing NaCl (10-20% W/V) (Fig.

2a.). The pure halophilic colonies were plated on tributyrin agar to isolate lipase

producers based on zone of hydrolysis (Fig.2b).

Fig. 2a. Isolated halophiles streaked on nutrient agar plate

Fig. 2b. Lipase activity of Halophiles on tributyrin agar plate



Cell Morphology and Gram Staining

The isolates exhibited variation in their cell morphology, cell arrangement and Gram’s

reaction. The microscopic observations revealed that majority of the isolates were Gram

negative. Only few were gram positive, overall results are summarized in Table 2.

Table 2

A typical Gram negative reaction of isolate K 1 (from saline samples of Kerala coast) is

shown in Fig. 3.

Fig 3. Gram’s staining of isolate K 1

Lipase Activity in broth

Site of sample collection Gram positive isolates Gram negative isolates

Kozhikhode beach, Kerala 3 9

Nagoa beach, Diu, Gujarat 4 8

Somanath, Gujarat 4 11

Triveni sangam, Gujarat 6 12saltern near old Goa (Goa) 1 7



To monitor lipase production in liquid broth, media with following composition was

used:

Peptone- 1.0%

Yeast extract- 0.5%

MgSo4,7H2O- 0.02%.

CaCl2, 2H2O- 0.01%

Olive oil- 1%

Salt (NaCl) – 10%.

Medium was inoculated with 4%, v/v of 24 h grown mother culture (OD~1.3) and

incubated at 30oC in orbital shaker maintained at 120 rpm. Samples were withdrawn at

various time intervals and centrifuged at 10,000 x g for 10 min at 4 oC. Lipase activity

was determined in the cell-free supernatant as per following procedure:

Lipase assay

p-nitrophenyl palmitate was used as substrate. Briefly, 1.8 ml of solution containing 0.15

M NaCl and 0.5% Triton X-100 in 0.05 M Tris-HCl buffer (pH 8.0) was preincubated at

40°C. To this solution, 200 µl of suitable dilution of culture supernatant and 20 µl of

substrate (50 mM p NPP in acetonitrile) were added followed by incubation at 40°C for

30 min. The amount of liberated p-nitrophenol ( p NP) was recorded at 400 nm. One unit

of lipase activity is defined as the amount of enzyme liberating 1 nmol of p NP under

standard assay conditions (Kilcawley et al ., 2002).

Level of lipase production in different isolates is summarized in Table 3. On the basis of

level of production isolate K 1 was selected for further study. Halophilic isolate S-15-9

was a generous gift, from Prof. S.P Singh (Saurashtra University) that was compared with

other isolates.



Table 3. Lipase production by halophilic isolates

Isolates Lipase production (U/ml)

K 1 129K 2 35

K 3 88

K 4 33

DL-20-1 51

DL-20-2 43

TL-20-2 65

SL-20-2 42

S-15-9 57

G4 32

G5 36

K- Isolate from sea water and sand (clear water) sample, Kozhikhode beach, Kerala

D- Isolate from sea water and sand. (Turbid water with soil particles) sample, Nagoa beach, Diu, Gujarat

T- Isolate from sea water, soil and sand. (Turbid water with soil particles) sample, Triveni sangam, Gujarat

S- Isolate from sea water, pebble and sand (clear water) sample, Somanath, Gujarat

G- Isolate from soil sample, saltern near old Goa (Goa)

Biochemical characterization of isolate K 1 was done by Hi25TM (HiMedia) kit. Results

are shown in Table. 4.

Table 4. Biochemical characteristics of isolate K 1



Parameters Observation Parameters Observation

Gram’s staining Negative Oxidase Positive

Cell shape Rod Methyl Red Negative

Pigmentation White Indole production Negative

Catalase Positive Xylose utilization Negative

ONPG Negative Serine utilization Positive

Lysine decarboxylase Positive Cellobiose utilization Positive

Ornithine decarboxylase Positive Melibiose utilization Negative

Urease Positive Fructose utilization Positive

Nitrate production Positive Raffinose utilization Negative

H2S production Negative Glucose utilization Positive

Citrate utilization Positive Lactose utilization Negative

Voges Proskauer’s Negative Glycerol utilization Positive

Identification of K 1 and S-15-9 by 16S rRNA gene sequencing

The full length gene of isolate K 1 showed ~99 % relatedness with Marinobacter sp.

Therefore, the new isolate is named as Marinobacter sp. EMB5. Isolate S-15-9 showed

~99 % relatedness with a Haloalkaliphilic bacterium. Therefore, this is named as

Haloalkaliphilic bacterium S-15-9. Phylogenetic tree of isolates are shown in Fig. 4.





Characterization of lipase

The preliminary characterization of Crude lipases from Marinobacter sp. EMB5 andHaloalkaliphilic bacterium S-15-9 was initiated. The results are shown in Table 5.

Table 5. Characterization of crude lipases

Isolates pH

optima

Salt

optima

(NaCl)

Temp.

optima

Solvent

stability

Thermal

stability

K1

( Marinobacter sp.

EMB5.)

9.0 2% 500C Highly

stable in

organic

solvents

600C for 30

minutes

S-15-9

(Haloalkaliphilic

bacterium S-15-9)

9.5 1.5% 650C Highly

stable in

organic

solvents

700C for 30

minutes

Optimization of conditions for lipase Production from Marinobacter sp. EMB5 is under

progress.

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