Research Plan On

Halophilic Enzymes and Their Biotechnological Applications

Submitted by of Sumit Kumar (2007CYZ8227)

Under the Supervision Dr. S. K. Khare

DEPARTMENT OF CHEMISTRY INDIAN INSTITUTE OF TECHNOLOGY, DELHI

Research Plan
Submitted by: Registered Topic:
Sumit Kumar (2007CYZ8227) Halophilic Enzyme and Their Biotechnological Applications Dr. S. K. Khare Completed 12 Credits CGPA: 9.5/10

Ph.D. Supervisor: Course Work:

Introduction
Hydrolases, especially proteases, lipases and amylases are the largest selling industrial 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 enzymes in 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 such conditions. 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 and function 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 hypersaline environment 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 50C. 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 75-80C and reported to be stable at high NaCl concentration. The biotechnological applications of halophiles and their enzymes are less explored. 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 in hypersaline 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 and Dordick, 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 protein folding 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 novel applications 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. The reasons 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 industrial importance. 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 by Response 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 broth can 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 apt for 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 most frequently 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 2.4 fold (Trodler et al., 2008). P. aeruginosa lipase has been purified using anion exchanger Q-Sepharose to 12.7 fold (Singh and Banerjee, 2007). DEAESepharose 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 HIC matrices have been employed for the purification of microbial lipases e.g. octylsepharose 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. strain S5 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 been cloned, 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 Source

Properties of some microbial lipases. Mol wt, pH, pH, temperature temperature stability optima 23 kDa, pH 7, 50C Stable at pH 4-8, temperatures lower than 45C Active at temperatures up to 70C Substrate specificity Comments Reference

Acinetobacter calcoaceticus LP009 Acinetobacte r sp. RAG-1

n.s. Hydrolyzes wide range of pNP esters, but preference for medium length acyl chains (C6, C8) Km 29 mM, Vmax 0.64 mM/mg/min

Enzyme inactivated with EDTA, enzyme stability enhanced with Triton X-100, Tween-80 or Tween-20

Pratuamgdejkul and Dharmsthiti, 2000

33 kDa, pH 9.0, 55C

Lipase stabilized by Ca2+, Snellman et al., strongly inhibited by EDTA, 2002 Hg2+ and Cu2+, retains 75% activity after exposure to organic solvents Activated by Ca2+ and Mg2+, Chakraborty inhibited by Co2+, Cu2+, Zn2+, and Raj, 2008 Fe2+, EDTA, PMSF, no hydrysis of triacylglycerols with more double bonds

B. licheniformis MTCC 6824

74.8 kDa, pH 8.0, 45oC

half-life of 82 and 48 min at

B. pumilus B26 (recombinant lipase) Burkholderia sp. lipase

n.s., pH 8.5, 35C

n.s.

Hydrolyzes various long Exhibits Ca2+ independent Kim et al., triacylglycerols, C14- thermostability and catalytic 2002 C18 and triolein (C18:1) activity High rate of hydrolysis towards linseed oil, neem oil, mustard oil and almond oil, preference for long chain (>C12) triacylglycerides High activity towards melted butter, castor, coconut oil Km 50 mM, Vmax 27.1 mmol/ min/ mg Stable in organic solvents, Rathi et al., activated in presence of 2001 CaCl2, MgCl2, BaCl2, stable to bleaches and proteases

30 kDa, pH 11.0.

Stable at pH 6 -12

P. aeruginosa LP 602 P. aeruginosa

n.s., pH 8, 55C

90% residual activity at pH 8 after 5 h; 50% residual activity at 55C after 2 h stable at pH 7-9

Insensitive towards EDTA

Dharmsthiti and Kuhasuntisuk, 1998 Singh and Banerjee, 2007

59.4 kDa, pI 5.5

Active in EDTA, Tween-80, and -Mercaptoethanol, sodium dodecyl sulphate and dithiothio-threitol inhibited the activity

P. aeruginosa LST-03

27.1 kDa, pH 6.0, 37oC

stable at pH 5-8 and and below 40C

Prefers tricaproin(C6), ethyl octanoate (C8) and coconut oil among triacylglycerols, fatty acid methyl esters and natural oils, random positional specificity

Ogino et al., 2000 n.s.

against triolein, high solvent stability

P. fluorescens AK 102 33 kDa, pH 4-10, stable pH 8.0- below 50 C for 1h. 10.0, 55C, pI 4.0 n.s., pH 9.0, 55C Stable over pH 311; stable at 0C with more than 70% residual activity for at least 2h Half-life of 116 min at 65C Broad specificity Enzyme stable in anionic surfactants Kojima et al., 1994

P. fluorescens NS2W

Kulkarni and Gadre, 2002 n.s. n.s.

P. luteola

n.s., 55C

Preference for medium chain saturated and unsaturated fatty acids Hydrolyzes pNP butyrate, Tween-80, olive oil

Inhibited by Sn and Zn

Litthauer et al., 2002

P. mendocina 3121-1

62 kDa, pH 7.29.5, 5065C 60 kDa, pH 9.0, 45C

Different for different substrates

pH and temperature kinetics, effect of various metal ions and EDTA depended on the nature of the substrate. Ca2+ and Mg2+ stimulated activity, EDTA had no effect, high solvent stability

Surinenaite et al., 2002

Pseudomon as sp. strain S5

stable at 45C and pH 6-9

highest activity in the presence of palm oil and triolein among natural oils and synthetic triglyceride,

Rahman et al., 2005

random positional specificity Serratia marcescens 52 kDa, pH 8.09.0, 37C n.s. Michelis-Menten constant 1.35 mM on tributyrin Abdou, 2003 n.s.

Halophilic Lipase Isolation and characterization of salt stable lipase from halophilic sources have attracted 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 salt concentrations (315% 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. Only 23% 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.(Martn et al., 2003). An extremely halophilic isolate, Salicola strain IC10, showing lipase and protease activities 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 (de Lourdes 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. Highest lipase 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 salty environments 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 extremely halophilic 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 the sample 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 on tributyrin/ 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 pnitrophenyl palmitate as substrate. The amount of liberated p-nitrophenol (pNP) 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 4C. Subculturing was done at monthly interval.

(10-20% NaCl in EM1 media containing (%): olive oil 2; MgSO 4.7H2O 0.04.; MgCl2.6H2O
0.07; CaCl2.2H2O 0.05; KH2PO4 0.03; K2HPO4 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 on agarose 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 ribosomal DNA 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 by centrifugation 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 Karnovskys fluid and processed as per the procedure of David et al. (1973). Micrographs were recorded 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. Scaleup 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 be determined 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 and estimating 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 and detergent 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 the recombinant 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 Grams 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

Site of sample collection Kozhikhode beach, Kerala Nagoa beach, Diu, Gujarat Somanath, Gujarat Triveni sangam, Gujarat saltern near old Goa (Goa)

Gram positive isolates 3 4 4 6 1

Gram negative isolates 9 8 11 12 7

A typical Gram negative reaction of isolate K 1 (from saline samples of Kerala coast) is shown in Fig. 3.

Fig 3. Grams staining of isolate K1

Lipase Activity in broth

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 40C. To this solution, 200 l of suitable dilution of culture supernatant and 20 l of substrate (50 mM pNPP in acetonitrile) were added followed by incubation at 40C for 30 min. The amount of liberated p-nitrophenol (pNP) was recorded at 400 nm. One unit of lipase activity is defined as the amount of enzyme liberating 1 nmol of pNP 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 K1 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 K1 K2 K3 K4 DL-20-1 DL-20-2 TL-20-2 SL-20-2 S-15-9 G4 G5 Lipase production (U/ml) 129 35 88 33 51 43 65 42 57 32 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 K1 was done by Hi25TM (HiMedia) kit. Results are shown in Table. 4.

Table 4. Biochemical characteristics of isolate K1

Parameters Grams staining Cell shape Pigmentation Catalase ONPG Lysine decarboxylase Ornithine decarboxylase Urease Nitrate production H2S production Citrate utilization Voges Proskauers

Observation Negative Rod White Positive Negative Positive Positive Positive Positive Negative Positive Negative

Parameters Oxidase Methyl Red Indole production Xylose utilization Serine utilization Cellobiose utilization Melibiose utilization Fructose utilization Raffinose utilization Glucose utilization Lactose utilization Glycerol utilization

Observation Positive Negative Negative Negative Positive Positive Negative Positive Negative Positive Negative Positive

Identification of K1 and S-15-9 by 16S rRNA gene sequencing The full length gene of isolate K1 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.

(a)

(b) Fig. 4. Phylogenetic analysis of 16S rRNA gene (a) Marinobacter sp. EMB5 (b) Haloalkaliphilic bacterium S-15-9

Transmission Electron Microscopy (TEM) The transmission electron micrograph of these two isolates is shown in Fig. 5 (a & b).Halophilic isolate Marinobacter sp. EMB5 was rod in shaped, whereas isolate Haloalkaliphilic bacterium S-15-9 was round in shape.

(a) (b) Fig.5. Transmission electron micrographs (a) Marinobacter sp. EMB5 cells (b) Haloalkaliphilic bacterium S-15-9 cells.

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

Table 5. Characterization of crude lipases

Isolates pH optima K1 (Marinobacter sp. EMB5.) S-15-9 (Haloalkaliphilic bacterium S-15-9) 9.5 1.5% 650C 9.0 Salt optima (NaCl) 2% Temp. optima 500C Solvent stability Highly stable in organic solvents Highly stable in organic solvents 700C for 30 minutes Thermal stability 600C for 30 minutes

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

References

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