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The extracellular thermoalkaline lipase from Geobacillus sp. Iso5 was puriï¬ed to homogeneity by ultrafiltration, 6% cross-linked agarose and Phenyl spehrose HIC column chromatography. The final purified lipase resulted in 8.7-fold with 6.2% yield. The relative molecular weight of the enzyme was determined to be a monomer of 47 kDa by SDS-PAGE and MALDI-TOF MS/MS spectroscopy. The puriï¬ed enzyme exhibit optimum activity at 70 °C and pH 8.0. The enzyme retained above 90% activity at temperatures of 70°C and about 35% activity at 85 °C for two hours. However, the stability of the enzyme decreased at the temperature over 90 °C. The enzyme activity was promoted in the presence of Ca2+ and Mg 2+ and strongly inhibited by HgCl2, PMSF, DTT, K+, Co2+and Zn 2+. EDTA did not affect the enzyme activity. The secondary structure of purified lipase contains 36% α-helix and 64% β- plated sheet which was determined by Circular dichromism, FTIR and Raman Spectroscopy
Geobacillus sp. Iso5, Lipase, MALDI-TOF MS/MS, CD, FTIR and Raman Spectroscopy.
Lipases (triacylglycerol acylhydrolases, 18.104.22.168) are the ubiquitous and indispensable class of hydrolases, which occupy a prominent place among biocatalysts. After proteases and carbohydrases, lipases are considered as the third largest group based on total sales about 5% of enzyme market [1, 2]. Lipase in turn need to develop novel drugs, surfactants, bioactive compounds, oleochemicals, leather, detergents, foods, perfumes and diagnostic products [3-7]. Like other hydrolases, microbial lipases have greater industrial importance, as they are more stable when compared to plant and animal lipases. The lipases being hydrolases, involved in the synthesis of a broad range of natural and non-natural esters . Lipases are catalyzed by inter-esterification, alcholysis, acidolysis, esterification, aminolysis, enantio and regioselective-hydrolysis. The conversion of triacylglycerides (TAG) is involved by the interfacial restriction of their catalytic activity by hydrolysis of ester bonds to interface between lipid and water on largely aggregated substrate [6, 9, 10].
The extensive application of thermophilic enzymes has gained lot of attention during recent years as industrial biocatalysts. Most of the biocatalysts are of inherently labile; therefore, their operational stability is of paramount importance for any bioprocess. The large-scale production due to remarkable thermostability, resistance to proteolysis, extreme pH and chemical agent have made them as a unique tool as industrial biocatalyst [11, 12]. As most of the industrial process, operate at the elevated range of temperature. Bioprocesses at high temperatures lead to higher diffusion rate, increased solubility of lipids, and reduced risk of contamination [13, 14]. Hence, thermostable lipases with their inherent stability, have received great potential in organic chemical processing, detergent formulations, synthesis of biosurfactants, oleochemicals industry, dairy industry, agrochemical industry, paper manufacture, nutrition, cosmetics and pharmaceutical processing [7, 15- 17]. The production of such lipases from microorganism has been focused into the optimal stability at high temperature and alkaline pH [14, 18- 20]. Since, the majority of the industrial process required a large-scale production of lipase. This was enabled by the enhancement of production capability by cloning and expression such as thermophilic lipase gene from thermophiles into more suitable mesophilic hosts is in practice [21- 23]
Recently, many thermostable lipases were isolated, purified, and its production was improved by cloning the several lipase gene from Geobacillus sp into suitable hosts [10, 24]. In this study, we have purified and characterized a thermoalklophilic extra cellular lipase from Geobacillus sp. Iso5 using conventional chromatography and spectroscopic methods.
Materials and Methods
The hyperthermophilic alkali resistant Geobacillus sp. Iso5, used in the study was previously isolated from Irde geothermal springs of southern India . In brief, the bacterium exhibited the typical morphological, physiological and biochemical characteristics of genus Geobacillus [26, 27]. The 16s rRNA gene sequence of the bacteria was assigned with the accession number DQ 140232. The bacteria can be able withstand the temperature up to 95 °C with optimum pH of 8.0.
The bacterial isolate was inoculated into enrichment medium containing (in g l-1) Tryptone, 6; Yeast Extract, 2; NaCl, 5; CaCl2, 0.1; olive oil, 2%; MgSO4 0.2% and supplemented with mineral salt [MgSO4. 7H2O (0.04%); MgCl2. 6H2O (0.07%); CaCl2. 2H2O (0.05%); KH2PO4 (0.03%); K2HPO4 (0.03%); (NH4)2SO4 (0.05%)] . The bacterium was incubated at 60°C under shaking condition (150 rpm) for two days at initial pH 7.0.
The lipase activity was assayed calorimetrically using copper soap method. In brief, one ml of culture ï¬ltrate was shaken with 2.5 ml of olive oil (70% oleate residues) emulsion (1:1, v/v) and 20 µl of 0.02 M CaCl2 in a water bath shaker at an agitation rate of 200 rpm. The emulsion was prepared by mixing an equal volume of olive oil (Bertoli, Italy) and 50 mM phosphate buï¬€er of pH 8.0 with a magnetic stirrer for 10 min. The reaction mixture was shaken for 30 min at 50 °C. The enzyme reaction in the emulsion system was stopped by adding 6 N HCl (1 ml) and isooctane (5 ml), followed by mixing using a vortex mixer for 30 s. The upper isooctane layer (4 ml) containing the fatty acid was transferred to a test tube for analysis. Copper reagent (1 ml) was added and again mixed with a vortex mixer for 30 s. The reagent was prepared by adjusting the solution of 5% (w/v) copper (II) acetate monohydrate to pH 6.1 with pyridine. The absorbance of the upper layer was read at 715 nm. Lipase activity was measured by measuring the amount of free fatty acids released from the standard curves of free fatty acids One unit of lipase activity was deï¬ned as the amount of enzyme releasing 1 µmole of fatty acid per minute.
Estimation of protein
Estimation of protein was performed using the method previously described . Bovine Serum Albumin (BSA) was used as standard protein.
Purification of Lipase
Lipase was purified by centrifuging the supernatant at 10000 rpm for 20 min. The supernatant was collected and concentrated on 10-kDa Amicon ultrafiltration membrane (Millipore, USA). The concentrated enzymes solution was precipitated to 75% using ammonium sulfate. The precipitate was collected by centrifugation at 6000 rpm for 10 min, dissolved in 10 mM potassium phosphate buffer, pH 8.0, and applied to a 6% cross linked agarose (12 X 1.5 cm) (GE, Pharmacia) gel filtration chromatography. The column was pre equilibrated with 10 mM phosphate buffer of pH 8.0; 100 mM NaCl; 1 mM EDTA and 7 mM 2- mercaptoethanol. The bound protein was eluted with the same equilibration buffer. Lipase active fractions were collected and the powdered ammonium sulfate was added to the collected solution to adjust the same concentration (final 1 M ammonium sulfate) with equilibration buffer of phenyl sepharose column. This solution was applied on Phenyl Sepharose fast flow HIC column (12 X 1.5) (GE, Pharmacia) pre-equilibrated with 10 mM phosphate buffer of pH 8.0; 100 mM NaCl; 1 mM EDTA; 7mM 2- mercaptoethanol and 1 M (NH4)2SO4. The enzyme was eluted by linearly decreasing gradient with 600 to 0 mM (NH4)2SO4 containing elution buffer. The Fractions containing high lipase activity (around 0.3 M and 0.5 M (NH4)2SO4) were pooled, desalted by dialysis (10 kDa cutoff membrane) against equilibration buffer.
Electrophoretic analysis and MALDI-TOF MS/MS
The lipase was tested for purity and molecular weight of purified enzyme was determined using 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) . The gels were stained with silver stain to visualize the protein bands. The apparent mass of the enzyme were estimated by interpolation from a logarithmic graph of molecular mass versus relative migrations of standard proteins; Bovine serum albumin (66 kDa), protease (48 kDa), Carbonic anhydrase (29 kDa) and Lysozyme (14 kDa) (Aristogene Bioscience, India). The exact molecular weight of enzyme was determined from the peak position in MALDI-TOF-MS (Ultraflex TOF-TOF Bruker Daltonics, Bremen, Germany) equipped with nitrogen laser (337 nm). The enzyme was mixed with equal volume of saturated matrix solution (2, 5-dihydroxybenzoic acid in 50% acetonitrile/H2O with 0.1% trifluroacetic acid) and dried. The mixture (1 µl) was spotted on a MALDI target plate and the spectrum was recorded in the linear positive ion mode. The spectral data were processed by Bruker Daltonics FLEX analysis software (version 2.0).
Effect of temperature and stability on the enzyme activity
The effect of temperature and thermal stability of the lipase enzyme was determined spectrophotometrically using copper soap method. The effect of temperature was determined by measuring the rate of reaction at temperature range of 35 to 90 °C. At various intervals of time from one minute to four hours in 50 mM potassium phosphate buffer, the thermal stability of the enzyme was determined at 60-85 °C.
Effect of pH on enzyme activity
The effect of pH on lipase activity was determined according to method previously described method . Three different buffer system for pH range 5.0 to 11; 50 mM acetate/Na-acetate buffer (pH 5.0-5.8), 50 mM Na2HPO4 / NaH2 PO buffer (pH 5.8-8.0), 50 mM Tris-HCl buffer (pH 7.5-9.0), or 50 mM glycine/NaOH buffer (pH 9.0-11.0) were used.
Effect of metal ions, Inhibitors and detergents on lipase activity
The effects of various metal ions on lipase activity was determined by pre incubating the enzymes with 5 mM concentration of various metal ion's K +, Ca2+, Mg2+, Fe2+, Co2+, Hg2+ and Na+ (chloride form) in 50 mM potassium phosphate buffer, pH 8.0. All the assay mixtures were incubated at 60°C for 30 min and residual activities were measured using spectrophotometrically by copper soap method (described above). The effects of various inhibitors on enzyme was assayed by incubating with 5mM concentration of PMSF, EDTA, β-mercpta ethanol and dithiotheriotol for 30 min in 50 mM Tris-HCl; pH 8.0. The effect of detergents on enzyme activity was also determined by incubating with 0.4% of Triton X-100, 80, 20 and 1 mM SDS under standard assay condition as described earlier.
Circular Dichroism (CD)
Circular dichroism analysis for enzyme secondary structure was performed at the protein concentration of 1 g/dl. The data was recorded over a wavelength range of 190 to 250 nm using Jasco-815 spectropolarimeter (Jasco UK) at room temperature. The analysis was recorded with data pitch of 0.2 nm, bandwidth of 1 nm, scanning speed of 50 nm.min− 1 and a response time of 1 second. Ultra pure water was used as a reference . At a given wavelength, the resultant spectra was expressed in molar ellipticity (Ï´) are (deg•cm2/dmol) and the percentage of conformation evidence was analyzed using the web service tool http://www.ogic.ca/projects/k2d2/.
Fourier transforms infrared spectroscopy (FTIR)
Nicolet 380 FTIR (Thermo Electron, USA) was used to determine the further secondary structure analysis of the lipase enzyme. The machine used a diamond crystal to measure absorbance from 30000cm-1-200cm-1. This study was carried out in the mid-infrared region of 4000cm-1 -1000cm-1, where the structural bonds appear. Each result was an average of 32 scans and subtraction from a pure water standard was made for all scans.
The Raman spectrum of the native enzyme was obtained by the method described previously [33, 34, 35]. The enzyme was diluted to 50 mM Tris-HCl, pH 7.0. The diluted sample was placed in the Raman sample cell at a spectral resolution of 5 cm-1. The sample was excited with 406.7 nm (Spectra Physics Model 170 Kr+) and 441.6 nm (Liconix, He-Cd) laser beams. Laser power at the sample was measured with a Coherent Model 201 power meter. Absorption spectrum was measured in a 1-mm path length optical cell using Uvikon 2000 UV-Vis spectrophotometer.
Purification of extracellular thermoalklophilic Lipase
Lipase was puriï¬ed by ultrafiltration, 6% cross-linked agarose size exclusion (Fig. 1), Phenyl Sepharose chromatography (Fig. 2). The size exclusion chromatography resulted in two peaks A and B. Where, peak A does not show any lipase activity. However, peak B was shown enhanced activity of lipase and determined to be 81 U/mg with a 36 % recovery, which was higher than the initial purification steps. The purification strategy was increased to about 34 fold with the specific activity of 126 U/mg on Phenyl-Sepharose HIC column using decreasing gradient of 600-0 mM (NH4)2SO4. The enzyme was eluted at 380 mM (NH4)2SO4 resulted in single lipase active peak. The detail purification steps are summarized in Table 1.
Characterization of Lipase
Molecular weight by SDS-PAGE and MALDI TOF MS/MS
The HIC column purified enzyme showed a single band at 47 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Fig. 3). Exact molecular weight purified enzyme was also confirmed by MALDI- TOF MS/MS, showed an intense peak corresponding to 47.162 kDa (Fig. 4).
Effect of temperature, stability and pH on the activity lipase
The thermostable lipase exhibited activities at temperatures range of 60-85 °C in 50 mM potassium phosphate buffer pH 8.0. After incubation at temperature range of 40-95°C for 20 min potassium phosphate buffer (pH 8.0), the optimal lipase enzyme activity was at 70 °C in 50mM potassium phosphate buffer (pH 8.0) (Fig. 5). The residual activity of the lipase was measured at 60-85 °C for four hours. The enzyme retained above 90% activity at temperatures 60°C and about 35% activity at 85 °C for two hours. However, the stability of the enzyme decreased at the temperature over 85 °C (Fig. 7). The pH profile of thermostable lipase was shown active over a broad pH range (pH 8.0-12) but exhibit optimum activity at pH 8.0 (Fig. 6). Results from this investigation revealed that, lipase enzyme was thermostable and resistant to slight alkaline pH range.
Effect of metal ions, Inhibitors and Detergents
The effect of CuCl2, and ZnCl2 on lipase activity was shown 62% and 65% inhibition respectively. However, other metal ions such as Co2+, Ca2+, Mg2+, Fe 2+ did not affect much on the activity. Maximum inhibition on residual activity of the enzyme was found with HgCl2 (09 %). Various inhibitors such as PMSF (12%) and DTT (31%) affects largely on the activity of the enzyme. Other inhibitors such as EDTA and β-Mercapta ethanol did not affect much on the enzyme reaction. The studies with the various Triton X family and SDS did not affect the lipase activity (Table 2).
The structural deconvolation by circular dichroism analysis was concluded that the native lipase covering of largest proportion of α -helical (36%) and áµ¦ -strand (64%) (Fig. 8). The spectral range for unordered structure did not appear for this enzyme. This uncertainty of composition was further concluded based on the vibrational bands of the protein and particularly the amide I band for carbonyl stretch for both FTIR (1630-1670 cm-1) and Raman (1600-1700 cm-1), which is sensitive to the secondary structure. In FT-IR and Raman spectrum, the characteristic amide I band arises principally from the C=O stretching vibration of the peptide group at 1645-1680 cm−1. The α- helical β- strands and random structure are attributed to the change of the shift in the normal frequency range 1645-1660 (α- helical) cm-1 for water soluble protein (Fig. 9). The higher shift (1702 cm-1) in frequency 1660-1680 cm-1 for β- strands, which contributed to the structural predominance in lipase enzyme. However, N-H deformation for amide II (1,550 cm−1) in FTIR has been contributed by absorbance at 1552 cm-1 to monitor 1H to 2H exchange in proteins. In contrary, the amide II frequencies are very sensitive to Raman spectroscopy. Hence, for globular protein mobility, it has little utility in backbone structural analysis. There is weak amide III frequency for random structure was observed in the IR spectrum. This is due to the amide III absorption is normally very weak in the infrared spectroscopy, arising primarily from N-H bending, C-N stretching and conformational shift in the protein bonds. The amide III frequencies was determined by Raman spectroscopy at 1230-1300 cm-1, which attributed by the presence of frequency at 1245 cm-1 (Fig. 10). It has been used in conjunction with amide I for estimating the relative contributions α-helix, β-sheet and random coil in a number of proteins. Results of these spectral analyses revealed that, the native enzyme composed of largest portion of α -helix and áµ¦ -strands.
Microorganisms thrive at elevated temperatures are able to produce thermo active enzymes, capable of being catalytically active at high temperature [16, 36]. The stability these biocatalysts are an important criterion when dealing with bioprocesses at the high temperatures for sustainable operation, which in turn determined by genetic and environmental factors . In addition, the thermozymes often showed higher stability toward organic solvents, detergents and other industrial harsh condition [2, 4, 5, 38, 39]. Many thermostable lipase producing microbial sources are available as wild and recombinant strains. In this relevant, the members of the genus Geobacillus are able to produce extracellular lipase enzymes, which have wide applications in biotechnology [10, 28, 24, 39]. Extracellular lipase enzyme production from this genus was shown high thermostable property, substrate specificity, resistant to mild alkaline condition, solvent degradation and optimization are less expensive [7, 10, 24]. It is reported that, the extracellular lipase from Geobacillus can be induced in the presence of natural oils, p-nitrophenyl palmate, triacylglycerols, fatty acids, hydrolyzable esters, tweens, bile salts and glycerol [4, 40- 42]. Certain long-chain fatty acids such as, oleic, linoleic and linolenic acids are known to support lipase production from various Bacilli . However, their production was significantly influenced by other carbon sources, such as sugars, sugar alcohol, polysaccharides, whey, casamino acids and other complex sources. Peptone and yeast extract, which have been used as a nitrogen source along with some metal ions such as KH2PO4, MgSO4, KCl, CaCl2, (NH4)2SO4 MgCl2 and K2HPO4 for improved production of lipase [16, 43]. Further, the expression of recombinant lipase enzyme from Geobacillus spp is much in practice [21, 44, 45].
The enzyme required at least two or three chromatographic steps to determine the absolute purity . The purification strategy of lipases was previously reported on G. stearothermophilus L1, G. stearothermophilus P1, G. thermocatenulatus and G. thermoleovorans [38, 44]. A 34 kDa lipase from B. thermoleovorans ID-1 was purified by three chromatographic purification steps . Two thermostable lipase enzymes from B. thermocatenulatus B.TL1, B.TL2 and Bacillus sp. J33 was shown to adsorbed on a hydrophobic support (octadecyl-Sepabeads) and phenyl Sepharose column exhibited a hyperactivation with respect to the soluble enzyme [48, 49]. Since, 43-44 kDa are the most commonly reported molecular weight for Geobacillus lipases [23, 46, 50-51]. However, the presence of low molecular weight lipases was also been reported . In particular, molecular sizes of lipases from thermophilic bacteria range from 16 kDa in Bacillus thermocatenulatus to 69 kDa in Bacillus sp THL027 were observed . The production of two or more of lipase from G. thermoleovorans CCR11 depending on the growing conditions are also reported . The Isoelectric point of purified lipase was 7; where, lipase from Bacillus subtilis 168, Bacillus sp A30-1, Bacillus stearothermophilus, and Bacillus sp H-257 has isoelectric points of 9.9, 5.15, 7.4 and 4.66, respectively .
The effect of temperature and pH on lipase from Geobacillus sp is higher when compared to other reported Bacillus sp. Extracellular lipase from Gb5 was shown optimum of 70°C at pH 8.0. However, the optimum catalytic temperature of Bacillus thermoleovorans CCR11 is 60°C and retaining more than 80% of activity after 26 h at 30 °C at a pH between 9 and 10 . On the other hand, the recombinant lipase of Bacillus sp. Tp10A.1 , L1 lipase  and P1 lipase  of Bacillus stearothermophilus and BTL2 lipase of Bacillus thermocatenulatus  had reported optimum temperatures of 55-600C. Similar to that, reported lipases from B. thermoleovorans ID1 (pH 9) and Bacillus A30-1 (pH 9.5) were higher to other lipases from thermophilic Bacillus which lie in the range of pH 5-11.
The effect of metal ions could attribute to the change in the solubility and the behavior of ionized fatty acids at interfaces. This may affect change in the catalytic properties of the P1 lipase . Lipase activity was not affected by the presence of EDTA. Whereas, Mg2+, K+ and Li+ salts decreased activity by 25, 24, and 23%, respectively, after 1 h of incubation at 30 °C. However, Zn2+, Cu2+, Hg2+ and Co2+ has the strong effect on lipase activity of several thermophiles [46, 51]. It has been known that Ca2+ salts increased activity immediately (59%) after one hour of incubation at 30 °C (35%).The influence of calcium ion was considered to be caused by their action on the release of fatty acids and on enzyme structure stabilization . The influence of detergents will greatly affect on the activity of lipase. Tween 80 completely inhibits the BTL2 lipase activity and while, P1 lipase showed a moderate inhibition in the presence of Tween 20 [46, 51]. On the other hand, SDS (0.1%) showed moderate inhibition on P1 lipase , because of a more rigid structure and closed lid conformation at lower treatment temperatures (370C). It was observed that, the inhibition by SDS might be due to binding of the ionic surfactant to hydrophobic and hydrophilic residues of Geobacillus sp T1 lipase. Thus, initiating unfolding of the tertiary structure and resulting in losing catalytic efficacy .
From the structural point of view, Geobacillus sp Iso5 lipase contains 64% β- strand and 36% α-helical contents. Certain cofactors are generally not required for lipase activity, but divalent cations like calcium often stimulate enzyme activity. However, lipase from P. fuorescens was determined to be 6% α-helix, 27% β-sheet, 0.6% β-turn, and 67% random coil in an absence of Ca2+ but an increase in α -helix and β -sheet content (from 33 to 55%) was observed in the presence of Ca2+ . Furthermore, a crystal structure of a homologous lipase P. aeruginoas contains high α-helix and β-sheet content (49%) which was inhibited by Ca2+ [51, 56]. In contrast, the mature GehD lipase from Staphylococcus epidermidis, the secondary structure composition was determined to be 26.5% α -helix, 20.6%, β -sheet, and 52.9% coil . Apart from bacterial enzymes, some other sources of enzymes were characterized for secondary structure using conventional vibrational spectroscopy such as IR and Raman. The presence of amide I, II and III are most important in predicting the secondary structure. In Infrared spectra of proteins in solution is intrinsically restricted by H20 and 2H20 absorption and are rather limited in range. Such spectra, even in the range of observation that is possible (e.g. in 2H20 from 1900 to 1200 cm-1), are relatively featureless compared with Raman spectra. Amide I, this is primarily characterized by carbonyl stretch at 1630-1670 cm-1and intense. Thus, α -helix is characterized by 1645-1660 cm-1, β -sheet at 1665-1680 cm-1 and random coil at 1660-1670 cm-1 are intense in both IR and Raman [58-62]. However, the presence of amide II at frequency 1530-1550 cm-1 and amide III (1230 cm-1), Raman-active occur at 1230-1300 cm-1. It has been used in conjunction with amide I for estimating the relative contributions of α-helix, β-sheet and random coil in a number of proteins at will also contribute to the secondary structure conformation
Critical analysis of current research shows that lipase from Geobacillus sp.Iso5 resulted in some interesting findings, such as its stability and structure. It has a molecular weight of 47 kDa and optimal activity at optimal activity at 70° C and pH 8.0. It constitutes highly ordered structural organization and stable towards some detergents. This indicating that, lipases from Geobacillus sp. Iso5 has significant potential for commercialization as a biocatalyst for industrial purposes. In comparison with other commercially available enzymes, we believe that, engineering present lipase will allow attainment of enzymes with new remarkable characteristics for a specific application.
This research was supported by the all the staffs and members of Department of Biochemistry, Kuvempu University. We would also like to thank Mr. Nuthan Kumar D, Department of Environmental science, who helped throughout the time course of research.
Conflict of Interest
No competing financial interests exist