File Name: production purification characterization and applications of lipases .zip
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Saovanee Dharmsthiti, Sudaporn Luchai, Production, purification and characterization of thermophilic lipase from Bacillus sp. The lipase was purified 2. Its molecular mass was 69 kDa. The purified enzyme was characterized for its general physical properties. Lipases have long been recognized to have potential for various industrial applications. Most of those produced commercially are currently obtained from fungi and yeasts .
However, interest in bacterial lipases has increased due to the fact that they are more stable than those from other organisms, especially when exposed to high temperatures and other severe conditions . Furthermore, salt tolerance has been reported to enhance the stability of thermophilic enzymes . This paper describes methods for production, purification and characterization of an extracellular lipase from THL for potential industrial applications.
Complex media were designed based on tests with minimal medium P MMP consisting of 2. Bacterial growth was determined by measuring the optical density at nm. Determination of lipase activity was done by the titrimetric method . One unit of lipase activity was defined as 1. Determination of reducing sugar concentration was done using the DNS method . The protein concentration in each fraction from column chromatography was determined by measuring the absorbance at nm Spectronic plus, Milton Roy Co.
For the pooled fractions obtained from each purification step, the protein concentration was determined using a Bio-Rad Protein Assay Kit according to the manufacturer's instruction.
THL lipase was purified to apparent homogeneity by a single Sephadex G gel filtration step. Specifically, 50 ml of cell-free supernatant of THL culture broth was filtered at 2. The concentrated solution of approximately 10 ml was applied to a Sephadex G gel filtration column 2. Fractions of 2. Fractions with high lipase activity were pooled and concentrated. All gels were run at 20 mA for min.
Proteins were stained using the silver staining procedure Bio-Rad. Molecular mass markers used in this study were purchased from Sigma Chemical Co.
Positional specificity of the lipase was determined by use of a method modified from that described by Lesuisse et al. Briefly, 40 mg of pure triolein was sonicated in 2 ml of 0. After incubation, the reaction products were extracted by the addition of 1.
The extract was concentrated by evaporation and applied to a silica gel plate Merck Co. A standard mixture of tri-, di-, and monoolein Sigma Chemical Co. The spots of glycerides and fatty acids were visualized by exposure to iodine vapor.
Based on local cost and availability, 1. To test the effect of pH, NBS cultivation medium was used. Oil inducers were omitted since they formed colloids at very low pH and saponification at high pH. Prior to chromatography of the concentrated crude enzyme, NaCl was eliminated by dialysis and this caused a However, This indicated that inhibition due to low salt was reversible. THL lipase was purified 2. The enzyme formed large aggregates which were easily separated from other proteins [8 , 9].
The aggregation also caused a slight reduction in lipolytic activity during the purification steps [4 , 7]. Polyacrylamide gel electrophoretic analysis of the Bacillus sp.
THL lipase. Purified THL lipase exhibited maximum lipolytic activity at pH 7. DV was the rate of activity decrease during 1-h storage. It was found that the enzyme was stable in the neutral pH range where the DV values were low Fig. Effect of pH a and temperature b on activity of the Bacillus sp. The effect of pH a was determined under the reaction condition of 0. However, the enzyme was sensitive to EDTA indicating that it was a metalloenzyme.
The effect of organic solvents was determined by mixing enzyme aliquots with tested solvents at a ratio at room temperature for 2 h. In the case of 1. These levels of stability were similar to or higher than those reported for other bacteria [9 , 10]. Substrate positional specificity of THL lipase was examined by thin layer chromatography. The hydrolytic products of THL from purified triolein after 2 h hydrolysis were 1,2 2,3 -diolein and monoolein Fig.
This is similar to the enzymes from other strains of Bacillus sp. Because of this position-specific property, the enzyme could potentially be used for inter- or transesterification reactions to produce novel substances. With respect to fatty acid specificity, the enzyme hydrolyzed triglycerides containing short- to medium-chain saturated fatty acids C4—C12 faster than it did saturated long-chain fatty acids C14—C18 Table 2.
Silica gel thin layer chromatography of the hydrolytic products obtained from the activity of Bacillus sp. THL lipase on triolein substrate. Spots were developed with iodine vapor. Specificity of lipase from Bacillus sp.
THL to various purified monoacid triglycerides. The enzyme was found to be active against all the lipids tested. The highest activity was found towards rice bran oil and olive oil Table 3. Potential applications of this enzyme should be further investigated. Complex media were designed based on tests with MMP. Higher growth and lipase production was obtained when the medium was supplemented with 0. Supplementing MGRS with 0. However, replacing yeast extract and soybean meal with the less expensive 0.
We are grateful to Professor T. Flegel for reviewing the manuscript. This research was sponsored by the National Research Council of Thailand. Jaeger K. Ransac S. Dijkstra B. Misset O. FEMS Microbiol. Google Scholar. Sugihara A. Ueshima M.
Shimada Y. Tsunasawa S. Tominaga Y. Reeve J. ASM News 60 , — Lotrakul P. Dharmsthiti S. Bernfeld P. In: Method in Enzymology Colowick S.
Kaplan N. Academic Press , New York. Google Preview. Laemmli U. Nature , — Lesuisse E. Schanck K. Colson C. Ammaranond P. Rua M. Schmidt-Dannert C. Wahl S.
This study was aimed at producing protease and lipase simultaneously on a common medium by Bacillus licheniformis VSG1, which was isolated from a tannery effluent. The effect of media composition with respect to protein source, lipid source and emulsifier on the production of protease and lipase was analysed. Both those enzymes were produced under optimized conditions like pH, temperature and incubation time. The enzyme mixture comprising of both protease and lipase was purified by ammonium sulphate precipitation, dialysis and gel filtration chromatography to obtain fold pure enzymes. The purified enzyme mixture was characterized to determine the optimum pH and temperature of protease and lipase, the response of the enzymes to inhibitors, additives and solvents. The concomitant production of protease and lipase and the purification of both the enzymes in a single mixture have industrial significance, as many industrial processes use both protease and lipase together. Of all the industrially important enzymes, proteases followed by carbohydrases and lipases have a wide range of applications.
Saovanee Dharmsthiti, Sudaporn Luchai, Production, purification and characterization of thermophilic lipase from Bacillus sp. The lipase was purified 2. Its molecular mass was 69 kDa. The purified enzyme was characterized for its general physical properties. Lipases have long been recognized to have potential for various industrial applications.
This article discusses the production, recovery, and use of microbial lipases. Issues of enzyme kinetics, thermostability, and bioactivity are addressed. Production.
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Subash C. Lipases are water-soluble enzymes that act on insoluble substrates and catalyze the hydrolysis of long-chain triglycerides. Lipases play a vital role in the food, detergent, chemical, and pharmaceutical industries. In the past, fungal lipases gained significant attention in the industries due to their substrate specificity and stability under varied chemical and physical conditions. Fungal enzymes are extracellular in nature, and they can be extracted easily, which significantly reduces the cost and makes this source preferable over bacteria. Soil contaminated with spillage from the products of oil and dairy harbors fungal species, which have the potential to secrete lipases to degrade fats and oils.
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