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1.
Lipases from Geotrichum candidum were produced in two different medium: A = 12 % (w/v) clarified corn steep liquor (CCSL) + 0.6 % (w/v) soybean oil (SO) and B = 3.5 % (w/v) yeast hydrolysate (YH) + 0.7 % (w/v) SO. Lipases were partially purified from both media by hydrophobic interaction chromatography using 3.0 mol L?1 of NaCl as mobile phase, and they were characterized in the crude and partially purified forms. The recovery of lipase activity from CCSL and YH via HIC were 96 and 94.3 %, and the purification factors were 44.3 and 86.7‐fold, respectively. All evaluated lipases had similar optimum pH (7.0–7.7), but, for the CCSL crude lipase, optimum temperature (47 °C) was 10 °C higher than others lipases evaluated. CCSL crude lipase possessed a higher thermo stability than YH crude lipase, e.g., at 37 °C (pH 7.0) the half‐life of CCSL crude lipase was 19.25 h and at pH 8.0 (30 °C) the half‐life was 48 h, which are five and ten times higher than with YH crude lipase, respectively. On the other hand, the YH crude lipase possessed a higher catalytic constant (kcat = 2.3 min?1) but with almost the same catalytic efficiency (Km/kcat = 32.12 mg mL min?1) in relation to CCSL crude lipase. The lipases differ in biocatalytic properties between substrates, suggesting that the two lipases can be employed for different applications.  相似文献   

2.
Lipase was isolated from bay laurel (Laurus nobilis L.) seeds, some biochemical properties were determined. The bay laurel oil was used as the substrate in all experiments. The pH optimum was found to be 8.0 in the presence of this substrate. The temperature optimum was 50 °C. The specific activity of the lipase was found to be 296 U mg protein−1 in optimal conditions. The enzyme activity is quite stable in the range of pH 7.0–10. The enzyme was stable for 1 h at its optimum temperature, and retained about 68% of activity at 60 °C during this time. K m and V max values were determined as 0.975 g and 1.298 U mg protein−1, respectively. Also, storage stability and metal effect on lipolytic activity were investigated. Enzyme activity was maintained for 9, 12, and 42 days at room temperature, 4 and −20 °C, respectively. Ca2+, Co2+, Cu2+, Fe2+, and Mg2+ lightly enhanced bay laurel lipase activity.  相似文献   

3.
Jiang Li  Xiumeng Liu 《Lipids》2017,52(7):619-627
A novel lipase gene lip256 was cloned and identified from the genomic library of hot spring strain Bacillus sp. HT19. The deduced amino acid sequence of lip256 has less than 32% identity to a predicted esterase (Cog1752) from Photobacterium leiognathi lrivu.4.1 and contains a novel motif (GTSAG) that differs from other clusters in the lipase superfamily. Following purification, a single band was obtained with a molecular mass of 33 kDa by SDS-PAGE, and the optimal temperature and pH for lipolytic activity of Lip25 were 70 °C and 9.0, respectively. Lip256 exhibited high activity at high temperatures, with 40% maximum activity at 80 °C and good stability at temperatures ranges between 50 and 80 °C. Additionally, the enzyme was highly stable in the presence of butyl-alcohol, glycerol, acetonitrile, pyridine, and urea. However, the presence of acetone, methanol, trichloromethane, petroleum ether, hexane, tert-butanol, isopropanol, dithiothreitol, ethylenediaminetetraacetic acid, polyhexamethylene biguanide, dimethyl sulfoxide, benzene, Triton X-100, Tween-20, Tween-80, and sodium dodecyl sulfate suppressed or absolutely inhibited enzyme activity. Furthermore, Ca2+, Mg2+, and Cu2+ suppressed enzyme activity, whereas Na+, Fe3+, K+, Fe2+, and Sr2+ enhanced enzyme activity. The unique characteristics of novel lipase Lip256, including its thermo-alkaliphilic performance, high tolerance toward metal ions, inhibitors, and detergents, and high stability in organic solvents, implied that this enzyme might be an interesting candidate for industrial processes.  相似文献   

4.
The lipase production of Burkholderia sp. GXU56 was influenced by carbon and nitrogen sources, inorganic salts, initial pH of the medium and cultivation temperature. The maximum lipase production was 580.52 U/mL and reached 5 times the level of the basic medium in the optimum medium at pH 8.0, 32 °C, 200 rpm and 40–48 h. The lipase was purified 53.6 fold to homogeneity and the molecular weight was 35 KDa on SDS‐PAGE. The optimum pH and temperature of the lipase were 8.0 and 40 °C, respectively, and it was stable in the range of pH 7–8.5 and at temperatures below 45 °C. The lipase activity was strongly inhibited by Zn2+, Cu2+, Co2+, Fe2+, Fe3+ ions and SDS, while it was stimulated by Li+ and Ca2+ ions and in presence of 0.1 % CTAB, 0.1 % Triton X‐100 and 10 % DMSO. Km and Vmax of the lipase were calculated to be 0.038 mmol/L, and 0.029 mmol/L min–1, respectively, with PNPB as the substrate. The GXU56 lipase showed enantioselective hydrolysis of (R,S)‐methyl mandelate to (R)‐mandelic acid, which is an important intermediate in the pharmaceutical industry.  相似文献   

5.
Lipolytic activity of California-laurel (Umbellularia californica) seeds   总被引:1,自引:1,他引:0  
The lipid content of dormant mature seeds of the California-laurel (Umbellularia califonnica) was 74% (mass basis) and decreased upon germination, reaching 43% 5 wk after germination. Dormant seeds contained only barely detectable lipase activity. Lipase activity rose upon germination, peaking 2 to 3 wk after the onset of visible germination. The combined addition of three detergents, 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate (1 mM), taurocholic acid (1 mM), and Tween-20 (0.05%, vol/vol) during homogenization increased the lipolytic activity of total seedling homogenates by about 60%. Following centrifugation of homogenates from seedlings of various ages, ca. 80% of the recovered lipolytic activity was located in the fat-free supernatant, with the remainder in the floating fat pad. The crude seed lipase did not show hydrolytic specificity for glycerol esters of lauric acid (the predominant fatty acid of the seed triglycerol): comparable lipolytic activities were seen toward olive oil, trilaurin, tripalmitin, and tristearin. Maximal lipolytic activity occurred at pH 8.5. This activity was stable over the pH range 6 to 9, and unstable at >40°C in a fashion that suggested the presence of multiple enzymes with different substrate specificities. Two lipolytic species, one of which showed some selectivity toward lauric acid esters, were partially separated from one another by ion-exchange chromatography.  相似文献   

6.
Lipase from Ficus carica L. (Moraceae) latex of the Zidi variety was purified 80.5-fold with 68.5 % recovery using silica gel chromatography. The molecular weight of the enzyme was 29 kDa as determined by SDS-PAGE. High lipolytic activity was found in the crude extract during the fruit ripening process. The activity of purified lipase (ZL) seemed to depend strongly on chain length and showed a preference to long chain triacylglycerols. Indeed, ZL specific activity was 370.3 UI/mg using olive oil as a substrate at 45 °C and pH 5.5. In contrast, activity towards short chain triacylglycerols (tributyrin) was 12-fold lower (32 UI/mg). The enzyme was quite stable in the pH range 4–8, and thermally stable at 60 °C displaying t 1/2 about 90 min using olive oil as a substrate. The values of K m app and V m were found to be 14.3 mM and 294.1 μmol/min/mg, respectively. ZL activity was strongly reduced by Fe2+, Mg2+ and Zn2+, while significantly increased by Ca2+ and Cu2+. The enzyme was stimulated by sodium dodecyl sulfate, and Tween-80, while Triton X-100 and EDTA had a slight inhibitory effect. No Effect was observed in addition of PMSF and iodoacetic acid.  相似文献   

7.
The lipolytic activity of free and immobilized whole cells of the pathogenic fungus Phoma glomerata was demonstrated, and several properties of the lipase involved were determined. Free fungal cells and small pieces of immobilized cells, prepared by spontaneous colonization on a solid surface or entrapped in calcium alginate, were incubated with triolein in buffered medium. Different incubation conditions were assayed to optimize the reaction, to determine the effects of heating and time on stability of the immobilized preparations and the time course of the reactions. Although the enzyme cleaves all ester bonds of triolein, it shows some preference for the outer bonds. An optimal pH of 7.5–8.0, optimal temperature of 45°C for free and 50°C for immobilized cell preparations, the necessity for substrate emulsifiers, and reaction independence from calcium and magnesium were demonstrated. Results suggest that immobilized whole cells of P. glomerata would be a suitable tool to study its lipid physiology and to explore further the possible biotechnological use of its lipase activity.  相似文献   

8.
Lipases from oilseeds have a great potential for commercial exploration as industrial enzymes. Lipases are used mixed with surfactants in cleaning and other formulated products, and accordingly, both components must be compatible with each other. This work presents the results of the effects of anionic, cationic and nonionic surfactants, polyethylene glycol and urea on the activity and stability of a lipase extracted of oilseeds from Pachira aquatica. The enzyme was purified and the spectrophotometric assays were done using p-nitrophenyl acetate (p-NPA) as substrate pH 7.5 and 25 °C. The activity was significantly enhanced by the cationic surfactant CTAB. Bile salts increased the lipase activity in the tested concentration range, whereas anionic and nonionic surfactants showed an inhibitory effect. Aqueous solutions of PEG activated the lipase and maximum activation (161%) occurred in PEG 12,000. This effect on lipase that can be due to exposition of some hydrophobic residues located in the vicinity of the active site or aggregation.  相似文献   

9.
BACKGROUND: As a new protein expression and self‐immobilization system, cell‐surface displayed enzymes have attracted increasing attention. In this study, Geotrichum sp. lipase (GSL), an important enzyme for the enrichment of polyunsaturated fatty acids (PUFAs), was first displayed on the cell surface of Saccharomyces cerevisiae. RESULTS: The activity of displayed GSL was higher (43.7 U g?1 dry cell) than that of Candida antarctica lipase B (26.26 U g?1 dry cell) and that of Rhizopus oryzae lipase (4.1 U g?1 dry cell). It also exhibited higher thermostability than the free lipase, and retained 89% of the original activity after incubation at 40 °C for 3 h, compared with 48% at 35 °C for the free lipase at pH 8.5. Interestingly, the displayed lipase had a wider pH range and better pH stability. It had higher activity at all pH values than the free GSL, and retained 86% of the original activity in the pH range 9.5 to 10.5, whereas the activity of the free GSL could not be detected at pH 10. CONCLUSION: This work presented a method to prepare a whole‐cell biocatalyst with better stability and broader pH tolerance which will provide a useful strategy for other cost‐effective self‐immobilized industrial lipases. Copyright © 2011 Society of Chemical Industry  相似文献   

10.
Lipase (E.C. 3.1.1.3) from walnut seed was purified 28.6-fold with 31% yield using Sephadex G-100 gel chromatography. Olive oil served as good substrate for the enzyme. The optimum pH and temperature were 9.0 and 70 °C, respectively. The lipase was stable between 30 and 80 °C for 5 min. K m and V max values were determined as 48 mM and 23.06 × 10−3 U/min mg for triolein as substrate. Lipase activity was slightly reduced by Cu2+, Ca2+, Hg2+, Mn2+, and Ni2+ ions, while Mg2+ and Zn2+ had no effects. Anionic surfactant sodium dodecyl sulfate stimulated lipase activity while non-ionic surfactants Tween-80 and Triton X-100 had negligible effects on enzymatic activity. The enzyme activity was not affected by 50 mM urea and thioacetamide. Potassium ferricyanide, n-bromosuccinamide and potassium cyanide reduced the enzyme activity. The enzyme showed a good stability in organic solvents, the best result being in n-hexane (113% residual activity). The activity of dialysate was maintained approximately 80% for 1 year at −20 °C.  相似文献   

11.
Germinating oilseeds have been explored as a possible source of lipases (glycerol ester hydrolase, EC.3.1.1.3) for the biotechnological processing of oils and fats. Seedlings of rape (Brassica napus) and mustard (Sinapis alba) at day 4 of germination and cotyledons of lupine (Lupinus albus) seedlings at day 3 of germination yield active crude lipase preparations upon homogenization with Tricine buffer (pH 7.5) followed by centrifugation at 23,000 g. The major portion of the lipase activity, determined with an emulsion of sunflower oil as substrate, is recovered in the supernatant fraction. These crude lipase preparations exhibit highest activity between pH 8 and 9, but they are inactive in acidic pH or at pH>10. Each of the crude lipase preparations is highly specific for thesn-1,3 positions of triacylglycerols. The crude lipase preparations exhibit excellent stability on storage at −10 C, but about 50–60% of their activity is lost upon freeze-drying. Dialysis of the crude lipase prior to freeze-drying does not prevent the loss of activity. However, acetone powder obtained from the seedlings exhibits a lipase activity as high as the undialyzed crude lipase preparation.  相似文献   

12.
BACKGROUND: Sal (Shorea robusta) deoiled seed cake extract (SDOCE) was assessed for its suitability as a cheap natural substrate for lipase production under submerged fermentation. The bacterial isolate Aeromonas sp. S1 isolated from dairy industry was used for lipase production. Both the isolate and its lipase were shown to be potential tools for treatment of dairy wastewater containing higher organic load. RESULTS: On substituting tributyrin with SDOCE, lipase production was enhanced 24‐fold (195 U mL?1) compared with the initial 8.13 U mL?1 lipase activity. Maximum lipase production was obtained at pH 8.0 and incubation temperature 30 °C. The lipase had pH and temperature optima of 10.0 and 55 °C, respectively. The isolate and its crude enzyme preparation were checked separately for applicability in dairy wastewater treatment. The isolate was able to reduce chemical oxygen demand (COD) by 93%, oil and grease (O&G) by 75%, and total suspended solids (TSS) by 47% after 96 h of treatment. Enzymatic preparation gave 86% reduction of COD after 12 h and 75 and 45% reduction of O&G and TSS, respectively, after 96 h of treatment. CONCLUSION: Overall, the study shows the usefulness of Sal seed deoiled cake, a cheap agro‐industrial by‐product for the production of lipase. The isolate and its lipase can also be used effectively for the treatment of dairy wastewater. Copyright © 2011 Society of Chemical Industry  相似文献   

13.
BACKGROUND: In this work we used Plackett–Burman statistical design and central composite design in order to optimize culture conditions for lipase production by Staphylococcus caseolyticus strain EX17 growing on raw glycerol, which was obtained as a by‐product of the enzymatic synthesis of biodiesel. The stability of lipase was verified over several organic solvents, such as methanol, ethanol and n‐hexane. RESULTS: Optimal culture conditions for lipase production were found to be 36 °C, initial pH 8.12, glycerol 30 g L?1, olive oil 3.0 g L?1, and soybean oil 2.5 g L?1, with 145.8 U L?1 of enzyme activity. When commercial glycerol was substituted by the raw glycerol from biodiesel synthesis, lipolytic activity was 127.3 U L?1. Experimental validation of enzyme production matched values predicted by the mathematical model, which was 138.3 U L?1. Stability tests showed that lipase from S. caseolyticus EX17 was stable in methanol, ethanol, and n‐hexane. CONCLUSIONS: Results obtained in this work suggest that raw glycerol can be used for lipase production by S. caseolyticus EX17 and that this enzyme has a potential application in the synthesis of biodiesel. Copyright © 2008 Society of Chemical Industry  相似文献   

14.
The aim of this work was to optimize the production of a new lipase by a halotolerant bacterial strain Halomonas sp. C2SS100, by means of the response-surface methodology (RSM). The process parameters having the most significant effect on lipase production were identified using the Plackett–Burman screening design-of-experiments. Then, Box–Behnken design was applied to optimize lipase activity and the quadratic regression model of the lipase production was built. Indeed, the lipase yield was increased, and the value obtained experimentally (39 ± 2 U/ml) was very close to the rate predicted by the model (40.3 U/ml). Likewise, optimization of parameters by RSM resulted in 2.78-fold increase in lipase activity. These findings provide the first report on lipase production and optimization by a halotolerant bacterial strain belonging to Halomonas genus. Afterward, the biochemical properties of the produced lipase were studied for apply in oil stains removal. The crude lipase showed a maximum activity at 60°C and at pH ranging from 7 to 10. It displayed an important stability at high temperature, pH, and NaCl. Interestingly, this bacterial lipase exhibited a prominent stability toward some commercial solid and liquid detergents after 30 min of incubation at 50°C. The capability of the crude lipase to eliminate stain was ascertained on polycotton fabric pieces stained with lubricating oil. Whether with the addition of hot water alone or of a commercially available detergent, lipase is able to considerably boost the elimination of oil stains. The actual findings highlight the capacity of Halomonas sp. lipase for energy-efficient biocatalytic application.  相似文献   

15.
The complete purification and characterization of an extracellular lipase (acylglycerol acylhydrolase, EC 3.1.1.3) fromR. delemar is described. The final product was homogeneous as judged by electrophoresis in denaturing polyacrylamide gels and by isoelectric focusing, and was shown by means of an activity stain to be lipolytic. The purified enzyme had a monomer molecular weight of 30,300, an isoelectric point of 8.6, and approximately one monosaccharide moiety per molecule.N-Terminal sequence data (28 residues) and the amino acid composition of the lipase indicated that it corresponds to the product of a lipase-encoding cDNA previously isolated fromR. delemar. Optimal activity occurred between pH 8.0 and 8.5. The activity and stability of the enzyme were maximum at 30°C. Divalent cations were required for activity, with barium, calcium and manganese conferring maximum activity. Activation by calcium was maximal at and above 10 mM. The lipase was not inactivated by reducing agents, sodium fluoride or phenylmethlsufonyl fluoride. It was resistant toN-ethylmaleimide, and inactivated byp-chloromercuribenzoic acid in a manner which was not reversed by cysteine. Mention of brand or firm names does not constitute an endorsement by the U.S. Department of Agriculture over others of a similar nature not mentioned.  相似文献   

16.
This work describes the extraction and back-extraction of a lipase from crude extract of Penicillium citrinum using AOT reversed micelles in isooctane. The effect of pH, ionic strength, AOT concentration on the protein forward and backward transfer at 20°C was studied. The maximum protein forward extraction (32·0%) was achieved at pH 4·0 with a 50 mmol dm−3 acetate buffer containing 100 mmol dm−3 KCl and 100 mmol dm−3 AOT in isooctane. Proteins were back-extracted (82·7%) to a new aqueous phase containing 100 mmol dm−3 pH 8·0 phosphate buffer and 1000 mmol dm−3 KCl. No enzyme activity could be detected either in the micellar phase or in the aqueous phase after protein back-extraction. However, the lipolytic activity was recovered after hydrophobic interaction chromatography on a Phenyl Superose column. The yield obtained for the overall process was 68% for activity, 26·4% for protein recovery and the purification factor was 810-fold. A single protein band at 33000 Da was obtained for SDS–PAGE analysis for the recovered and purified enzyme. © 1997 SCI.  相似文献   

17.
Enzymes can decrease the environmental and economic load of detergent products by reducing the amount of chemicals used in detergents and by allowing washing at ambient temperatures. In this study, Streptomyces cellulosae AU‐10 (GenBank accession number: MG780240) lipase was purified 7.08‐fold with 68% yield using an aqueous 2‐phase system. The Streptomyces sp. AU‐10 lipase showed maximal activity at pH 9.0 and 40 °C. Hundred percent activities were measured in the pH range from 9.0 to 11.0 for 1 h. The enzyme was also highly stable at 30–50 °C. The values of Km and Vmax were calculated as 0.34 mM and 0.83 mM min?1, respectively. The lipase has high hydrolytic activity for olive oil and sunflower oil. The effect of ethylenediamine tetraacetic acid on the enzyme has shown that the lipase is a metalloenzyme. The activity increased in the presence of Fe2+, Cu2+, and various boron compounds. The enzyme has shown a good stability not only with surfactants but also with oxidizing agents. In addition, activities in the presence of Omo, Ariel, Tursil, Pril, and Fairy were measured as 108.8%, 115.6%, 98.35%, 140.4%, and 107.6%, respectively. Considering its remarkable ability, the S. cellulosae AU‐10 lipase can be considered as a potential additive in the detergent industry.  相似文献   

18.
Lipase was extracted and purified from Pseudomonas aeruginosa SRT9. Culture conditions were optimized and highest lipase production amounting to 147.36 U/ml was obtained after 20 h incubation. The extracellular lipase was purified on Mono QHR5/5 column, resulting in a purification factor of 98-fold with specific activity of 12307.81 U/mg. Lipase was immobilized on tri (4-formyl phenoxy) cyanurate to form Schiff’s base. An immobilization yield of 85% was obtained. The native and immobilized lipases were used for catalyzing the hydrolysis of olive oil in aqueous medium. Comparative study revealed that immobilized lipase exhibited a shift in optimal pH from 6.9 (free lipase) to 7.5 and shift in optimal temperature from 55 °C to 70 °C. The immobilized lipase showed 20–25% increase in thermal stability and retained 75% of its initial activity after 7 cycles. It showed good stability in organic solvents especially in 30% acetone and methanol. Enzyme activity was decreased by ∼60% when incubated with 30% butanol. The kinetic studies revealed increase in K M value from 0.043 mM (native) to 0.10 mM for immobilized lipase. It showed decrease in the V max of immobilized enzyme (142.8 μmol min−1 mg−1), suggesting enzyme activity decrease in the course of covalent binding. The immobilized lipase retained its initial activity for more than 30 days when stored at 4 °C in Tris-HCl buffer pH 7.0 without any significant loss in enzyme activity.  相似文献   

19.
Lipases are highly demanded enzymes, used to synthesize high commercial value products in food, pharmaceutical, cosmetic, textile, paper, and detergent industries. The effect of high pressure processing (HPP) on Thermomyces lanuginosus lipase is studied at 0.1, 100, and 300 MPa and 25 °C. Kinetics is assessed after HPP treatment in three sets of time series: 0–25, 51–75, and 101–112 min. Conformational change of lipase is studied by infrared attenuated total reflectance (ATR)–FTIR spectra deconvolution and 1D 1H NMR. Pressure treatment lead to changes in enzymatic activity from 51 to 75 and from 101 to 112 min. Treated enzyme at 300 MPa has the highest activity (≈twofold higher than untreated lipase). Statistical difference in activity (p  > 0.05) is not found during the first 25 min. Lipase activity is best described by first‐order model (0–25 min) and zero‐order model (51–75 and 101–112 min). Lipase unfolding is evident at 100 MPa due to an increase of β‐turns and β‐sheet components. Pressurized enzyme at 300 MPa enhances its activity from 51 to 112 min, due to an increase of β‐sheet and α‐helix structures (refolding). These findings impact directly on cost‐effectiveness of lipase catalyzed products manufacture. Practical Applications: Enzyme enhanced activity and stability improves the performance of manufacture processes. Assessment of structure changes of lipase induced by HPP lead to accurate parameter selection to increase stability and profitability of lipase catalyzed products processing. Pressure levels of 100 MPa led to enzyme unfolding, whereas 300 MPa causes improved activity and stability provoked by lipase refolding. Pressure treatment at 300 MPa of Thermomyces lanuginosus lipase is recommended for long term bioprocessing (≥112 min), which require increased activity and stability of the enzyme.  相似文献   

20.
BACKGROUND: The investigation of enzymes from extremophilic microorganisms has generated great scientific interest in recent decades owing to their ability to operate in harsh conditions. Since lipolytic enzymes are one of the three groups most commercially demanded, the study of novel thermostable lipolytic enzymes is of much interest. RESULTS: Lipolytic enzymes produced by Thermus aquaticus YT1 have been investigated. Very interesting properties against thermal inactivation, with a half‐life time of 1 h at 95 °C were found. The use of compounds such as polyols is a strategy used to further improve the thermostability of these enzymes, and the modelling of the deactivation process with a series‐type equation, allows one to quantitatively ascertain the free energy of deactivation. Finally, the application to dibutyl adipate hydrolysis was investigated, as an indicator of the potential usefulness of the enzymes in polymer biodegradation. CONCLUSION: The crude lipolytic enzymes possess outstanding properties of thermostability (high lipolytic activity after 12 days at 80 °C, and a half‐life time of 1 h at 95 °C), and they can be significantly improved by adding polyols (addition of glycerol entailed activity losses of just 3% after 1 h at 95 °C). These enzymes are able to catalyze ester cleavage, showing a zero‐order kinetics. Copyright © 2011 Society of Chemical Industry  相似文献   

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