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1.
Mikko Uusi-Oukari Christian Ehnholm Matti Jauhlainen 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1996,682(2):233
Phenylboronates are competitive inhibitors of serine hydrolases including lipases. We studied the effect of m-aminophenylboronate on triglyceride-hydrolyzing activity of hepatic lipase (EC 3.1.1.3). m-Aminophenylbo ronate inhibited hepatic lipase activity with a K1 value of 55 μM. Furthermore, m-aminophenylboronate protected hepatic lipase activity from inhibition by di-isopropyl fluorophosphate, an irreversible active site inhibitor of serine hydrolases. Inhibition of hepatic lipase activity by m-aminophenylboronate was pH-dependent. The inhibition was maximal at pH 7.5, while at pH 10 it was almost non-existent. These data were used to develop a purification procedure for postheparin plasma hepatic lipase and lipoprotein lipase. The method is a combination of m-aminophenylboronate and heparin-Sepharose affinity chromatographies. Hepatic lipase was purified to homogeneity as analyzed on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The specific activity of purified hepatic lipase was 5.46 mmol free fatty acids h−1 mg−1 protein with a total purification factor of 14 400 and a final recovery of approximately 20%. The recovery of hepatic lipase activity in m-aminophenylboronate affinity chromatography step was 95%. The purified lipoprotein lipase was a homogeneous protein with a specific activity of 8.27 mmol free fatty acids h−1 mg−1 The purification factor was 23 400 and the final recovery approximately 20%. The recovery of lipoprotein lipase activity in the m-aminophenylboronate affinity chromatography step was 87%. The phenylboronate affinity chromatography step can be used for purification of serine hydrolases which interact with boronates. 相似文献
2.
Andrea Salis Enrico Sanjust Vincenzo Solinas Maura Monduzzi 《Biocatalysis and Biotransformation》2013,31(5):381-386
Three commercial lipases (CLs), A Amano 6 (from Aspergillus niger), M Amano 10 (from Mucor javanicus), and R Amano (from Penicillium roqueforti) – called lipase A, M and R respectively – were characterized in terms of carbohydrate content, protein content and enzymatic activity (p-nitrophenylacetate assay). All the CL preparations contained different proteins as observed from electrophoresis. Lipases were immobilized on Accurel MP1004 porous polypropylene by physical adsorption.The Immobilization process caused a loss of enzymatic activity. The retained activity was similar for lipase M and R (about 15%). In contrast, lipase A retained only the 1.3% of the specific activity of the free lipase. The retained activity of lipases M and R seems to be due to a feature of the support, while the lower activity a of lipase A may be attributed to a strong structure distortion caused by lipase–support interaction. 相似文献
3.
Seven kinds of lipase were immobilized to Sepharose 4 B, porous glass beads and ion-exchange resins. Lipase immobilized to porous glass beads was most suitable for the treatment of yolk-contaminated egg white. Optimum pH and temperature were not varied by the immobilization. The immobilized lipoprotein lipase from Pseudomonas fluorescens and lipase from Rhizopus delemar were applied to the treatment of yolk-contaminated egg white. The foam stability of the spray-dried egg white solution containing 0.05 % yolk was completely recovered by the 30 min’s treatment with both immobilized lipases. This demonstrated the feasibility of improving the foaming properties of yolk-contaminated egg white by the immobilized lipase treatment. 相似文献
4.
Chien Wei Ooi Beng Ti Tey Siew Ling Hii Siti Mazlina Mustapa Kamal John Chi Wei Lan Arbakariya Ariff Tau Chuan Ling 《Process Biochemistry》2009,44(10):1083-1087
Alcohol/salt-based aqueous two-phase systems (ATPSs) were used to recover lipase derived from Burkholderia pseudomallei (B. pseudomallei). Nine biphasic systems, comprised of an alcohol-based top phase (ethanol, 2-propanol and 1-propanol) and a salt-based bottom phase (ammonium sulfate, potassium phosphate and sodium citrate), were evaluated for their effectiveness in lipase recovery. The stability of lipase in each of the solutions was tested, and phase diagrams were constructed for each system. The optimum partition efficiency for the purification of lipase was obtained in an ATPS of 16% (w/w) 2-propanol and 16% (w/w) phosphate in the presence of 4.5% (w/v) NaCl. The purified lipase had a purification factor of 13.5 and a yield of 99%. 相似文献
5.
M. L. Camarinha Vicente M. R. Aires-Barros J. M. S. Cabral 《Biotechnology Techniques》1990,4(2):137-142
Summary Two upases produced by Chromobacterium viscosum were isolated and purified by liquid-liquid extraction using organic solvents containing reversed micelles. One lipase (lipase A) was purified from the original crude enzyme preparation by 4,3-fold with a recovery of 91% and the other (lipase B) by 3,7-fold with a recovery of 76%. 相似文献
6.
Kenthorai Raman Jegannathan Eng-Seng Chan Pogaku Ravindra 《Journal of Molecular Catalysis .B, Enzymatic》2009,58(1-4):78-83
In this work, a novel approach for lipase immobilization was exploited. Lipase from Burkholderia cepacia was encapsulated into κ-carrageenan by co-extrusion method to form a liquid core capsule. The diameter of the encapsulated lipase was found to be in the range of 1.3–1.8 mm with an average membrane thickness of 200 μm and 5% coefficient of variance. The encapsulation efficiency was 42.6% and 97% moisture content respectively. The encapsulated lipase was stable between pH 6 and 9 and temperature until 50 °C. The encapsulated lipase was stable until disintegration of the carrier when stored at 27 °C and retained 72.3% of its original activity after 6 cycles of hydrolysis of p-NPP. The encapsulated lipase was stable in various organic solvents including methanol, ethanol, iso-propanol, n-hexane and n-heptane. Kinetic parameters Km and Vmax were found to be 0.22 mM and 0.06 μmol/min for free lipase and 0.25 mM and 0.05 μmol/min for encapsulated lipase respectively. 相似文献
7.
Joo Ling Loo Oi Mlng Lai Kamariah Long Hasanah Mohd Ghazali 《World journal of microbiology & biotechnology》2007,23(12):1771-1778
Mycelium-bound lipase (MBL) was prepared using a strain of Geotrichum candidum isolated from local soil. At the time of maximum lipase activity (54 h), the mycelia to which the lipase was bound were harvested
by filtration and centrifugation. Dry MBL was prepared by lyophilizing the mycelia obtained. The yield of MBL was 3.66 g/l
with a protein content of 44.11 mg/g. The lipase activity and specific lipase activity were 22.59 and 510 U/g protein, respectively.
The moisture content of the MBL was 3.85%. The activity of free (extracellular) lipase in the culture supernatant (after removal
of mycelia) was less than 0.2 U/ml. The MBL showed selectivity for oleic acid over palmitic acid during hydrolysis of palm
olein, indicating that the lipase from G. candidum displayed high substrate selectivity for unsaturated fatty acid containing a cis-9 double bond, even in crude form. This unique specificity of MBL could be a direct, simple and inexpensive way in the fats
and oil industry for the selective hydrolysis or transesterification of cis-9 fatty acid residues in natural triacylglycerols. 相似文献
8.
The extracellular lipase structural gene was isolated from cDNA of Aureobasidium pullulans HN2-3 by using SMARTTM RACE cDNA amplification kit. The gene had an open reading frame of 1245 bp long encoding a lipase. The coding region of the
gene was interrupted by only one intron (55 bp). It encodes 414 amino acid residues of a protein with a putative signal peptide
of 26 amino acids. The protein sequence deduced from the extracellular lipase structural gene contained the lipase consensus
sequence (G-X-S-X-G) and three conserved putative N-glycosylation sites. According to the phylogenetic tree of the lipases,
the lipase from A. pullulans was closely related to that from Aspergillus fumigatus (XP_750543) and Neosartorya fischeri (XP_001257768) and the identities were 50% and 52%, respectively. The mature peptide encoding cDNA was subcloned into pET-24a
(+) expression vector. The recombinant plasmid was expressed in Escherichia coli BL21(DE3). The expressed fusion protein was analyzed by SDS-PAGE and western blotting and a specific band with molecular
mass of about 47 kDa was found. Enzyme activity assay verified the recombinant protein as a lipase. A maximum activity of
0.96 U/mg was obtained from cellular extract of E. coli BL21(DE3) harboring pET-24a(+)LIP1. Optimal pH and temperature of the crude recombinant lipase were 8.0 and 35 °C, respectively and the crude recombinant lipase
had the highest hydrolytic activity towards peanut oil. 相似文献
9.
Novel magnetic cross‐linked lipase aggregates were fabricated by immobilizing the cross‐linked lipase aggregates onto magnetic particles with a high number of ‐NH2 terminal groups using p‐benzoquinone as the cross‐linking agent. At the optimal fabrication conditions, 100% of immobilization efficiency and 139% of activity recovery of the magnetic cross‐linked lipase aggregates were achieved. The magnetic cross‐linked lipase aggregates were able to efficiently resolve (R, S)‐2‐octanol, and retained 100% activity and 100% enantioselectivity after 10 cycles of reuse, whereas the cross‐linked lipase aggregates only retained about 50% activity and 70% enantioselectivity due to insufficient cross‐linking. These results provide a great potential for industrial applications of the magnetic cross‐linked lipase aggregates. Chirality 27:199–204, 2015. © 2014 Wiley Periodicals, Inc. 相似文献
10.
Sánchez-Otero MG Valerio-Alfaro G García-Galindo HS Oliart-Ros RM 《Journal of industrial microbiology & biotechnology》2008,35(12):1687-1693
A partially purified lipase produced by the thermophile Geobacillus thermoleovorans CCR11 was immobilized by adsorption on porous polypropylene (Accurel EP-100) in the presence and absence of 0.1% Triton X-100.
Lipase production was induced in a 2.5% high oleic safflower oil medium and the enzyme was partially purified by diafiltration
(co. 500,000 Da). Immobilization conditions were established at 25 °C, pH 6, and a protein concentration of 0.9 mg/mL in the
presence and absence of 0.1% Triton X-100. Immobilization increased enzyme thermostability but there was no change in neither
the optimum pH nor in pH resistance irrelevant to the presence of the detergent during immobilization. Immobilization with
or without Triton X-100 allowed the reuse of the lipase preparation for 11 and 8 cycles, respectively. There was a significant
difference between residual activity of immobilized and soluble enzyme after 36 days of storage at 4 °C (P < 0.05). With respect to chain length specificity, the immobilized lipase showed less activity over short chain esters than
the soluble lipase. The immobilized lipase showed good resistance to desorption with phosphate buffer and NaCl; minor loses
with detergents were observed (less than 50% with Triton X-100 and Tween-80), but activity was completely lost with SDS. Immobilization
of G. thermoleovorans CCR11 lipase in porous polypropylene is a simple and easy method to obtain a biocatalyst with increased stability, improved
performance, with the possibility for re-use, and therefore an interesting potential use in commercial conditions. 相似文献
11.
This study was designed to investigate the stability of a lipase fused with a cellulose-binding domain (CBD) to cellulase.
The fusion protein was derived from a gene cluster of a CBD fragment of a cellulase gene inTrichoderma hazianum and a lipase gene inBacillus stearothermophilus L1. Due to the CBD, this lipase can be immobilized to a cellulose material. Factors affecting the lipase stability were divided
into the reaction-independent factors (RIF), and the reaction-dependent factors (RDF). RIF includes the reaction conditions
such as pH and temperature, whereas substrate limitation and product inhibition are examples of RDF. As pH 10 and 50°C were
found to be optimum reaction conditions for oil hydrolysis by this lipase, the stability of the free and the immobilized lipase
was studied under these conditions. Avicel (microcrystal-line cellulose) was used as a support for lipase immobilization.
The effects of both RIF and RDF on the enzyme activity were less for the immobilized lipase than for the free lipase. Due
to the irreversible binding of CBD to Avicel and the high stability of the immobilized lipase, the enzyme activity after five
times of use was over 70% of the initial activity. 相似文献
12.
Wen-Hsiung Liu Teruhiko Beppu Kei Arima 《Bioscience, biotechnology, and biochemistry》2013,77(11):2493-2499
Some physical and chemical properties of the extracellular lipase from the thermophilic fungus, Humico la lanuginosa S–38, were investigated. The results were as follows: Sedimentation coefficient was 2.4 × 10?13 (cm-g/sec-dyne); diffusion coefficient was 8.8 × 10?7 (cm2/sec); and frictional coefficient was 1.22. Molecular weight was 27,500±500 and α-helix content was 18.9%. The number of amino acid residues contained in 1 mole of protein of Humicola lipase was 224. Sugar and lipid were not detected. The effect of calcium ion and denaturing reagents, such as urea, sodium dodecyl sulfate and dithiothreitol, on the thermostability of Humicola lipase was examined. It was concluded that the thermostability of Humicola lipase was not influenced by protective cofactors but was attributable to the enzyme itself. Some properties of enzyme structure which were concerned with the thermostability of Humicola lipase are also discussed. 相似文献
13.
Dolores Reyes Duarte Edmundo Castillo Eduardo Bárzana Agustín López-Munguía 《Biotechnology letters》2000,22(22):1811-1814
Capsaicin was hydrolysed by lipase B from Candida antarctica into vanillylamine and 8-methyl-6-trans-nonenoic acid. Conversions of 70% were obtained after 72 h at 70 °C in water but decreased to only 15% when capsaicin was solubilized in 15% (v/v) ethanol/water after 72 h at 45 °C. No activity occurred in chloroform/water mixtures. According to our knowledge, this is the first report concerning amide hydrolysis by a lipase. 相似文献
14.
Summary The effects of residual enzyme activity, stepwise addition of lipase at different reaction times, and enzyme quantity in direct polyesterification of sebacic acid and 1,4-butanediol catalyzed by a lipase from Rhizomucor miehei were investigated. Although the lipase activity dropped sharply in the beginning period of the reaction, the molar mass of the polyester increased rapidly, up to 39,000 g mol–1 in 72 h. The residual lipase activity (hydrolytic) was only 14 %. Stepwise addition of lipase did not improve polyester synthesis. Highest mass average molar mass of 56,000 g mol–1 was obtained with 0.125 g of lipase (28.5%, w/w) in 5 days. 相似文献
15.
Chien Wei Ooi Beng Ti Tey Siew Ling Hii Arbakariya Ariff Ho Shing Wu John Chi Wei Lan Ruey Shin Juang Siti Mazlina Mustapa Kamal Tau Chuan Ling 《Biotechnology and Bioprocess Engineering》2009,14(6):811-818
An aqueous two-phase purification process was employed for the recovery of Burkholderia pseudomallei lipase from fermentation broth. The partition behavior of B. pseudomallei lipase was investigated with various parameters such as phase composition, tie-line length (TLL), volume ratio (VR), sample loading, system pH, and addition of neutral salts. Optimum conditions for the purification of lipase were obtained
in polyethylene glycol (PEG) 6000-potassium phosphate system using TLL of 42.2% (w/w), with VR of 2.70, and 1% (w/w) NaCl
addition at pH 7 for 20% (w/w) crude load. Based on this system, the purification factor of lipase was enhanced to 12.42 fold,
with a high yield of 93%. Hence, the simplicity and effectiveness of aqueous two-phase systems (ATPS) in the purification
of lipase were proven in this study. 相似文献
16.
Extracellular lipase of <Emphasis Type="Italic">Aspergillus niger</Emphasis> NRRL3; production,partial purification and properties 总被引:1,自引:0,他引:1
Four strains of Aspergillus niger were screened for lipase production. Each was cultivated on four different media differing in their contents of mineral components
and sources of carbon and nitrogen. Aspergillus niger NRRL3 produced maximal activity (325U/ml) when grown in 3% peptone, 0.05% MgSO4.7H2O, 0.05% KCl, 0.2% K2HPO4 and 1% olive oil:glucose (0.5:0.5). A. niger NRRL3 lipase was partially purified by ammonium sulphate precipitation. The majority of lipase activity (48%) was located
in fraction IV precipitated at 50–60% of saturation with a 18-fold enzyme purification. The optimal pH of the partial purified
lipase preparation for the hydrolysis of emulsified olive oil was 7.2 and the optimum temperature was 60°C. At 70°C, the enzyme
retained more than 90% of its activity. Enzyme activity was inhibited by Hg2+ and K+, whereas Ca2+ and Mn2+ greatly stimulated its activity. Additionally, the formed lipase was stored for one month without any loss in the activity. 相似文献
17.
A constitutive level of a mycelium-bound lipolytic activity from Aspergillus niger MYA 135 was strongly increased by 97% in medium supplemented with 2% olive oil. The constitutive lipase showed an optimal
activity in the pH range of 3.0–6.5, while the mycelium-bound lipase activity produced in the presence of olive oil had two
pH optima at pH 4 and 7. Interestingly, both lipolytic sources were cold-active showing high catalytic activities in the temperature
range of 4–8°C. These mycelium-bound lipase activities were also very stable in reaction mixtures containing methanol and
ethanol. In fact, the constitutive lipase maintained almost 100% of its activity after exposure by 1 h at 37°C in ethanol.
A simple methodology to evaluate suitable transesterification activities in organic solvents was also reported. 相似文献
18.
C. Marcin L. Katz R. Greasham M. Chartrain 《Journal of industrial microbiology & biotechnology》1993,12(1):29-34
Summary The production ofPseudomonas aeruginosa MB 5001 extracellular lipase was optimized by batch cultivation employing shake flasks and 23-L bioreactors. This enzyme efficiently and selectively bioconverts dimethyl 5-(3-(2-(7-chloroquinolin-2-yl)ethyl)phenyl)4,6-dithianonanedioate (diester) to its (S)-ester acid. Process development studies focused on the identification and optimization of the physicochemical parameters required to achieve maximum lipase production. Of the media evaluated, a peptonized milk-based medium was found to support excellent lipase production and stability. Medium composition and process parameters that supported optimal lipase production were different from those supporting maximum biomass formation. Of the parameters investigated, dissolved oxygen tension had the most significant and unexpected impact on lipase production. Elevated lipase production was achieved whenP. aeruginosa MB 5001 was cultivated in a dissolved oxygen limited environment. Overall, these process development studies resulted in a 100% increase in lipase production when compared to the original shake flask process employing skim milk. 相似文献
19.
Cloning and expression of gene, and activation of an organic solvent-stable lipase from Pseudomonas aeruginosa LST-03 总被引:1,自引:0,他引:1
Ogino H Katou Y Akagi R Mimitsuka T Hiroshima S Gemba Y Doukyu N Yasuda M Ishimi K Ishikawa H 《Extremophiles : life under extreme conditions》2007,11(6):809-817
Organic solvent-tolerant Pseudomonas aeruginosa LST-03 secretes an organic solvent-stable lipase, LST-03 lipase. The gene of the LST-03 lipase (Lip9) and the gene of the
lipase-specific foldase (Lif9) were cloned and expressed in Escherichia coli. In the cloned 2.6 kbps DNA fragment, two open reading frames, Lip9 consisting of 933 nucleotides which encoded 311 amino
acids and Lif9 consisting of 1,020 nucleotides which encoded 340 amino acids, were found. The overexpression of the lipase
gene (lip9) was achieved when T7 promoter was used and the signal peptide of the lipase was deleted. The expressed amount of the lipase
was greatly increased and overexpressed lipase formed inclusion body in E. coli cell. The collected inclusion body of the lipase from the cell was easily solubilized by urea and activated by using lipase-specific
foldase of which 52 or 58 amino acids of N-terminal were deleted. Especially, the N-terminal methionine of the lipase of which
the signal peptide was deleted was released in E. coli and the amino acid sequence was in agreement with that of the originally-produced lipase by P. aeruginosa LST-03. Furthermore, the overexpressed and solubilized lipase of which the signal peptide was deleted was more effectively
activated by lipase-specific foldase. 相似文献
20.
Anita Eberl Sonja Heumann Tina Brückner Rita Araujo Artur Cavaco-Paulo Franz Kaufmann Wolfgang Kroutil Georg M. Guebitz 《Journal of biotechnology》2009,143(3):207-212
A lipase from Thermomyces lanuginosus and cutinases from Thermobifida fusca and Fusarium solani hydrolysed poly(ethylene terephthalate) (PET) fabrics and films and bis(benzoyloxyethyl) terephthalate (3PET) endo-wise as shown by MALDI-Tof-MS, LC–UVD/MS, cationic dyeing and XPS analysis. Due to interfacial activation of the lipase in the presence of Triton X-100, a seven-fold increase of hydrolysis products released from 3PET was measured. In the presence of the plasticizer N,N-diethyl-2-phenylacetamide (DEPA), increased hydrolysis rates of semi-crystalline PET films and fabrics were measured both for lipase and cutinase. The formation of novel polar groups resulted in enhanced dye ability with additional increase in colour depth by 130% and 300% for cutinase and lipase, respectively, in the presence of plasticizer. 相似文献