首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 15 毫秒
1.
The extracellular β-agarase LSL-1 produced by an agar-liquefying, soil bacterium Acinetobacter sp., AG LSL-1 was purified to homogeneity by combination of ion-exchange and size exclusion chromatography with final yield of 44%. The enzyme has a specific activity of 397 U mg−1 protein and with a molecular mass of 100 kDa. The agarase was active in the pH range of 5.0–9.0, optimally at pH 6.0 and temperature between 25 °C and 55 °C and optimal at 40 °C. The enzyme retained 63% of native activity at 50 °C suggesting it is a thermostable. The activity of the agarase was completely inhibited by metal ions, Hg2+, Ag+ and Cu2+, whereas 25–40% of native activity was retained in the presence of Zn2+, Sn2+ and SDS. Neoagarobiose was the final product of hydrolysis of both agarose and neoagarohexaose by the purified agarase LSL-1. Based on the molecular mass and final products of agarose hydrolysis, the β-agarase LSL-1 may be further grouped under group III β-agarases and may be a member of GH-50 family. This is the first report on the purification and biochemical characterization of β-agarase from an agar-liquefying Acinetobacter species.  相似文献   

2.
Alkalophilic Bacillus licheniformis NH1 strain produced at least five major extracellular proteases and a unique amylase as showed by zymography technique. The optimum pH and temperature for the proteolytic activity were 10.0 and 70 °C, respectively, while those of amylolytic activity were 6.5 and 90 °C, respectively. The alkaline proteases and thermostable α-amylase showed extreme stability towards non-ionic and anionic surfactants after pre-incubation for 1 h at 40 °C, and relative stability towards oxidizing agents. Additionally, the crude enzyme showed excellent stability and compatibility with various solid and liquid detergents. Wash performance analysis revealed that the NH1 crude enzyme could effectively remove a variety of stains, such as blood, chocolate and barbecue sauce. Considering its promising properties, B. licheniformis NH1 crude enzyme containing both α-amylase and proteases activities may be considered a potential candidate for future use in detergent processing industries.  相似文献   

3.
Silanized palygorskite for lipase immobilization   总被引:2,自引:0,他引:2  
Lipase from Candida lipolytica has been immobilized on 3-aminopropyltriethoxysilane-modified palygorskite support. Scanning electron micrographs proved the covalently immobilization of C. lipolytica lipase on the palygorskite support through glutaraldehyde. Using an optimized immobilization protocol, a high activity of 3300 U/g immobilized lipase was obtained. Immobilized lipase retained activity over wider ranges of temperature and pH than those of the free enzyme. The optimum pH of the immobilized lipase was at pH 7.0–8.0, while the optimum pH of free lipase was at 7.0. The retained activity of the immobilized enzyme was improved both at lower and higher pH in comparison to the free enzyme. The immobilized enzyme retained more than 70% activity at 40 °C, while the free enzyme retained only 30% activity. The immobilization stabilized the enzyme with 81% retention of activity after 10 weeks at 30 °C whereas most of the free enzyme was inactive after a week. The immobilized enzyme retains high activity after eight cycles. The kinetic constants of the immobilized and free lipase were also determined. The Km and Vmax values of immobilized lipase were 0.0117 mg/ml and 4.51 μmol/(mg min), respectively.  相似文献   

4.
Curtobacterium luteum, a gram-positive psychrotrophic bacterium, secreting an extracellular protease was isolated from the soil of Gangotri glacier, Western Himalaya. The maximum enzyme production was achieved when isolate was grown in a pH-neutral medium containing skim milk at 15oC over 120 hour. The metal ions such as Zn2+ and Cr2+ enhanced enzyme production. The specific activity of purified enzyme was 8090 u/mg after 34.1 fold purification. The 115 kD enzyme was a metalloprotease (activity inhibited by EDTA and EGTA) and showed maximum activity at 20oC and pH 7. The enzyme was active over a broad pH range and retained 84% of its original activity between pH 6–8. There was no loss in enzyme activity when exposed for 3 hours at 4oC-20oC. However, lost 65% of activity at 30oC, and was almost inactivated at 50oC, but was resistant to repeated freezing and thawing. The enzyme activity was stimulated by manganese ions; however, it was inactivated by copper ions.  相似文献   

5.
A putative lysophospholipase (PF0480) encoded by the Pyrococcus furiosus genome has previously been cloned and expressed in Escherichia coli. Studies involving crude extracts established the enzyme to be an esterase; however, owing presumably to its tendency to precipitate into inclusion bodies, purification and characterization have thus far not been reported. Here, we report the overexpression and successful recovery and refolding of the enzyme from inclusion bodies. Dynamic light scattering suggests that the enzyme is a dimer, or trimer, in aqueous solution. Circular dichroism and fluorescence spectroscopy show, respectively, that it has mixed beta/alpha structure and well-buried tryptophan residues. Conformational changes are negligible over the temperature range of 30–80 °C, and over the concentration range of 0–50% (v/v) of water mixtures with organic solvents such as methanol, ethanol and acetonitrile. The enzyme is confirmed to be an esterase (hydrolyzing p-NP-acetate and p-NP-butyrate) and also shown to be a lipase (hydrolyzing p-NP-palmitate), with lipolytic activity being overall about 18- to 20-fold lower than esterase activity. Against p-NP-palmitate the enzyme displays optimally activity at pH 7.0 and 70 °C. Remarkably, over 50% activity is retained at 70 °C in the presence of 25% acetonitrile. The high organic solvent stability and thermal stability suggest that this enzyme may have useful biodiesel-related applications, or applications in the pharmaceutical industry, once yields are optimized.  相似文献   

6.
Phosphotriesterase homology protein (PHP) is a member of a recently discovered family of proteins related to phosphotriesterase. Phosphotriesterase is a hydrolytic, bacterial enzyme with unusual substrate specificity for synthetic organophosphate triesters, common constituents of chemical warfare agents and agricultural pesticides. PHP may belong to the family of proteins from which phosphotriesterase evolved. The PHP gene from the thermophilic bacterium Geobacillus caldoxylosilyticus TK4 was cloned and overexpressed in Escherichia coli with 6×His tag in the N-terminal. The recombinant protein was purified with nickel affinity chromatography and characterized in detail. The enzyme did not have any activity against paraoxon. The highest activities were observed with p-nitrophenyl acetate (pNPA) and p-nitrophenyl butyrate. pH and temperature optima were determined as 8.0 and 50 °C, respectively, with pNPA. The enzyme activity was not largely affected by the incubation of the enzyme at 50 °C in the different buffer solutions (pHs between 3.0 and 9.0) for 7 days. After the incubation at 90 °C for 7 days, G. caldoxylosilyticus TK4 PHP retained 62% of its original activity. The enzyme was also resistant to some metal ions and organic solvents. These results suggest that this is the first reported PHP having an extremely pH- and thermo-stable esterase activity.  相似文献   

7.
The effects of temperature, pH, and various components in a detergent on the activity and stability of a lipase produced by a soda lake fungus strain Fusarium solani N4-2 were studied in a preliminary evaluation for use in detergent formulation. The molecular mass of the lipase was 31.6 kDa by SDS-PAGE after purification using acetone fractionation and Q-sepharose ion exchange technique. In aqueous solutions, the lipase showed maximal activity at pH 9.0 in a glycine–NaOH buffer at 30 °C. At 0 °C and 10 °C, 52.3% and 82.6% of its maximum activities were detained, respectively. Among the metal ions tested, additions of Cu2+, Ca2+, Mg2+, Al3+ and Mn2+ ions were found to enhance the enzyme activity while Zn2+, Ba2+ and Hg2+ ions showed inhibitory effects. Compared with the commercial lipase Lipolase®, the enzyme showed better stability towards selected surfactants, commercial detergents, oxidizing agents and proteases. The remarkable resistance capability of the lipase makes it a potential additive for better detergent formulation.  相似文献   

8.
An endonuclease from Xanthomonas oryzae pathovar oryzae (Xoo) KACC10331, XorKII, was recombinantly produced in Escherichia coli by applying the stationary state induction method, which was necessary to prevent the unwanted lysis of E. coli cells. XorKII was purified by immobilized metal affinity chromatography on an FPLC system. The yield was 3.5 mg of XorKII per liter of LB medium. The purified recombinant XorKII showed that it recognized and cleaved to the same site as PstI. It behaved as a dimer as evidenced by the size exclusion chromatography. The specific activity of the purified XorKII was determined to be 31,300 U/mg. The enzyme activity was monitored by cleaving lambda DNA or YEp24 plasmid as substrates. The enzyme was the most active at 10 mM Tris–HCl pH 7.0, 10 mM MgCl2, 1 mM dithiothreitol at 37 °C. XorKII was easily inactivated by heating at 65 °C for 5 min, but retained most of the original activity after incubation at 37 °C for 24 h.  相似文献   

9.
The stability of crude extracellular protease produced by Bacillus licheniformis RP1, isolated from polluted water, in various solid laundry detergents was investigated. The enzyme had an optimum pH and temperature at pH 10.0–11.0 and 65–70 °C. Enzyme activity was inhibited by PMSF, suggesting that the preparation contains a serine-protease. The alkaline protease showed extreme stability towards non-ionic (5% Tween 20% and 5% Triton X-100) and anionic (0.5% SDS) surfactants, which retained 100% and above 73%, respectively, of its initial activity after preincubation 60 min at 40 °C.

The RP1 protease showed excellent stability and compatibility with a wide range of commercial solid detergents at temperatures from 40 to 50 °C, suggesting its further application in detergent industry. The enzyme retained 95% of its initial activity with Ariel followed by Axion (94%) then Dixan (93.5%) after preincubation 60 min at 40 °C in the presence of 7 mg/ml of detergents. In the presence of Nadhif and New Det, the enzyme retained about 83.5% of the original activity. The effects of additives such as maltodextrin, sucrose and PEG 4000 on the stability of the enzyme during spray-drying and during subsequent storage in New Det detergent were also examined. All additives tested enhanced stability of the enzyme.  相似文献   


10.
An alkaline protease from marine Engyodontium album was characterized for its physicochemical properties towards evaluation of its suitability for potential industrial applications. Molecular mass of the enzyme by matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) analysis was calculated as 28.6 kDa. Isoelectric focusing yielded pI of 3–4. Enzyme inhibition by phenylmethylsulfonyl fluoride (PMSF) and aprotinin confirmed the serine protease nature of the enzyme. K m, V max, and K cat of the enzyme were 4.727 × 10−2 mg/ml, 394.68 U, and 4.2175 × 10−2 s−1, respectively. Enzyme was noted to be active over a broad range of pH (6–12) and temperature (15–65°C), with maximum activity at pH 11 and 60°C. CaCl2 (1 mM), starch (1%), and sucrose (1%) imparted thermal stability at 65°C. Hg2+, Cu2+, Fe3+, Zn2+, Cd+, and Al3+ inhibited enzyme activity, while 1 mM Co2+ enhanced enzyme activity. Reducing agents enhanced enzyme activity at lower concentrations. The enzyme showed considerable storage stability, and retained its activity in the presence of hydrocarbons, natural oils, surfactants, and most of the organic solvents tested. Results indicate that the marine protease holds potential for use in the detergent industry and for varied applications.  相似文献   

11.
An intracellular β-xylosidase from the thermophilic fungus Sporotricum thermophile strain ATCC 34628 was purified to homogeneity by Q-Sepharose and Mono-Q column chromatographies. The protein properties correspond to molecular mass and pI values of 45 kDa and 4.2, respectively. The enzyme is optimally active at pH 7.0 and 50 °C. The purified β-xylosidase is fully stable at pH 6.0–8.0 and temperatures up to 50 °C and retained over 58% of its activity after 1 h at 60 °C. The enzyme hydrolyzes β-1,4-linked xylo-oligosaccharides with chain lengths from 2 to 6, releasing xylose from the non-reducing end, but is inactive against xylan substrates. The apparent Km and Vmax values from p-nitrophenyl β-d-xylopyranoside are 1.1 mM and 114 μmol p-nitrophenol min−1 mg−1, respectively. Alcohols inactivate the enzyme, ethanol at 10% (v/v) yields a 30% decrease of its activity. The enzyme is irreversibly inhibited by 2,3-epoxypropyl β-d-xylobioside while alkyl epoxides derived from d-xylose were not inhibitors of the enzyme. The enzyme catalyses the condensation reaction using high donor concentration, up to 60% (w/v) xylose.  相似文献   

12.
Porcine trypsin was glycated with glucose and covalently immobilized through its carboxyl groups onto aminated glass beads to produce porcine immobilized glycated-trypsin (IGT). On incubation at 60 °C and pH 8, IGT retained its full activity for 8 h and 50% of its activity after 24 h. In comparison, under the same conditions porcine native trypsin lost 80% of its activity in 2 h and was completely inactivated in less than 4 h. The rate of autolysis of porcine glycated-trypsin at 37 °C was 40% that of native trypsin and with IGT there was no significant autolysis, even at elevated temperatures as high as 60 °C. Glycation significantly increased the stability of trypsin and immobilization also significantly increased the stability of trypsin. The remarkable thermostability of IGT is attributed to a synergistic effect when these two modifications are combined. Tryptic fragmentation of denatured proteins with IGT can be performed at 60 °C for shorter digestion times and with smaller amounts of enzyme than normally employed to achieve complete digestion with soluble forms of trypsin. Prior denaturation of proteins for tryptic digestion is not required with IGT as in situ denaturation and digestion can be achieved simultaneously at 60 °C with an enzyme:protein mass ratio as low as 1:1000.  相似文献   

13.
A gene encoding glutamate racemase (GluRA) was found in a thermophilic Bacillus strain named SK-1. The gene was cloned and expressed in Escherichia coli WM335, a -glutamate auxotroph. It consists of 792 bp with a start codon, TTG. The amino acid sequence deduced from the gene indicates that the GluRA has two cysteines and their surrounding regions are well conserved. The GluRA produced in the recombinant E. coli was purified to homogeneity by heat-treatment and Resource Q and Phenyl sepharose column chromatographies. The enzyme, which was determined to be a monomeric protein with a molecular weight of 29,000, did not require a cofactor such as pyridoxal 5′-phosphate, nicotinamide, or flavin for its activity. The enzyme was stable after incubation at 55 °C and retained 60% of its original activity after incubation at 60 °C. It was found to be stable in the region of pH 6.0–11.5. The thermostable GluRA was used as a catalyst in a multi-enzyme system composed of four enzyme reactions for the production of -phenylalanine. By running the multi-enzyme system for 35 h, 58 g l−1 of -phenylalanine was produced with 100% of optical purity from equimolar amount of phenylpyruvate.  相似文献   

14.
A new strain of Bacillus sp. I-3, isolated from natural soil samples, showed a high raw starch digesting activity towards potato starch. Upon optimization of various environmental and cultural conditions, the yield of α-amylase reached 642 U/mL. The kinetic characterization of partially purified enzyme exhibited the maximum activity at 70 °C, pH 7.0 and revealed a high thermostability in the presence of 10 mM CaCl2·2H2O where it could retain more than 90% residual activity at 70 °C after 3.5 h. At 80, 90 and 100 °C, the enzyme retained 80, 59 and 26% of its maximum activity after 2.5, 0.5 and 0.5 h, respectively. The enzyme preparation had a strong affinity towards raw potato starch granules and was almost completely adsorbed onto it. It also hydrolyzed raw potato starch at a concentration of 12.5% significantly in a short period of time of 12 h.  相似文献   

15.
The dynamics of β-xylosidase biosynthesis from Aspergillus niger B 03 was investigated in laboratory bioreactor. Maximum xylosidase activity 5.5 U/ml was achieved after 80 h fermentation at medium pH 4.0. The isolated β-xylosidase was immobilized on polyamide membrane support and the basic characteristics of the immobilized enzyme were determined. Maximum immobilization and activity yield obtained was 30.0 and 6.8%, respectively. A shift in temperature optimum and pH optimum was observed for immobilized β-xylosidase compared to the free enzyme. Immobilized enzyme exhibited maximum activity at 45 °C and pH 4.5 while its free counterpart at 70 °C and pH 3.5, respectively. Thermal stability at 40 and 50 °C and storage stability of immobilized β-xylosidase were investigated at pH 5.0. Kinetic parameters Km, Vmax and Ki were determined for both enzyme forms. Free and immobilized β-xylosidase were tested for xylose production from birchwood xylan. The substrate was preliminarily depolymerized with xylanase to xylooligosaccharides and the amount of xylose obtained after their hydrolysis with free and immobilized β-xylosidase was determined by HPLC analysis. Continuous enzyme hydrolysis of birchwood xylan was performed with xylanase and free or immobilized β-xylosidase. The maximum extent of hydrolysis was 25 and 30% with free and immobilized enzyme, respectively. Immobilized preparation was also examined for reusability in 20 consecutive cycles at 40 °C.  相似文献   

16.
We have evaluated for the first time the impact of a solvent/detergent (S/D) treatment on the quality and in vivo neutralization potency of horse-derived whole IgG antivenom used in the treatment of viperid snake bite envenoming in Central America. The S/D treatment by 1% tri (n-butyl) phosphate (TnBP) – 1% Triton X-45 at 22–25 °C was applied either on starting plasma or on purified immunoglobulins. The S/D agents were removed from both fractions by extractions with oil. S/D-treated plasma was subjected to caprylic acid precipitation to purify the immunoglobulins. Products were formulated, sterile-filtered, and filled into 10-mL vials, stored at 5 ± 3 °C, and subjected to routine quality controls, SDS-PAGE, determination of anti-Bothrops asper venom antibody titre by ELISA, in vivo B. asper venom-neutralization potency tests, and safety test, comparatively with an antivenom manufactured by caprylic acid fractionation without S/D treatment. Results indicate that these conditions of S/D treatment on purified immunoglobulin yielded an antivenom of high turbidity that induced weight loss in animals. In contrast, antivenom fractionated from the S/D-treated plasma had physico-chemical and biological characteristics indistinguishable from those of the non-S/D-treated antivenom. S/D treatment of horse plasma may be considered to increase the viral safety of antivenoms.  相似文献   

17.
A bleach-stable, thermotolerant, alkaline protease for detergent formulation from a newly isolated Bacillus SB5 is reported. Most (85%) activity of the enzyme was retained in the presence of 10% (v/v) H2O2 and 1% SDS (w/v) at 40°C, after 1 h. The enzyme was optimal at pH 10 and 60°C to 70°C. Enzyme activity was enhanced 30 to 80% in presence of ionic and non-ionic detergents, surfactants and commercial detergents or bleach.  相似文献   

18.
Preimaginal development and adult longevity and reproduction of Dichochrysa prasina Burmeister were studied at six constant temperatures (15, 20, 25, 27, 30 and 33 °C) and a photoperiod of 16:8 (L:D). Eggs of the flour moth Ephestia kuehniella (Zeller) were used as food throughout preimaginal development, whereas the adults of D. prasina fed on a liquid diet of water, yeast hydrolysate, sugar and honey. At the highest tested temperature of 33 °C no larvae completed their development. At the rest of the tested temperatures the egg to adult developmental period ranged from approximately 92 days at 15 °C to 25 days at 30 °C. Percentages of adult emergence ranged from 36% at 15 °C to 84% at 30 °C. Both adult longevity and fecundity were significantly affected by temperature and the intrinsic rate of increase (rm) reached its maximum value at 27 °C. These results could be useful for the establishment of a small scale rearing and mass production of D. prasina.  相似文献   

19.
An extracellular thermostable alkaline protease isolated from Bacillus laterosporus-AK1 was purified by sephadex G-200 gel filtration and DEAE cellulose ion-exchange chromatography techniques. The purified protease showed a maximum relative activity of 100% on casein substrate and appeared as a single band on SDS-PAGE with the molecular mass of 86.29 kDa. The protease was purified to 11.1-folds with a yield of 34.3%. Gelatin zymogram also revealed a clear hydrolytic zone due to proteolytic activity, which corresponded to the band obtained with SDS-PAGE. The protease enzyme had on optimum pH of 9.0 and exhibited highest activity at 75°C. The enzyme activity was highly susceptible to the specific serine protease inhibitor PMSF, suggesting the presence of serine residues at the active sites. Enzyme activity strongly enhanced by the metal ions Ca2+ and Mg2+ and this enzyme compatible with aril detergent stability retained 75% even 1-h incubation. The purified protease remove bloodstain completely when used with Wheel detergent.  相似文献   

20.
N-Succinyl-chitosan (NSC), a pH-sensitive polymer of reversibly soluble–insoluble characteristics with pH change, was prepared by modification of the chitosan backbone with succinic anhydride and employed as carrier for alliinase immobilization. The obtained NSC is soluble at pH above 4.8 and insoluble at pH below 4.4. The characteristics of NSC were evaluated using Fourier transform IR spectrophotometer, the X-ray diffraction spectrometry and thermogravimetric analyzer. Under an optimized condition (glutaraldehyde 0.8% (v/v), 31.2 U alliinase), the enzyme immobilization yield was 75.6%. The maximum activity of NSCA was achieved at 40 °C, pH 7, while the free enzyme exhibited maximum activity at 30 °C, pH 6. The Michaelis–Menten constant of NSCA was lower than that of free alliinase, indicating higher affinity of immobilized enzyme toward its substrate. The NSCA retained 85% of its initial activity even after being recycled 5 times. The immobilized alliinase in reversibly soluble NSC is suitable to catalyze the conversion of alliin to allicin, as active ingredient of pharmaceutical compositions and food additive.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号