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1.
The Antarctic marine environment provides a good source of novel lipolytic enzymes that possess beneficial properties, i.e., resistance to extreme physical and chemical conditions. We found a lipolytic Escherichia coli colony that was transformed using genomic DNA from Marinobacter lipolyticus 27-A9 isolated from the Antarctic Ross Sea. DNA sequence analysis revealed an open reading frame of lipolytic enzyme gene. The gene translates a protein (LipA9) of 404 amino acids with molecular mass of 45,247 Da. Recombinant LipA9 was expressed in E. coli BL21 (DE3) cells and purified by anion exchange and gel filtration chromatography. The kcat/Km of LipA9 was 175 s−1 μM−1, and the optimum temperature and pH were 70 °C and pH 8.0, respectively. LipA9 had quite high organic solvent stability; it was stable toward several common organic solvents up to 50% concentration. Substrate specificity studies showed that LipA9 preferred a short acyl chain length of p-nitrophenyl ester and triglyceride. Sequence analysis showed that LipA9 contained catalytic Ser72 and Lys75 in S-x-x-K motif, like family VIII esterases. Homology modeling and site-directed mutagenesis studies revealed that Tyr141 and Tyr188 residues were located near the conserved motif and played an important role in catalytic activity. 相似文献
2.
A new organic solvent-tolerant strain Bacillus megaterium AU02 which secretes an organic solvent-tolerant protease was isolated from milk industry waste. Statistical methods were employed to achieve optimum protease production of 43.6 U/ml in shake flask cultures. The productivity of the protease was increased to 53 U/ml when cultivated under controlled conditions in a 7-L fermentor. The protease was purified to homogeneity by a three-step process with 24 % yield and specific activity of 5,375 U/mg. The molecular mass of the protease was found to be 59 kDa. The enzyme was active over a wide range of pH (6.0–9.0), with an optimum activity at pH 7.0 and temperature from 40 to 70 °C having an optimum activity at 50 °C. The thermal stability of the enzyme increased significantly in the presence of CaCl 2, and it retained 90 % activity at 50 °C for 3 h. The K m and V max values were determined as 0.722 mg/ml and 0.018 U/mg respectively. The metalloprotease exhibited significant stability in the presence of organic solvents with log P values more than 2.5, nonionic detergents and oxidising agent. An attempt was made to test the synthesis of aspartame precursor (Cbz-Asp-Phe-NH 2) which was catalysed by AU02 protease in the presence of 50 % DMSO. These properties of AU02 protease make it an ideal choice for enzymatic peptide synthesis in organic media. 相似文献
3.
The aim of this present study was to produce a microbial enzyme that can potentially be utilized for the enzymatic transesterification of waste cooking oil. To that end, an extracellular lipase was isolated and purified from the culture broth of Streptomyces sp. CS273. The molecular mass of purified lipase was estimated to be 36.55 kDa by SDS PAGE. The optimum lipolytic activity was obtained at alkaline pH 8.0 to 8.5 and temperature 40 °C, while the enzyme was stable in the pH range 7.0?~?9.0 and at temperature ≤40 °C. The lipase showed highest hydrolytic activity towards p-nitrophenyl myristate (C14). The lipase activity was enhanced by several salts and detergents including NaCl, MnSo 4, and deoxy cholic acid, while phenylmethylsulfonyl fluoride at concentration 10 mM inhibited the activity. The lipase showed tolerance towards different organic solvents including ethanol and methanol which are commonly used in transesterification reactions to displace alcohol from triglycerides (ester) contained in renewable resources to yield fatty acid alkyl esters known as biodiesel. Applicability of the lipase in transesterification of waste cooking oil was confirmed by gas chromatography mass spectrometry analysis. 相似文献
4.
An organic solvent-tolerant lipase from Serratia marcescens ECU1010 (rSML) was overproduced in Escherichia coli in an insoluble form. High concentrations of both biomass (50 g cell wet weight/L culture broth) and inclusion bodies (10.5 g/L) were obtained by applying a high-cell-density cultivation procedure. Activity assays indicated that the enzymatic activity of rSML reached 600 U/L. After treatment with isopropyl ether for 12 h, the maximum lipase activity reached 6,000 U/L. Scanning electron microscopy and Fourier transform infrared microspectroscopy revealed the activation mechanism of rSML in the presence of organic solvents. rSML was stable in broad ranges of temperatures and pH values, as well as in a series of organic solvents. Besides, rSML showed the best enantioselectivity for the kinetic resolution of (±)- trans-3-(4-methoxyphenyl)glycidic acid methyl ester. These features render the S. marcescens ECU1010 lipase attractive for biotechnological applications in the field of organic synthesis and pharmaceutical industry. 相似文献
5.
An extracellular lipase secreted by Pseudomonas aeruginosa CS-2 was purified to homogeneity about 25.5-fold with an overall yield of 45.5%. The molecular mass of the lipase was estimated
to be 33.9 kDa by SDS-PAGE and 36 kDa by gel filtration. The optimum temperature and pH were 50 °C and 8.0. The lipase was
found to be stable at pH 4–10 and below 50 °C. Its hydrolytic activity was highest against p-nitrophenyl palmitate ( p-NPP) among p-nitrophenyl esters of fatty acids with various chain lengths. The lipase was activated in the presence of Ca 2+, while it was inactivated by other metal ions more or less. EDTA significantly reduced the lipase activity, indicating the
lipase was a metalloenzyme. Gum Arabic and polyvinyl alcohol 124 enhanced lipase activity but Tween-20, Tween-80, and hexadecyltrimethyl
ammonium bromide strongly inhibited the lipase. It exhibited stability in some organic solvents. The lipase was activated
in the presence of acetonitrile. Conversely, it was drastically inactivated by methanol and ethanol. 相似文献
6.
A novel lipase lipB from Serratia marcescens ECU1010 is highly stable in the presence of organic solvents. By sequence and structure comparison with homologous lipase lipA, three amino acid residues were found to be different between them. To identify the residues which increase the organic solvent stability of lipB, residues that potentially provide this stability were mutated to the ones of lipA at equivalent positions. The replacement of Gly at position 33 by Asp obviously decreased its stability in organic solvents. Molecular modeling and structural analysis also suggested that the Gly33 residue is important for the organic solvent stability of lipB. 相似文献
7.
The genes of CS-2 lipase and its cognate foldase were cloned from Pseudomonas aeruginosa CS-2. A stop codon was not found in the lipase gene. The amino acid sequence deduced from the lipase gene from P. aeruginosa CS-2 showed 97.8%, 71.3%, and 71.2% identity with lipases from P. aeruginosa LST-03, P seudomonas mendocina ymp, and Pseudomonas stutzeri A1501, respectively. The co-expression of CS-2 lipase and its cognate foldase of P. aeruginosa CS-2 in E scherichia coli BL21 (DE3) resulted in the formation of a soluble lipase. The recombinant lipase and foldase were purified to homogeneity using nickel affinity chromatography and about 10.2-fold with 40.9% recovery was achieved for the purification of the recombinant lipase. The molecular masses of the lipase and the foldase were estimated to be 35.7 and 38.3 kDa in SDS-PAGE, respectively. The recombinant lipase showed stability in the presence of some organic solvents. The recombinant CS-2 lipase was immobilized and subsequently used for the synthesis of butyl acetate in heptane. The conversion of substrate decreased from 98.2% to 87.4% after 5 cycles in reuse of the immobilized lipase. 相似文献
8.
Abstract A simple device for isolation of organic compounds from aqueous samples has been designed and its operating parameters tested during bioth periodic and continuous operation using isolation of organochlorine compounds as an example. A stream of an aqueous sample is pumped at elevated temperature by a piston pump to an unit for expransion of the liquid phase surface, where the liquid is sprayed on the walls of the unit and flows down freely. Organochlorine compounds passing to the gaseous phase are purged with a stream of purified air, oxidied and the chlorides formed are determined coulometrically. The designed device, due to its simplicity, can be built and employed in each averagely equipped laboratory. 相似文献
9.
A novel cold-adapted lipase (designated as LipYY31) was obtained from a psychrotrophic Pseudomonas sp. YY31. The strain YY31 was gram-negative, rod shaped, motile by means of one polar flagellum, and exhibited chemotaxis toward oil droplets under a microscope. The strain displayed remarkable degradation of edible oil and fat even at 5 °C. The LipYY31 DNA fragment contains an open reading frame of 1,410 bp which encoded a protein of 470 amino acids with an estimated molecular mass of 49,584 Da. LipYY31 showed high sequence similarity to those of subfamily Ι.3 lipase and had a conserved GXSXG motif around the catalytic Ser residue. Its optimal temperature was 25–30 °C, and it retained 20–40 % of its activity at 0–5 °C. The optimal pH value was 8.0. The activity was strongly inhibited by Cd 2+, Zn 2+, EDTA and was highly dependent on Ca 2+. Tricaprin and p-nitrophenyl caprate were the most favorable substrates among the triglycerides and p-nitrophenyl esters, respectively. LipYY31 also had high activity towards natural substrates including edible vegetable oils and animal fat. Furthermore, LipYY31 was very active and stable in the presence of several detergents and organic solvents. In particular, the lipase exhibited high stability against organic solvents such as methanol, ethanol, and isopropanol. 相似文献
10.
Organic solid-state semiconductor lasers are attracting ever-increasing interest for their potential application in future photonic circuits. Despite the great progress made in recent years, an organic laser from 3D chiral structures has not been achieved. Now, the first example of an organic nano-laser from the micro-helix structure of an achiral molecule is presented. Highly regular micro-helixes with left/right-handed helicity from a distyrylbenzene derivative (HM-DSB) were fabricated and characterized under microscope spectrometers. These chiral micro-helixes exhibit unique photonic properties, including helicity-dependent circularly polarized luminescence (CPL), periodic optical waveguiding, and length-dependent amplified spontaneous emission (ASE) behavior. The successful observation of laser behavior from the organic micro-helix extends our understanding to morphology chirality of organic photonic materials and provides a new design strategy towards chiral photonic circuits. 相似文献
12.
In this article we review how molecular modeling techniques can be used to shed some light on the influence of organic solvents on the molecular characteristics of proteins and enzymes. Molecular dynamic simulations on bovine pancreas trypsin inhibitor, chymotrypsin, and subtilisin make it possible to get a deeper understanding into how increased intramolecular interactions improve conformational rigidity, thus explaining the lower reactivity and the higher thermostability of enzymes in non-aqueous media. The application of thermodynamics-based models allows first qualitative predictions on the selectivity of many reaction types; however, the application of quantum mechanical/molecular mechanical methods is required for the development of quantitative models of actual reactivity patterns. 相似文献
13.
Summary. In this article we review how molecular modeling techniques can be used to shed some light on the influence of organic solvents
on the molecular characteristics of proteins and enzymes. Molecular dynamic simulations on bovine pancreas trypsin inhibitor,
chymotrypsin, and subtilisin make it possible to get a deeper understanding into how increased intramolecular interactions
improve conformational rigidity, thus explaining the lower reactivity and the higher thermostability of enzymes in non-aqueous
media. The application of thermodynamics-based models allows first qualitative predictions on the selectivity of many reaction
types; however, the application of quantum mechanical/molecular mechanical methods is required for the development of quantitative
models of actual reactivity patterns.
Received November 18, 1999. Accepted (revised) February 8, 2000 相似文献
15.
The role of the enzyme source was studied in the reaction of hydrogen peroxide decomposition by immobilised catalase in acetonitrile. Enzymes isolated from bacterial and mammalian sources were conveniently immobilised on a spectroscopic graphite to obtain an organic-phase enzyme electrode (OPEE). Amperometry at constant potential was employed as basic analytical approach in this study. 相似文献
16.
Summary. The role of the enzyme source was studied in the reaction of hydrogen peroxide decomposition by immobilised catalase in acetonitrile. Enzymes isolated from bacterial and mammalian sources were conveniently immobilised on a spectroscopic graphite to obtain an organic-phase enzyme electrode (OPEE). Amperometry at constant potential was employed as basic analytical approach in this study. 相似文献
17.
Vaccinium dunalianum Wight, usually processed as a traditional folk tea beverage, is widely distributed in the southwest of China. The present study aimed to investigate the antioxidant, α-glucosidase and pancreatic lipase inhibitory activities of V. dunalianum extract and isolate the bioactive components. In this study, the crude extract (CE) from the buds of V. dunalianum was prepared by the ultrasound-assisted extraction method in 70% methanol and then purified with macroporous resin D101 to obtain the purified extract (PM). Five fractions (Fr. A–E) were further obtained by MPLC column (RP-C18). Bioactivity assays revealed that Fr. B with 40% methanol and Fr. D with 80% methanol had better antioxidant with 0.48 ± 0.03 and 0.62 ± 0.01 nM Trolox equivalent (TE)/mg extract for DPPH, 0.87 ± 0.02 and 1.58 ± 0.02 nM TE/mg extract for FRAP, 14.42 ± 0.41 and 19.25 ± 0.23 nM TE/mg extract for ABTS, and enzyme inhibitory effects with IC 50 values of 95.21 ± 2.21 and 74.55 ± 3.85 for α-glucosidase, and 142.53 ± 11.45 and 128.76 ± 13.85 µg/mL for pancreatic lipase. Multivariate analysis indicated that the TPC and TFC were positively related to the antioxidant activities. Further phytochemical purification led to the isolation of ten compounds (1–10). 6- O-Caffeoylarbutin (7) showed significant inhibitory effects on α-glucosidase and pancreatic lipase enzymes with values of 38.38 ± 1.84 and 97.56 ± 7.53 µg/mL, and had the highest antioxidant capacity compared to the other compounds. 相似文献
18.
Organic solid‐state semiconductor lasers are attracting ever‐increasing interest for their potential application in future photonic circuits. Despite the great progress made in recent years, an organic laser from 3D chiral structures has not been achieved. Now, the first example of an organic nano‐laser from the micro‐helix structure of an achiral molecule is presented. Highly regular micro‐helixes with left/right‐handed helicity from a distyrylbenzene derivative (HM‐DSB) were fabricated and characterized under microscope spectrometers. These chiral micro‐helixes exhibit unique photonic properties, including helicity‐dependent circularly polarized luminescence (CPL), periodic optical waveguiding, and length‐dependent amplified spontaneous emission (ASE) behavior. The successful observation of laser behavior from the organic micro‐helix extends our understanding to morphology chirality of organic photonic materials and provides a new design strategy towards chiral photonic circuits. 相似文献
20.
A novel antimicrobial polypeptidc was isolated and charaeterized from loach,Misgurmis anguillieaudatus, The polypcptide, named MAPP, is a single-chain polypcptide with Mw about 9,800 Dalton and p1 about 4.78; the N-tag ofMAPP was CFGWN. MAPP showed good inhibition against various bacteria including Bacillus subtilis, Eseherichia coli and Staphylococcus aurens. MAPP could be used as a lead compound in antibiotics drug discovery. 相似文献
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