共查询到20条相似文献,搜索用时 546 毫秒
1.
Chao-Hsun Yang Yu-Chun Huang Cheng-Yu Chen Chia-Ying Wen 《Journal of industrial microbiology & biotechnology》2010,37(4):401-406
A gene encoding the thermostable raw starch digesting α-amylase in Thermobifida fusca NTU22 was amplified by PCR, sequenced and cloned into Pichia pastoris X-33 host strain using the vector pGAPZαA, allowing constitutive expression and secretion of the protein. Recombinant expression
resulted in high levels of extracellular amylase production, as high as 510 U/l in the Hinton flask culture broth. The purified
amylase showed a single band at about 65 kDa by SDS-polyacrylamide gel electrophoresis after being treated with endo-β-N-acetylglycosaminidase H, and this agrees with the predicted size based on the nucleotide sequence. About 75% of the original
activity remained after heat treatment at 60°C for 3 h. The optimal pH and temperature of the purified amylase were 7.0 and
60°C, respectively. The purified amylase exhibited a high level of activity with raw sago starch. After 48-h treatment, the
DPw of raw sago starch obviously decreased from 830,945 to 378,732. The surface of starch granules was rough, and some granules
displayed deep cavities. 相似文献
2.
Gamma-aminobutyric acid (GABA) is a non-proteinaceous amino acid that is widely distributed in nature and acts as the major
inhibitory neurotransmitter in the mammalian brain. This study aimed to find a separation method for getting high-purity GABA
from a fermented broth. Firstly, a fermented broth with a high content of GABA (reaching 997 ± 51 mM) was prepared by fermentation
with Lactobacillus brevis NCL912. GABA purification was conducted by successive centrifugation, filtration, decoloration, desalination, ion-exchange
chromatography (IEC), and crystallization. Inorganic salt (Na2SO4) was removed from the both by desalination with 70% ethanol solution. A ninhydrin test strip was designed for the real-time
detection of GABA during IEC. The recovery rate for the whole purification process was about 50%. The purified product was
characterized by thin-layer chromatography and HPLC, and its purity reached 98.66 ± 2.36%. 相似文献
3.
Chen Y Jiang P Liu S Zhao H Cui Y Qin S 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2011,879(13-14):993-997
Zinc-decorated silica-coated magnetic nanoparticles (ZnSiMNPs) were prepared by adsorbing zinc onto colloidal silica. These nanoparticles were used for the rapid purification of 6×His-tagged recombinant phycobiliprotein. The surface changes in the magnetic nanoparticles after zinc adsorption were characterized by transmission electron microscopy. The adsorption of the 6×His-tagged phycobiliprotein onto ZnSiMNPs in 10 mM PBS at 25°C was found to follow the Langmuir isotherm. ZnSiMNPs could be used to extract 6×His-tagged phycobiliprotein from lysate to single-band purity, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. No spectral variation was observed in the purified phycobiliprotein. Thus, ZnSiMNPs served as a useful tool for the magnetic separation and delivery of the 6×His-tagged phycobiliprotein. 相似文献
4.
Chao-Hsun Yang Yu-Chun Huang Cheng-Yu Chen Chia-Ying Wen 《Journal of industrial microbiology & biotechnology》2010,37(9):953-960
A gene encoding the thermostable α-amylase in Thermobifida fusca NTU22 was amplified by PCR, sequenced, and cloned into Yarrowia lipolytica P01g host strain using the vector pYLSC1 allowing constitutive expression and secretion of the protein. Recombinant expression
resulted in high levels of extracellular amylase production, as high as 730 U/l in the Hinton flask culture broth. It is higher
than that observed in P. pastoris expression system and E. coli expression system. The purified amylase showed a single band at about 65 kDa by SDS-polyacrylamide gel electrophoresis and
this agrees with the predicted size based on the nucleotide sequence. About 70% of the original activity remained after heat
treatment at 60°C for 3 h. The optimal pH and temperature of the purified amylase were 7.0 and 60°C, respectively. The purified
amylase exhibited a high level of activity with raw sago starch. After 72-h treatment, the DP
w
of raw sago starch obviously decreased from 830,945 to 237,092. The boiling stable resistant starch content of the sago starch
increased from 8.3 to 18.1%. The starch recovery rate was 71%. 相似文献
5.
Tony Marcio Silva André Ricardo de Lima Damásio Alexandre Maller Michele Michelin Fabio M. Squina João Atílio Jorge Maria de Lourdes Teixeira de Moraes Polizeli 《Folia microbiologica》2013,58(6):495-502
An extracellular amylase secreted by Aspergillus niveus was purified using DEAE fractogel ion exchange chromatography and Sephacryl S-200 gel filtration. The purified protein migrated as a single band in 5 % polyacrylamide gel electrophoresis (PAGE) and 10 % sodium dodecyl sulfate (SDS-PAGE). The enzyme exhibited 4.5 % carbohydrate content, 6.6 isoelectric point, and 60 and 52 kDa molar mass estimated by SDS-PAGE and Bio-Sil-Sec-400 gel filtration column, respectively. The amylase efficiently hydrolyzed glycogen, amylose, and amylopectin. The end-products formed after 24 h of starch hydrolysis, analyzed by thin layer chromatography, were maltose, maltotriose, maltotetraose, and maltopentaose, which classified the studied amylase as an α-amylase. Thermal stability of the α-amylase was improved by covalent immobilization on glyoxyl agarose (half-life of 169 min, at 70 °C). On the other hand, the free α-amylase showed a half-life of 20 min at the same temperature. The optima of pH and temperature were 6.0 and 65 °C for both free and immobilized forms. 相似文献
6.
Park H Park HJ Kim JA Lee SH Kim JH Yoon J Park TH 《Journal of microbiological methods》2011,84(1):41-45
The primary goal of this study was to develop a new strategy to inactivate bacterial biofilms using the thermal stress derived from superparamagnetic iron oxide nanoparticles (SPIONs) in an alternating current (AC) magnetic field. A large number of studies have examined the inactivation of bacterial biofilms using antimicrobial agents; however, there have been no attempts to inactivate biofilms by hyperthermia using SPIONs. In this study, a SPION solution was added to Pseudomonas aeruginosa (P. aeruginosa) PA01 biofilm, and heat was generated by placing the nanoparticle-containing biofilm in an AC magnetic field. The heating temperature was dependent on the concentration of the added SPION solution. More than 4 log inactivation of the PA01 biofilm was obtained using a 60 mg mL−1 SPION solution in 8 min, and this resulted in a dramatic disintegration of the bacterial cell membrane in the biofilm. This inactivation was largely due to the thermal effect. Local heating of a specific area is also possible using this method, and the heating temperature can be easily adjusted by controlling the concentration of the SPION solution. Therefore, hyperthermia using magnetic nanoparticles holds promise as an effective tool for inactivating the bacterial biofilm. 相似文献
7.
《Bioscience, biotechnology, and biochemistry》2013,77(12):2054-2055
An extracellular enzyme that produces di-D-fructofuranose 2′,1;2,1′-dianhydride (difructose anhydride I= DFA I) from inulin was purified from the culture broth of Streptomyces sp. MCI-2524. The purification enhanced the specific activity 7-fold with an overall yield of 17%. The purified enzyme, when electrophoresed on a SDS polyacrylamide gel, gave a single band corresponding to a molecular weight of 36 kDa. Gel filtration chromatography gave a single peak that eluted at a position corresponding to 70 kDa. The enzyme was active from pH 3.0 to pH 9.0, and at temperatures up to 65°C. Maximal activity was observed at pH 6.0, at 55°C. The enzyme was inhibited by Cu2+. 相似文献
8.
Application of superparamagnetic nanoparticles in purification of plasmid DNA from bacterial cells 总被引:1,自引:0,他引:1
Chiang CL Sung CS Wu TF Chen CY Hsu CY 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2005,822(1-2):54-60
The aim of this study was to develop a simple and rapid method for purification of ultrapure supercoiled plasmid DNA with high yields from bacterial cultures. Nanosized superparamagnetic nanoparticles (Fe3O4) were prepared by chemical precipitation method using Fe2+, Fe3+ salt, and ammonium hydroxide under a nitrogen atmosphere. The surface of Fe3O4 nanoparticles was modified by coating with the multivalent cationic agent, polyethylenimine (PEI). The nanoparticles were characterized by transmission electron microscopy, X-ray diffraction, Fourier transformation infrared spectroscopy and superconducting quantum interference device magnetometer. The PEI-modified magnetic nanobeads were employed to simplify the purification of plasmid DNA from bacterial cells. We demonstrated a useful plasmid, pRSETB-EGFP, encoding the green fluorescent protein with T7 promoter, was amplified in DE3 strain of Escherichia coli. The loaded nanobeads are recovered by magnetically driven separation and regenerated by exposure to the elution buffer with optimal ionic strength (1.25 M) and pH (9.0). Up to approximately 35 microg of high-purity (A260/A280 ratio=1.87) plasmid DNA was isolated from 3ml of overnight bacterial culture. EGFP expression was detected by fluorescent microscopy in the transformed E. coli cells, indicating the biological activities of DNA fragments were retained after purified from magnetic nanobeads. The protocol, starting from the preparation of bacterial lysate and ending with purified plasmids takes less than 10 min. Thus, the separation and purification qualities of PEI-modified magnetic nanobeads as well as its ease of use surpass those of conventional anion-exchange resins. 相似文献
9.
Waleed Ahmad Khattak Minkyung Kang Mazhar Ul-Islam Joong Kon Park 《Bioprocess and biosystems engineering》2013,36(6):737-747
A number of hydrolyzing enzymes that are secreted from malt during brewing, including cell wall-hydrolyzing, saccharide-hydrolyzing, protein-degrading, lipid-hydrolyzing, and polyphenol and thiol-hydrolyzing enzymes, are expected to exist in an active form in waste from beer fermentation broth (WBFB). In this study, the existence of these enzymes was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, after which enzyme extract was partially purified through a series of purification steps. The hydrolyzing enzyme activity was then measured under various conditions at each purification step using carboxymethyl cellulose as a substrate. The best hydrolyzing activities of partially purified enzymes were found at pH 4.5 and 50 °C in a citrate buffer system. The enzymes showed highest thermal stability at 30 °C when exposed for prolonged time. As the temperature increased gradually from 25 to 70 °C, yeast cells in the chemically defined medium with enzyme extract lost their cell wall and viability earlier than those without enzyme extract. Cell wall degradation and the release of cell matrix into the culture media at elevated temperature (45–70 °C) in the presence of enzyme extract were monitored through microscopic pictures. Saccharification enzymes from malt were relatively more active in the original WBFB than supernatant and diluted sediments. The presence of hydrolyzing enzymes from malt in WBFB is expected to play a role in bioethanol production using simultaneous saccharification and fermentation without the need for additional enzymes, nutrients, or microbial cells via a cell-free enzyme system. 相似文献
10.
Mohammad Shafiei Abed-Ali Ziaee Mohammad Ali Amoozegar 《Journal of industrial microbiology & biotechnology》2011,38(2):275-281
A halophilic α-amylase produced by Nesterenkonia sp. strain F was purified to homogeneity by 80% ethanol precipitation, Q-Sepharose anion exchange, and Sephacryl S-200 gel
filtration chromatography. The purified amylase exhibited specific activity of 357 unit/mg protein that corresponds to twofold
purification. The molecular mass of the amylase was determined to be 57 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE) and gel filtration chromatography. The optimal pH and temperature for enzyme activity were 6.5 and 45°C, respectively.
The amylase was active over a wide range of salt concentrations (0–4 M) with maximum activity at 0.75–1 M NaCl. The α-amylase
activity was stimulated by Ca2+ and inhibited by ethylenediamine tetraacetic acid (EDTA), suggesting that this enzyme is a metalloenzyme. The purified enzyme
showed remarkable stability towards surfactants (Tween 20, Tween 80, and Triton X-100), and its activity was increased by
β-mercaptoethanol. The halophilic α-amylase was stable in the presence of various organic solvents such as benzene, chloroform,
toluene, and cyclohexane. These properties indicate wide potential applications of this α-amylase in starch-processing industries. 相似文献
11.
Naohiro Yoshigi Takahide Chikano Minora Kamimura 《Bioscience, biotechnology, and biochemistry》2013,77(12):3369-3376
An extracellular amylase was obtained from a culture medium of Bacillus cereus NY-14. This enzyme was purified to show a single band on disc gel electrophoresis by ammonium sulfate fractionation and Sephadex G-100 gel filtration to 1101-fold of the activity of the original culture liquor. The purified enzyme had a molecular weight of 55,000, an isoelectric point of 6.13, an optimum pH of 6.0, and an optimum temperature of 55°C. The pH-stability range was wide; the enzyme retained more than 80% of its initial activity in the range of pH 5.5 to 12. It was stable below 35°C and required calcium ions for the stabilization. The action pattern of this enzyme on amylaceous polysaccharides was unique in that the predominant product was maltopentaose. The purified amylase could also digest starch granules of such plants as rice, barley, corn, and kuzu to produce maltopentaose as the main product. 相似文献
12.
Yasuhide Ota Teruaki Nakamiya Koichi Yamada 《Bioscience, biotechnology, and biochemistry》2013,77(9):1368-1374
The lipase from Candida paralipolytica was purified, as judged by disc electrophoresis. The purification was about 132 fold, based on protein, with a recovery of 32% from the acetone precipitate of the cultivated broth.After purification, modification of the enzyme was performed by dialyzing its solution against 1 m sodium chloride in acetate buffer at room temperature and by separating the modified enzyme from an unknown substance(s) with a Sephadex G–75 column.The optimum pH for lipolysis of the purified lipase was 8.0, while that of the modified one was 7.0. Sodium taurocholate was required essentially by the purified enzyme, but not by the modified one. The purified lipase was stable below 37°C and in the pH range from 3.5 to 9.0 at 5°C. 相似文献
13.
嗜热菌来源的生淀粉酶分离纯化及其酶学性质 总被引:2,自引:0,他引:2
从嗜热菌库中分离到两株能水解生淀粉的菌株173和174,通过扩增和测定两株菌的16S rDNA序列并进行比对结果表明,所分离两株菌属于Geobacillus属的细菌.液体摇瓶发酵菌株173、174,其产生的生淀粉酶(简称RSDE173、RSDE174)活力分别达14.5 U/mL和12.9 U/mL.通过生淀粉吸附-熟淀粉洗脱系统和TOYOPEARL HW-55F系统进行分离纯化,得到纯化的RSDE173和RSDE174,纯化倍数分别为50和29,活力回收率分别为34%和41%.有关RSDE173和RSDE174酶学性质研究显示.对熟淀粉水解的最适作用温度均为70℃,而对生淀粉水解则分别在50℃~60℃和40℃~60℃下表现出高水解活力;对不同底物的最适作用pH值均为5.0~5.5;它们对大多数试验离子的敏感性较低,但个别离子如Co2 、Cu'2 对RSDE173或u'2 对RSDE174的酶活力有一定的抑制作用.纯化的这两种生淀粉酶对不同来源生淀粉的底物专一性并不相同.RSDE173底物专一性顺序为红薯淀粉>小麦淀粉>玉米淀粉>木薯淀粉>糯米淀粉;而RSDE174的糯米淀粉>小麦淀粉>红薯淀粉>玉米淀粉>木薯淀粉.RSDE173对生红薯淀粉有很好的降解,其水解糊化淀粉与生红薯淀粉的比值为1.48;而RSDE174优先降解生糯米淀粉,其相应比值为1.69. 相似文献
14.
S. K. Soni I. K. Sandhu K. S. Bath U. C. Banerjee P. R. Patnaik 《Folia microbiologica》1996,41(3):243-248
A strain of starch-assimilating yeast,Saccharomycopsis capsularis, isolated from Indian cereal-based fermented foods, produced significant levels of extracellular α-amylase and glucoamylase.
The enzymes reached their peak activities during the stationary phase at the end of the 5th and 4th day of cultivation, respectively.
The amylase yields were maximized by a proper choice of carbon and nitrogen sources, starting pH of the culture medium and
growth temperature. High activities of the enzymes were obtained through inexpensive agricultural commodities, such as wheat
bran and corn meal as carbon sources, and defatted soybean meal and peanut meal as nitrogen sources. A temperature of 28–32°C
and an initial pH of 4.5–5.0 were optimum. The crude amylase mixture could liquefy and saccharify a 1% starch solution completely
in 24 h at 50°C. 相似文献
15.
The amylase of Bacillus subtilis NRRL B3411 has been purified and partially characterized. The specific activity can be increased from 300,000 units/g to 6,000,000 units/g with a 60% recovery of total units. The purified material consists of one major and one trace anodic component as determined by disc gel electrophoresis. The molecular weight was 48,000 as determined by bio-gel filtration; the molecular weight was 44,900 ± 2400 as determined by sedimentation equilibrium methods. This purified enzyme is stable at, 70°C in the presence of 0.01 M Ca++ and 0.1 M NaCl over a broad pH range from 5.5–9.5. The pH activity profile indicates optimum activity at pH 6.0. This amylase exhibits maximum activity at 60°C. The enzyme is a liquefying α-amylase as determined by analysis of hydrolysis products and immunological studies. 相似文献
16.
从Bacillus pumilus M-26发酵液中分离纯化碱性木聚糖酶,进行酶学性质研究,同时制备工业用碱性木聚糖酶制剂。首先将M-26发酵液进行硫酸铵盐析,制备工业用碱性木聚糖酶干品;然后进行sephadexG-25层析脱盐和cellulose DE-52层析得以纯化。硫酸铵的饱和度50%,酶制剂的酶活可达9 000 IU/g,收率为85%;分离纯化使酶的比活为126.32 IU/mg蛋白,纯化倍数为19.89,酶的回收率12.83%;分子量约为20 ku;M-26碱性木聚糖酶的最适温度和pH分别是55℃和pH 8.0,具有一定的耐碱性;该酶无纤维素酶活性,Fe2+对其有激活作用;Mn2+、Zn2+、Fe3+、Cu2+对其具有抑制作用。短小芽胞杆菌M-26碱性木聚糖酶具有纸浆生物漂白应用前景。 相似文献
17.
Luís Borlido Leila Moura Ana M. Azevedo Ana C. A. Roque Dr. Maria R. Aires-Barros Dr. José Paulo S. Farinha 《Biotechnology journal》2013,8(6):709-717
Monoclonal antibodies (mAbs) are important therapeutic proteins. One of the challenges facing large-scale production of monoclonal antibodies is the capacity bottleneck in downstream processing, which can be circumvented by using magnetic stimuli-responsive polymer nanoparticles. In this work, stimuli-responsive magnetic particles composed of a magnetic poly(methyl methacrylate) core with a poly(N-isopropylacrylamide-co-acrylic acid) (P(NIPAM-co-AA)) shell cross-linked with N, N'-methylenebisacrylamide were prepared by miniemulsion polymerization. The particles were shown to have an average hydrodynamic diameter of 317 nm at 18°C, which decreased to 277 nm at 41°C due to the collapse of the thermo-responsive shell. The particles were superparamagnetic in behavior and exhibited a saturation magnetization of 12.6 emu/g. Subsequently, we evaluated the potential of these negatively charged stimuli-responsive magnetic particles in the purification of a monoclonal antibody from a diafiltered CHO cell culture supernatant by cation exchange. The adsorption of antibodies onto P(NIPAM-co-AA)-coated nanoparticles was highly selective and allowed for the recovery of approximately 94% of the mAb. Different elution strategies were employed providing highly pure mAb fractions with host cell protein (HCP) removal greater than 98%. By exploring the stimuli-responsive properties of the particles, shorter magnetic separation times were possible without significant differences in product yield and purity. 相似文献
18.
Shin-ichiro Suye 《Current microbiology》1997,34(6):374-377
Alcohol oxidase from Candida methanosorbosa was
purified about sixfold with a yield of 37.6% from the culture broth of
Candida methanosorbosa M-2003. The enzyme preparation was homogeneous
on slab gel electrophoresis. The purified enzyme had an optimal pH from 6.0
to 9.0 and was stable in the range 6.0–8.5. Its optimal temperature of
reaction was 50°C, and it was stable below 50°C. In the presence of
NaN3, the enzyme retained its initial activity at 30°C for 35
days, indicating stability for a long term, so far. The isoelectric point was
pH 4.3. Its molecular weight was 620,000 by gel filtration chromatography and
80,000 by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. These
results indicate that the enzyme consists of 8 subunits.
Received: 1 October 1996 / Accepted: 12 December 1996 相似文献
19.
Yu-Liang Jiao Shu-Jun Wang Ming-Sheng Lv Jin-Li Xu Yao-Wei Fang Shu Liu 《Current microbiology》2011,62(1):222-228
The gene encoding a new extracellular amylopullulanase (type II pullulanase) was cloned from an extremely thermophilic anaerobic
archaeon Thermococcus siculi strain HJ21 isolated previously from a deep-sea hydrothermal vent. The functional hydrolytic domain of the amylopullulanase
(TsiApuN) and its MalE fusion protein (MTsiApuN) were expressed heterologously. The complete amylopullulanase (TsiApu) was
also purified from fermentation broth of the strain. The pullulanase and amylase activities of the three enzymes were characterized.
TsiApu had optimum temperature of 95°C for the both activities, while MTsiApuN and TsiApuN had a higher optimum temperature
of 100°C. The residual total activities of MTsiApuN and TsiApuN were both 89% after incubation at 100°C for 1 h, while that
of TsiApu was 70%. For all the three enzymes the optimum pHs for amylase and pullulanase activities were 5.0 and 6.0, respectively.
By analyzing enzymatic properties of the three enzymes, this study suggests that the carboxy terminal region of TsiApu might
interfere with the thermoactivity. The acidic thermoactive amylopullulanases MTsiApuN and TsiApuN could be further employed
for industrial saccharification of starch. 相似文献
20.
Ashraf F. Elbaz Ahmed Sobhi Ahmed ElMekawy 《Bioprocess and biosystems engineering》2015,38(4):767-776
The discovery of novel bacterial cyclodextrin glucanotransferase (CGTase) enzyme could provide advantages in terms of its production and relative activity. In this study, eight bacterial strains isolated from soils of a biodiversity-rich vegetation in Egypt based on their hydrolyzing activity of starch, were screened for CGTase activity, where the most active strain was identified as Bacillus lehensis. Optimization process revealed that the using of rice starch (25 %) and a mixture of peptone/yeast extract (1 %) at pH 10.5 and 37 °C for 24 h improved the bacterial growth and enzyme activity. The bacterial CGTase was successively purified by acetone precipitation, gel filtration chromatography in a Sephadex G-100 column and ion exchange chromatography in a DEAE-cellulose column. The specific activity of the CGTase was increased approximately 274-fold, from 0.21 U/mg protein in crude broth to 57.7 U/mg protein after applying the DEAE-cellulose column chromatography. SDS-PAGE showed that the purified CGTase was homogeneous with a molecular weight of 74.1 kDa. Characterization of the enzyme exhibited optimum pH and temperature of 7 and 60 °C, respectively. CGTase relative activity was strongly inhibited by Mg2+, Zn2+, Al3+ and K+, while it was slightly enhanced by 5 and 9 % with Cu2+ and Fe2+ metal ions, respectively. 相似文献