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1.
Yoshiki Kumada Tomio Takeuchi Hamao Umezawa 《Bioscience, biotechnology, and biochemistry》2013,77(5):869-876
A dehydrodicaffeic acid dilactone-forming enzyme was purified from the mycelia of a mushroom, Inonotus sp. K-1410 by calcium acetate treatment, ammonium sulfate precipitation and column chromatography on Sephadex G-100, DEAE-Sephadex A-50 and caffeic acid-bound AH-Sepharose 4B. The enzyme was purified about 1200-fold from a crude extract and shown to be almost completely homogeneous by polyacrylamide gel electrophoresis. The molecular weight of this enzyme was estimated by gel filtration on Sephadex G-100 to be approximately 39,000. The optimal pH for the enzymic conversion of caffeic acid to dehydrodicaffeic acid dilactone is around 6.0. The enzyme is stable up to 60°C and preincubation of the enzyme at 40°C for 10 min gives 1.5-fold activation compared with preincubation at 0°C. The optimal temperature for the enzyme reaction is 40°C. 相似文献
2.
Xylaria regalis, a wood-grown ascomycete isolated in Taiwan, produces β-glucosidase (EC 3.2.1.21) extracellularly. The β-glucosidase was
purified to homogeneity by ammonium sulfate precipitation, ion-exchange, and gel filtration chromatography. The molecular
mass of the purified enzyme was estimated to be 85 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. With
p-nitrophenyl β-D-glucopyranoside (PNPG) as the substrate at pH 5.0 and 50°C, the K
m
was 1.72 mM and V
max
was 326 μmol/min/mg. Optimal activity with PNPG as the substrate was at pH 5.0 and 50°C. The enzyme was stable at pH 5.0
at temperatures up to 50°C. The purified β-glucosidase was active against PNPG, cellobiose, sophorose, and gentiobiose, but
did not hydrolyze lactose, sucrose, Avicel, and o-nitrophenyl β-D-galactopyranoside. The activity of β-glucosidase was stimulated by Ca2+, Mg2+, Mn2+, Cd2+ and β-mercaptoethanol, and inhibited by Ag+, Hg2+, SDS, and p-chloromercuribenzoate (PCMB).
Received: 30 March 1996 / Accepted: 3 May 1996 相似文献
3.
Huang HK Tokashiki M Maeno S Onaga S Taira T Ito S 《Journal of industrial microbiology & biotechnology》2012,39(1):55-62
A heat-labile phenolic acid decarboxylase from Candida guilliermondii (an anamorph of Pichia guilliermondii) was purified to homogeneity by simple successive column chromatography within 3 days. The molecular mass was 20 kDa by sodium
dodecyl sulfate–polyacrylamide gel electrophoresis and 36 kDa by gel-filtration chromatography, suggesting that the purified
enzyme is a homodimer. The optimal pH and temperature were approximately 6.0 and 25°C. Characteristically, more than 50% of
the optimal activity was observed at 0°C, suggesting that this enzyme is cold-adapted. The enzyme converted p-coumaric acid, ferulic acid, and caffeic acid to corresponding products with high specific activities of approximately 600,
530, and 46 U/mg, respectively. The activity was stimulated by Mg2+ ions, whereas it was completely inhibited by Fe2+, Ni2+, Cu2+, Hg2+, 4-chloromericuribenzoate, N-bromosuccinimide, and diethyl pyrocarbonate. The enzyme was inducible and expressed inside the cells moderately by ferulic
acid and p-coumaric acid and significantly by non-metabolizable 6-hydroxy-2-naphthoic acid. 相似文献
4.
Tadao Oikawa Toshinori Kamatani Takeshi Kaimura Minoru Ameyama Kenji Soda 《Current microbiology》1997,34(5):309-313
A cellulose-producing acetic acid bacterium,
Acetobacter xylinum KU-1, abundantly produces an extracellular
endo-β-glucanase (EC 3.2.1.4) in the culture broth. The enzyme was purified
to homogeneity by DEAE- and CM- Toyopearl 650M ion-exchange chromatography,
Butyl-Toyopearl 650M hydrophobic chromatography, and Toyopearl HW-50 gel
filtration. The purified enzyme showed the maximum activity at pH 5 and
50°C: it was stable up to 50°C at pH 5, activated by Co2+, and
competitively inhibited by Hg2+; the apparent
K
i
was 7 μM. The molecular weight of the enzyme was
determined to be about 39,000 by sodium dodesyl sulfate/polyacrylamide gel
electrophoresis, and about 41,000 by Toyopearl HW-50 gel filtration; the
enzyme is monomeric. The enzyme hydrolyzed carboxymethylcellulose with an
apparent K
m
of 30 mg/ml and V
max of 1.2
μM/min. It hydrolyzed cellohexaose to cellobiose, cellotriose and
cellotetraose, and also cellopentaose to cellobiose and cellotriose, but did
not act on cellobiose, cellotriose, or cellotetraose.
Received: 3 October 1996 / Accepted: 5 November 1996 相似文献
5.
An intracellular glucoamylase (E.C. 3.2.1.3) was purified
to homogeneity from Lactobacillus amylovorus on a Fast Protein Liquid
Chromatography System (FPLC) with a Mono Q ion-exchanger and two Superose 12
gel filtration columns arranged in series. The enzyme activity was quantified
with a specific, chromogenic substrate, p-nitrophenyl-β-maltoside.
Preparative gel electrophoresis was then used to further purify active enzyme
fractions. Native polyacrylamide gel electrophoresis (Native-PAGE) and sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified
enzyme showed a single protein band of molecular weight 47 kDa. Glucoamylase
activity of the purified protein was confirmed by its ability to degrade
starch on a 0.025% starch-polyacrylamide gel stained with
I2/KI. Glucoamylase exhibited optimum catalytic activity at pH 6.0
and 45°C, and the enzyme had an isoelectric point near 4.39. The
glucoamylase contained high levels of hydrophilic amino acids, comparable to
fungal glucoamylases.
Received: 12 July 1996 / Accepted: 10 September 1996 相似文献
6.
Masaru Sakurada Diego P. Morgavi Kenji Komatani Yoshifumi Tomita Ryoji Onodera 《Current microbiology》1997,35(1):48-51
An autolysis chitinase was purified from the cultural medium of the anaerobic fungus Piromyces communis OTS1 by ammonium sulfate precipitation, affinity chromatography with regenerated chitin, chromato-focusing, gel filtration,
and chromato-focusing again. The optimal pH and temperature were 6.0 and 50°C, respectively, for a 20-min assay. The chitinase
was stable from pH 6.0 to 8.0, but was unstable at 70°C for 20 min. The molecular mass of chitinase was estimated by SDS-PAGE
to be 44.9 kDa, and its pI was 4.4. The enzyme activity, which was of the ‘endo’ type, was inhibited by Hg2+ and allosamidin. The chitinase hydrolyzes chitin powder and fungal cell walls at a higher rate than an artificial chitin
substrate. It can be concluded that extracellular chitinase is similar to cytosolic chitinase, but they are not the same protein.
Received: 3 December 1996 / Accepted: 28 January 1997 相似文献
7.
S. L. Khoo A.-A. Amirul M. Kamaruzaman N. Nazalan M. N. Azizan 《Folia microbiologica》1994,39(5):392-398
Aspergillus flavus produced approximately 50 U/mL of amylolytic activity when grown in liquid medium with raw low-grade tapioca starch as substrate.
Electrophoretic analysis of the culture filtrate showed the presence of only one amylolytic enzyme, identified as an α-amylase
as evidenced by (i) rapid loss of color in iodine-stained starch and (ii) production of a mixture of glucose, maltose, maltotriose and maltotetraose as starch digestion products. The enzyme was
purified by ammonium sulfate precipitation and ion-exchange chromatography and was found to be homogeneous on sodium dodecyl
sulfate— polyacrylamide gel electrophoresis. The purified enzyme had a molar mass of 52.5±2.5 kDa with an isoelectric point
at pH 3.5. The enzyme was found to have maximum activity at pH 6.0 and was stable in a pH range from 5.0 to 8.5. The optimum
temperature for the enzyme was 55°C and it was stable for 1 h up to 50°C. TheK
m andV for gelatinized tapioca starch were 0.5 g/L and 108.67 μmol reducing sugars per mg protein per min, respectively. 相似文献
8.
Hiroshi Shinmoto Hiroki Murakami Shun'ichi Dosako Koji Yamada Hirohisa Omura 《Bioscience, biotechnology, and biochemistry》2013,77(10):2653-2654
The amylomaltase from Escherichia coli IFO 3806 was purified to homogeneity seen by SDS- polyacrylamide gel electrophoresis after DEAE-Sephadex, Ultrogel AcA 44, hydroxylapatite, and 1,6- hexane-diamine-Sepharose 4B column chromatographies. The molecular weight of the purified enzyme was 93,000 by SDS-polyacrylamide gel electrophoresis. The enzyme was most active at pH 6.5 and at 35°C, and stable up to 45°C at pH 7.0 and from pH 6.0 —7.3 at 40°C on 30min incubation. The enzyme acted on maltotetraitol, maltopentaitol, and maltosylsucrose besides maltooligosaccharides, but did not act on maltitol, maltotriitol, glucosylsucrose, isomaltose, panose, isopanose, or isomaltosyl- maltose. This enzyme did not catalyze hydrolytic action on maltotetraitol, maltopentaitol, or maltosylsucrose. 相似文献
9.
Jia Ouyang Shen Wang Yan Wang Xin Li Mu Chen Qiang Yong Shiyuan Yu 《World journal of microbiology & biotechnology》2011,27(4):751-758
The purpose of this study was to produce a Trichoderma reesei xylanase (XYN2) in Pichia pastoris and to test its potential application for pulp bleaching. The recombinant xylanase was purified by a two-step process of
ultrafiltration and gel filtration chromatography. The molecular mass of the recombinant enzyme was 21 and 25 kDa by SDS–PAGE
analysis, due to different glycosylation of the native protein. The optimum pH and temperature of the recombinant XYN2 was
5.0 and 50 °C. Enzyme activity was stable at 50 °C and at pH 5.0–7.0. The bleaching ability of the recombinant xylanase was
also studied at 50 °C and pH 6.0, using wheat straw pulp. Biobleaching of the xylanase produced chlorine dioxide savings of
up to 60%, while retaining brightness at the control level and led to a lower kappa number and small enhancements in tensile,
burst and tear strength of pulp fibers. 相似文献
10.
An extracellular polygalacturonase was isolated from 5-day culture filtrates of Thermoascus aurantiacus CBMAI-756 and purified by gel filtration and ion-exchange chromatography. The enzyme was maximally active at pH 5.5 and 60–65°C.
The apparent K
m with citrus pectin was 1.46 mg/ml and the V
max was 2433.3 μmol/min/mg. The apparent molecular weight of the enzyme was 30 kDa. The enzyme was 100% stable at 50°C for 1 h
and showed a half-life of 10 min at 60°C. Polygalacturonase was stable at pH 5.0–5.5 and maintained 33% of initial activity
at pH 9.0. Metal ions, such as Zn+2, Mn+2, and Hg+2, inhibited 50, 75 and 100% of enzyme activity. The purified polygalacturonase was shown to be an endo/exo-enzyme, releasing
mono, di and tri-galacturonic acids within 10 min of hydrolysis. 相似文献
11.
Purification was conducted on polyvinyl alcohol (PVA) degrading enzyme produced and secreted into the culture medium by Pseudomonas O–3 strain. The enzyme was found to separate into several fractions by ion-exchange chromatography and gel filtration. Among these fractions, a fraction adsorbed to SP-Sephadex C–50 at pH 6.0 was purified to homogeneity by polyacrylamide gel electrophoresis. Some properties of this purified enzyme were examined. Optimum pH and temperature were 9.0 and 40°C, respectively. The enzyme was stable up to 50°C and in a pH range between 5 and 11 at 5°C. The enzyme activity was inhibited by Co2+, Ni2+, EDTA, hydroxylamine and salicylaldoxime. In substrate specificity, this enzyme oxidized several kinds of modified PVA, as well as normal PVA, but did not oxidize other synthetic polymers, such as vinylon, polyacrylamide and polyvinyl acetate. The effect of oxygen on this enzyme was examined, and without oxygen, PVA was not broken down by this enzyme. The molecular weight of this enzyme was estimated by gel filtration on Sephadex G–100 to be approximately 26,000. 相似文献
12.
Alkaline protease production by a newly isolated Bacillus species from laundry soil was studied for detergent biocompatibility. From its morphological and nucleotide sequence (about
1.5 kb) of its 16S rDNA it was identified as Bacillus species with similarity to Bacillus species Y (Gen Bank entry: ABO 55095), and close homology with Bacillus cohnii YN-2000 (Gen Bank entry: ABO23412). Partial purification of the enzyme by ammonium sulfate (50–70% saturation) yielded 8-fold
purity. Casein zymography and Sodium dodecylsulphate-Polyacrylamide gel electrophoresis (SDS-PAGE) of the partially purified
enzyme revealed two isozymes of molecular sizes approximately 66 kDa and 18 kDa, respectively. The enzyme was most active
at pH 12 and 50°C. At pH 12 the enzyme was stable for 5 h and retained 60% activity. The enzyme retained 44% activity at 50°C
up to 2 h. The protease showed good hydrolysis specificity with different substrates tested. The presence of Mn2+, Co2+ and ethylenediaminetetracetic acid (EDTA) showed profound increase in protease activity. The protease of Bacillus species Y showed excellent stability and compatibility with three locally available detergents (Kite, Tide and Aerial) up
to 3 h retaining almost 70–80% activity and 10–20% activity at room temperature (30°C) and 50°C, respectively, indicating
the potential role of this enzyme for detergent application. 相似文献
13.
Lu L Zhao M Zhang BB Yu SY Bian XJ Wang W Wang Y 《Applied microbiology and biotechnology》2007,74(6):1232-1239
The white rot fungus Pycnoporus sanguineus produced high amount of laccase in the basal liquid medium without induction. Laccase was purified using ultrafiltration,
anion-exchange chromatography, and gel filtration. The molecular weight of the purified laccase was estimated as 61.4 kDa
by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme oxidized typical substrates of laccases including
2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonate), 2,6-dimethoxyphenol, and syringaldazine. The optimum pH and temperature for the purified
laccase were 3.0 and 65°C, respectively. The enzyme was stable up to 40°C, and high laccase activity was maintained at pH 2.0–5.0.
Sodium azide, l-cysteine, and dithiothreitol strongly inhibited the laccase activity. The purified enzyme efficiently decolorized Remazol
Brilliant Blue R in the absence of added redox mediators. The high production of P. sanguineus laccase as well as its decolorization ability demonstrated its potential applications in dye decolorization. 相似文献
14.
《Bioscience, biotechnology, and biochemistry》2013,77(9):1401-1405
An enzyme hydrolyzing nigeran (alternating α-l,3-and α-l,4-linked glucan) was purified from the culture filtrate of Streptomyces sp. J-13-3, which lysed the cell wall of Aspergillus niger, by precipitation with ammonium sulfate and column chromatographies on DEAE-Sephadex A-50, CM-Sephadex C-50, chromatofocusing, and Sephadex G-I00. The final preparation was homogenous in polyacrylamide gel electrophoresis (PAGE). The molecular weight of the enzyme was 68,000 by SDS–PAGE and gel filtration. The optimum pH and temperature for the enzyme activity were 6.0 and 50°C, respectively. The enzyme was stable in the pH range from 6.0 to 8.0 and up to 50°C. The enzyme activity was inhibited significantly by Hg+, Hg2+, and p-chloromercuribenzoic acid. The Km (mg/ml) for nigeran was 3.33. The enzyme specifically hydrolyzed nigeran into nigerose and nigeran tetrasaccharide by an endo-type of action, indicating it to be a mycodextranase (EC 3.2.1.61) that splits only the α-l,4-glucosidic linkages in nigeran. 相似文献
15.
Paecilomyces variotii IRI017 was isolated as a formaldehyde-resistant fungus from wastewater containing formaldehyde. The fungus grew in a medium
containing 0.5% formaldehyde and had consumed formaldehyde completely after 5 days. Alcohol oxidase was purified from the
fungus grown on methanol. A 20-fold purification was achieved with a yield of 44%. The molecular mass of the purified enzyme
was estimated to be 73 and 450 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography,
respectively, suggesting that the enzyme consists of six identical subunits. The N-terminal amino acid sequence of the subunit
was TIPDEVDIII. The enzyme showed an absorption spectrum typical of a flavoprotein and had a noncovalently bound flavin different
from FAD, FMN, and riboflavin. The pH optimum of the enzyme activity was pH 6–10. The enzyme was stable in the pH range of
pH 5–10. The enzyme retained full activity after incubation at 50°C for 30 min. The enzyme oxidized not only methanol but
also lower primary alcohols and formaldehyde. The K
m values for methanol, ethanol, and formaldehyde were 1.9, 3.8, and 4.9 mmol l−1, respectively. 相似文献
16.
The dermatophyte Trichophyton mentagrophytes var. erinacei isolated from patients infected with tinea cruris was cultured in Sabouraud dextrose broth, from which an exocellular kenitinase
extract was obtained. The keratinase was partially purified with sephadix G-100 gel filtration. Some biochemical characteristics
of the purified enzyme were examined. Its molecular weight was estimated to be 38000 dalton on sodium dodecyle sulfate polyacrylmide
gel electrophoresis (SDS-PAGE). The optimal pH was 5.5 and optimal temperature for the highest keratinase activity was 50°C.
The enzyme activity was specifically increased against guinea pig hair and fibrous protein and inhibited by phenylmethylsulfonylfloride.
This revised version was published online in June 2006 with corrections to the Cover Date. 相似文献
17.
Dodia MS Rawal CM Bhimani HG Joshi RH Khare SK Singh SP 《Journal of industrial microbiology & biotechnology》2008,35(2):121-131
An alkaline protease secreting Haloalkaliphilic bacterium (Gene bank accession number EU118361) was isolated from the Saurashtra
Coast in Western India. The alkaline protease was purified by a single step chromatography on phenyl sepharose 6 FF with 28%
yield. The molecular mass was 40 kDa as judged by SDS-PAGE. The enzyme displayed catalysis and stability over pH 8–13, optimally
at 9–11. It was stable with 0–4 M NaCl and required 150 mM NaCl for optimum catalysis at 37 °C; however, the salt requirement
for optimal catalysis increased with temperature. While crude enzyme was active at 25–80 °C (optimum at 50 °C), the purified
enzyme had temperature optimum at 37 °C, which shifted to 80 °C in the presence of 2 M NaCl. The NaCl not only shifted the
temperature profile but also enhanced the substrate affinity of the enzyme as reflected by the increase in the catalytic constant
(K
cat). The enzyme was also calcium dependent and with 2 mM Ca+2, the activity reached to maximum at 50 °C. The crude enzyme was highly thermostable (37–90 °C); however, the purified enzyme
lost its stability above 50 °C and its half life was enhanced by 30 and sevenfold at 60 °C with 1 M NaCl and 50 mM Ca+2, respectively. The activity of the enzyme was inhibited by PMSF, indicating its serine type. While the activity was slightly
enhanced by Tween-80 (0.2%) and Triton X-100 (0.05%), it marginally decreased with SDS. In addition, the enzyme was highly
stable with oxidizing-reducing agents and commercial detergents and was affected by metal ions to varying extent. The study
assumes significance due to the enzyme stability under the dual extremities of pH and salt coupled with moderate thermal tolerance.
Besides, the facts emerged on the enzyme stability would add to the limited information on this enzyme from Haloalkaliphilic
bacteria. 相似文献
18.
In vitro transgenic hairy root cultures provide a rapid system for physiological, biochemical studies and screening of plants
for their phytoremediation potential. The hairy root cultures of Brassica juncea L. showed 92% decolorization of Methyl orange within 4 days. Out of the different redox mediators that were used to achieve
enhanced decolorization, 2, 2′-Azinobis, 3-ethylbenzothiazoline-6-sulfonic acid (ABTS) was found to be the most efficient.
Laccase activity of 4.5 U mg−1 of protein was observed in hairy root cultures of Brassica juncea L., after the decolorization of Methyl orange. Intracellular laccase produced by B. juncea root cultures grown in MS basal medium was purified up to 2.0 fold with 6.62 U mg−1 specific activity using anion-exchange chromatography. Molecular weight of the purified laccase was estimated to be 148 kDa
by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purified enzyme efficiently oxidized ABTS which was also
required for oxidation of the other tested substrates. The pH and temperature optimum for laccase activity were 4.0 and 40°C,
respectively. The purified enzyme was stable up to 50°C and was stable in the pH range of 4.0–6.0. Laccase activity was strongly
inhibited by sodium azide, EDTA, dithiothreitol and l-cysteine. The purified enzyme decolorized various textile dyes in the presence of ABTS as an efficient redox mediator. These
findings contribute to a better understanding of the enzymatic process involved in phytoremediation of textile dyes by using
hairy roots. 相似文献
19.
Akira Endo 《Bioscience, biotechnology, and biochemistry》2013,77(9):639-645
Exopolygalacturonase from Coniothyrium diplodiella has been purified by ammonium sulfate fractionation, chromatography on DEAE-cellulose and column zone electrophoresis. The enzyme was concentrated about 5-fold with a yield of 0.24% on the basis of polygalacturonase activity per weight of total nitrogen. The purified enzyme was homogenous On free-boundary electrophoresis. The enzyme was most active in the pH range 4.0~4.5. The enzyme was stable at 50°C and pH range of 3.5~6.0, but inactivated at higher than 55°C. Hydrolysis of pectic acid by the enzyme went to completion via galacturonic acid liberation from the end of the chain, but pectin was little affected by the enzyme. 相似文献
20.
Erkang Yin Yilin Le Jianjun Pei Weilan Shao Qiyin Yang 《World journal of microbiology & biotechnology》2008,24(2):275-280
According to the amino acid sequence, a codon-optimized xylanase gene (xynA1) from Thermomyces lanuginosus DSM 5826 was synthesized to construct the expression vector pHsh-xynA1. After optimization of the mRNA secondary structure in the translational initiation region of pHsh-xynA1, free energy of the 70 nt was changed from −6.56 to −4.96 cal/mol, and the spacing between AUG and the Shine-Dalgarno sequence
was decreased from 15 to 8 nt. The expression level was increased from 1.3 to 13% of total cell protein. A maximum xylanase
activity of 47.1 U/mL was obtained from cellular extract. The recombinant enzyme was purified 21.5-fold from the cellular
extract of Escherichia coli by heat treatment, DEAE-Sepharose FF column and t-Butyl-HIC column. The optimal temperature and pH were 65 °C and pH 6.0,
respectively. The purified enzyme was stable for 30 min over the pH range of 5.0–8.0 at 60 °C, and had a half-life of 3 h
at 65 °C. 相似文献