共查询到20条相似文献,搜索用时 46 毫秒
1.
Mona A. Esawy Yannick Combet-Blanc 《World journal of microbiology & biotechnology》2006,22(3):197-200
Milk-clotting enzyme from Bacillus licheniformis 5A1 was immobilized on Amberlite IR-120 by ionic binding. Almost all the enzyme activity was retained on the support. The
immobilized milk-clotting enzyme was repeatedly used to produce cheese in a batch reactor. The production of cheese was repeated
5 times with no loss of activity. The specific activity calculated on a bound-protein basis was slightly higher than that
of free enzyme. The free and immobilized enzyme were highly tolerant to repeated freezing and thawing. The optimum temperature
for milk-clotting activity was 70 °C with the free enzyme whereas, it was ranged from 70 to 80 °C with the immobilized milk-clotting
enzyme. The activation energy (E
A) of the immobilized milk-clotting enzyme was lower than the free enzyme (E
A = 1.59 and 1.99 Kcal mol−1 respectively). The immobilized milk-clotting enzyme exhibited great thermal stability. The milk-clotting optimum pH was 7.0
for both free and immobilized enzyme. The Michaelis constant K
m of the immobilized milk-clotting enzyme was slightly lower than the free enzyme. 相似文献
2.
《Bioscience, biotechnology, and biochemistry》2013,77(11):1705-1710
Chlorogenic acid oxidase was extensively purified to homogeneity from apple flesh (Malus pumila cv. Fuji). The enzyme was purified 470-fold, with a total yield close to 70% from the plastid fraction by ammonium sulfate precipitation, gel filtration and ion-exchange chromatography. The molecular weight was determined to be 65,000 by both SDS-PAGE and gel filtration chromatography. The optimum pH for the enzyme activity was around 4.0, and the enzyme was stable in the range of pH 6–8. The pI obtained by isoelectrofocusing was 5.4, and the N-terminal amino acid sequence was N-Asp-Pro-Leu-Ala-Pro-Pro-. The reaction rate of the purified enzyme was much larger for chlorogenic acid than for other o-diphenols such as (+)-catechin, (?)-epicatechin and 4-methylcatechol, and the enzyme lacked both cresolase activity and p-diphenol oxidase activity. The Km value for the enzyme was found to be 122μM toward chlorogenic acid. The purified enzyme had far less thermal stability than the enzyme of the plastid fraction. Diethyl-dithiocarbamate, sodium azide, o-phenanthroline and sodium fluoride markedly inhibited the enzyme activity. 相似文献
3.
SYNOPSIS Dihydrofolate reductase (E.C. 1.5.1.3) from Plasmodium falciparum and from its host, the owl monkey (Aotus trivirgatus). were partially purified and characterized. The molecular weight of the parasite enzyme was estimated to be over 10 times as high as that of the host enzyme. The host enzyme had 2 pH optima whereas the parasite enzyme only one. The activity of the host enzyme was greatly stimulated by KCI and urea, while that of the parasite enzyme was inhibited at high concentrations of such chaotropic agents. Km of the parasite enzyme was significantly higher than that of the host enzyme. The parasite enzyme had much lower Ki for pyrimethamine than the host enzyme. Dihydrofolate reductases isolated from pyrimethamine-resistant and pyrimethaminesensitive strains of P. falciparum were found to be similar. 相似文献
4.
Tokuko Haraguchi Keiko Abe Soichi Arai Seiichi Homma Masao Fujimaki 《Bioscience, biotechnology, and biochemistry》2013,77(8):1951-1952
The production of pectolytic enzyme in the genus Kluyveromyces was investigated. The production of the enzyme was dependent on the strain, and some strains belonging to K. fragilis, K. Marxianus, and K. Wickerhamii produced this enzyme among 11 species (29 strains) of the genus Kluyveromyces. K. Fragilis IFO 0288 produced at least four endo-polygalacturonases which have different molecular weights. The dominant endo-polygalacturonase in the culture filtrate of the strain was purified and isolated as crystals. The purified enzyme was homogeneous based on analysis by polyacrylamide gel electrophoresis and ultracentrifugation. The enzyme was a glycoprotein having an isoelectric point around pH 5.6. The sedimentation coefficient (s2o,w) was 3.77S, and the molecular weight was around 33,000. The enzyme contained aspartic acid (asparagine), serine,threonine, and glycine at relatively high levels. The enzyme showed the highest activity around pH 5.0 and was stable at pH 5.0 up to 30°C. With the enzyme, and activity which releases highly polymerized pectin from various protopectins (protopectinase activity) was found. 相似文献
5.
Takemitsu Mizunaga Tadahiro Oshida Akiko Takasaki Yoshiharu Maruyama 《Bioscience, biotechnology, and biochemistry》2013,77(6):1445-1450
To find the function of the mannan chains covalently attached to yeast repressible acid phosphatase, the N-glycosidic carbohydrate chains were removed by endo-β-N-acetyl-glucosaminidase H under native conditions. Almost all of the N-glycosidic mannan chains were cleaved off by the glycosidase. The deglycosylated enzyme was shown to be a dimer structure as is the native enzyme. The deglycosylated enzyme retained enzyme activity, the same Km, and the same circular dichroism spectra as the native enzyme. These results indicate that the carbohydrate chains are not essential for maintaining the active enzyme structure, but the deglycosylated enzyme was shown to be more sensitive to acidic pH and high temperature. 相似文献
6.
Fusako Kawai Hideaki Yamada Koichi Ogata 《Bioscience, biotechnology, and biochemistry》2013,77(2):184-190
The enzyme activity to synthesize pyridoxine glucoside was demonstrated in intact cells and cell extracts of genera, Sarcina and Micrococcus. The isolated and identified strain, Micrococcus sp. No. 431 was found to have high activity of this enzyme in its cell extract.The enzyme activity reached to a maximum after 20 hr of cultivation.The enzyme which synthesized pyridoxine glucoside via transglucosidation from sucrose to pyridoxine was purified from Micrococcus sp. No. 431 by means of ammonium sulfate fractionation, DEAE-Sephadex, hydroxylapatite and Sephadex G–100 column chromatographies. The enzyme was purified about 354–fold and confirmed to be homogenous in polyacrylamide-gel electrophoresis and ultracentrifugation. 相似文献
7.
Ko Sawai Toshikatsu Okuno Fumihito Seito Takashi Fujita 《Bioscience, biotechnology, and biochemistry》2013,77(12):3151-3152
Two enzymes, nitrile hydratase and amidase, which participate in the conversion of trans-1,4- dicyanocyclohexane (t-DCC) to frans-4-cyanocyclohexane-l-carboxylic acid (t-MCC), a tranexamic acid intermediate, were purified and characterized. Nitrile hydratase was obtained in a homogeneous state. The molecular weight of the native enzyme was 61,400 and that of the subunit 26.900, indicating a dimer structure. Valeronitrile and butyronitrile were good substrates for the enzyme. The enzyme could also hydrate benzonitrile, p-hydroxybenzonitrile and 4-cyanobenzoic acid. t-DCC was ex-clusively hydrated to fnzws-4-cyanocycIohexane-l-car boxy amide (t-MCMA), further hydration of the nitrile group of t-MCMA and t-MCC not being observed. The presence of pyrroloquinoline quinone in the enzyme was confirmed. The presence of iron was also confirmed. The amidase of the strain was also purified. The latter enzyme could hydrate t-MCMA, yielding t-MCC. The enzyme was highly resistant to SH reagents. 相似文献
8.
Osamu Yamada Yoji Kaise Fumio Futatsuya Shuichi Ishida Kensaku Ito Hiroshi Yamamoto 《Bioscience, biotechnology, and biochemistry》2013,77(11):2013-2015
A new enzyme, 2-methylisocitrate dehydratase, isolated from Yallowia lipolytica, functioning in the methylcitric acid cycle for propionate metabolism, had a pI of 4.4 and a Mr of 69,500. The enzyme was composed of 624 residues of amino acids per molecule. No cofactor was required for full enzyme activity. The enzyme was competitively inhibited by threo-Ds-isocitrate (Ki =68mM), but not by any other tested metabolites. The enzyme was weakly inhibited by some thiol reagents, but not by any metal-chelating reagents, differing from aconitase, which dehydrates 2-methylisocitrate. This difference between the enzymes made it possible to estimate the activity of the new enzyme even in crude cell-free extracts. The enzyme was constitutively synthesized, but had no regulatory function in the methylcitric acid cycle. The enzyme was supposed to have evolutionarily developed from a hypothetical and prototypical isocitrate dehydratase. 相似文献
9.
Lei Kai Xiao-Hang Ma Xue-Lai Zhou Xiao-Ming Jia Xia Li Kang-Ping Guo 《World journal of microbiology & biotechnology》2008,24(3):401-406
In order to study the properties of a thermostable uricase produced by Microbacterium sp. strain ZZJ4-1, the enzyme was purified by ammonium sulfate precipitation and DEAE-cellulose ion exchange, hydrophobic
and molecular sieve chromatography. The molecular mass of the purified enzyme was estimated to be 34 kDa by SDS-PAGE. The
enzyme was stable between pH 7.0 and 10.00. The optimal reaction temperature of the enzyme was 30 °C at pH 8.5. The K
m
and K
cat
of the enzyme were 0.31 mM and 3.01 s−1, respectively. Fe3+ could enhance the enzyme activity, whereas Ag+, Hg2+, o-phenanthroline and SDS inhibited the activity of the enzyme considerably. After purification, the enzyme was purified 19.7-fold
with 31% yield. As compared with uricases from other microbial sources, the purified enzyme showed excellent thermostability
and other unique characteristics. The results of this work showed that strains of Microbacterium could be candidates for the production of a thermostable uricase, which has the potential clinical application in measurement
of uric acid. 相似文献
10.
Eiko Matsumura Eiko Yamamoto Tatsu Kawano Takashi Shin Sawao Murao 《Bioscience, biotechnology, and biochemistry》2013,77(6):1563-1571
A novel enzyme, which was named Nα-benzyloxycarbonyl amino acid urethane hydrolase II, was purified from a cell-free extract of Lactobacillus fermenti 36 ATCC 9338. The enzyme catalyzed the stoichiometric hydrolysis of Nα-benzyloxycarbonyl arginine to form benzyl alcohol and arginine. The enzyme was purified 106-fold with an activity yield of 3%. The purified enzyme was homogeneous by disc gel electrophoresis. The molecular weight of the native enzyme is about 200,000 by gel filtration, and a molecular weight of 27,000 was found for the reduced and denaturated enzyme by gel electrophoresis in sodium dodecyl sulfate. The isoelectric point of the enzyme was 5.0, it was inhibited by disodium ethylenediamine tetraacetate and p-chloromercuribenzoate, and the presence of a divalent cation, i.e. Co2+, is essential for its activity. 相似文献
11.
A thiosulfate-oxidizing enzyme was partially purified fromChromatium vinosum, and some of its properties were studied. The enzyme rapidly reducede HiPIP (high-potential nonheme iron protein) in the
presence of thiosulfate. Cytochromesc of yeast and tuna and ferricyanide also acted well as electron acceptors for the enzyme; horse cytochromec was a poor electron acceptor. Cytochromec-552, cytochromec′, and cytochromec-553 did not act as electron acceptors. The enzyme was inhibited by cyanide and sulfite. On the basis of the stoichiometry
in reduction of ferricyanide catalyzed by the enzyme in the presence of thiosulfate, the oxidized product of thiosulfate was
inferred to be tetrathionate. 相似文献
12.
Purification and biochemical characterization of a fibrinolytic enzyme from Bacillus subtilis BK-17 总被引:1,自引:0,他引:1
Jeong Yong Kee Park Jeong Uck Baek Hyun Park Sung Hoon Kong In Soo Kim Dong Wan Joo Woo Hong 《World journal of microbiology & biotechnology》2001,17(1):89-92
A fibrinolytic enzyme from Bacillus subtilis BK-17 has been purified to homogeneity by gel-filtration and ion-exchange chromatography. Compared to the crude enzyme extract, the specific activity of the enzyme increased 929-fold with a recovery of 29%. The subunit molecular mass of the purified enzyme was estimated to be 31 kDa by SDS–PAGE. The N-terminal amino acid sequence of the purified fibrinolytic enzyme was: A-Q-S-V-P-Y-G-V-S-Q-I-K-A-P-A-A-H-N. The sequence was highly homologous to the fibrinolytic enzymes nattokinase, subtilisin J and subtilisin E from Bacillus spp. However, there was a substitution of three amino acid residues in the N-terminal sequence. The amidolytic activity of the purified enzyme for several substrates was assessed. In comparison with nattokinase and CK (fibrinolytic enzyme from a Bacillus spp.), which showed strong fibrinolytic activity, the amidolytic activity of the enzyme for the synthetic substrate, kallikrein (H-D-Val-Leu-Arg-pNA, S-2266) increased 2.4- and 11.8-fold, respectively. 相似文献
13.
Takamitsu Yorifuji Ichiro Sugai Hideki Matsumoto Akira Tabuchi 《Bioscience, biotechnology, and biochemistry》2013,77(5):1361-1367
3-Guanidinopropionate amidinohydrolase, a new enzyme (EC class 3.5.3), was purified 220-fold from Pseudomonas aeruginosa PAO 1 grown on 3-guanidinopropionate. The enzyme was found to be essentially homogeneous on polyacrylamide gel electrophoresis. The molecular weight of the native enzyme was estimated to be 195,000-215,000. The subunit molecular weight was estimated to be 36,000. The optimal pH was 9.0. The Km value for 3-guanidinopropionate was 45 mm. Incubation of the enzyme with EDTA in potassium phosphate buffer, pH 7.0, at 40°C resulted in almost complete inactivation, and the inactive enzyme was specifically reactivated by Mn2+. Taurocyamine (11%) and 4-guanidinobutyrate (3%) were hydrolyzed as fast as 3-guanidinopropionate at the relative rates indicated. The enzyme was inactivated by p-chloromercuribenzoic acid and the inactive enzyme was reactivated by incubation with 2-mercaptoethanol. Coelectrophoresis of the enzyme with 4-guanidinobutyrate amidinohydrolase purified from Pseudomonas sp. ATCC 14676 in polyacrylamide gels in the presence and absence of sodium dodecyl sulfate demonstrated their close mobilities. 4-Aminobutyrate, propionate, and n-butyrate were common competitive inhibitors of these enzymes. The evolutionary relationship between the two enzymes was discussed. 相似文献
14.
Shinjiro Iwasaki Tsuneo Yasui Gakuzo Tamura Kei Arima 《Bioscience, biotechnology, and biochemistry》2013,77(12):1421-1433
The purification of the milk clotting enzyme from Mucor pusillus Lindt could be achieved by column chromatography on Amberlite IRC-50 by raising pH from 3.5 to 4.5 and about 70% of activity was recovered after this treatment. After the treatment through the column of DEAE-Sephadex A-25, the trace cellulase activity could be eliminated.The homogeneity of the purified preparation was proved by ultracentrifugal analysis and electrophoretic patterns at various pH values.Isoelectric point of this enzyme is considered to lie between pH 3.5 and 3.8.The enzyme activity was inhibited by Hg++ or Fe+++.Trypsinogenkinase activity was not contained in this enzyme.The antiserum against the milk clotting enzyme from Mucor pusillus reacted with the purified and crude enzyme preparations in precipitin test and inhibited their enzyme activities, but did not react with other enzymes such as rennin, pepsin, acid proteases from Aspergillus saitoi and Aspergillus oryzae, or the culture filtrates of some strains of Mucor and Rhizopus.The antigen-antibody reaction was so specific that it might be possible with this antibody to identify this enzyme and also the strain itself.Normal sera from some mammals inhibited this enzyme activity too, but the degree was less than that with rennin. 相似文献
15.
Mahmood Vessal Seyed Mohammad Bagher Tabei 《Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology》1996,113(4):757-763
Cytoplasmic malate dehydrogenase from ovine liver Echinococcus granulosus protoscolices was purified 22-fold by QAE- and SP-Sephadex chromatography. The pH optimum of the enzyme was 8.0 in either Tris-HCl or barbital buffer. The κm values of oxaloacetate and NADH were 0.400 ± 0.018 and 0.410 ± 0.038 mM, respectively. The enzyme lost about 90% of its activity when heated for 2 min at 65°C. A 61.4% inhibition of the enzyme was noted at 4 mM concentration of diethyl pyrocarbonate. A 3 mM concentration of fructose 1,6-diphosphate inhibited the enzyme by 76.5%. The inhibition was non-competitive with respect to NADH with a κi value of 0.85 mM. A 75% inhibition of the enzyme was noted at 1 mM concentration of mebendazole that inhibited the enzyme upon competing with NADH with a κi value of 0.176 mM. A 2-mM concentration of citrate almost doubled the enzyme activity. The enzyme was inhibited at high concentrations of either substrate. The enzyme was not inhibited by p-hydroxymercuribenzoate or fumarate. The enzyme was absolutely specific for NADH as a cofactor. The properties of this enzyme are compared with those of the enzyme from the host liver, the cyst fluid and some other animal sources. The results are discussed in terms of the differences among the properties of the host liver, the cyst fluid and the protoscolices enzymes. The biochemical basis for the use of mebendazole in the treatment of echinococcosis is also elucidated. 相似文献
16.
Gunki Funatsu Shun Miyauchi Masaru Funatsu 《Bioscience, biotechnology, and biochemistry》2013,77(3):639-640
A maltotetraose-forming amylase from Pseudomonas stutzeri was highly purified by adsorption on starch granules and by chromatographies on Sephadex G-100 and DEAE-cellulose. The purified enzyme showed a single band in polyacrylamide gel electrophoreses with or without sodium dodecylsulfate. The optimum pH for enzyme action on starch was 6.0-6.5, and the optimum temperature was 45°C. The purified enzyme attacked starch from the non-reducing end to produce α-anomer oligosaccharides. This indicated that the enzyme was an exo-α-amylase which had not hitherto been found. The enzyme activity was markedly inhibited by the addition of Cu2+, Hg2+, N-bromosuccinimide and 2,3-butanedione. The molecular weight of the enzyme determined by the method of Weber and Osborn was about 5.7 × 104. The isoelectric point of the enzyme was estimated to be 5.3 by polyacrylamide gel electrofocusing. The Km and k0 values of this enzyme for starch, glycogen, short chain amylose and some maltooligosaccharides were calculated from Lineweaver-Burk plots. 相似文献
17.
Miyagawa Eiji Junko Yano Toshinari Hamakado Yasuji Kido Keiji Nishimoto Yoshinobu Motoki 《Bioscience, biotechnology, and biochemistry》2013,77(10):2881-2886
N-Benzoylgiycine amidohydrolase (hippurate hydrolase EC 3.5.1.32), which catalyzes the hydrolysis of hippuric acid to benzoic acid and glycine, was found in a cell-free extract of Pseudomonas putida C692-3 grown on a medium containing hippuric acid. The enzyme was purified from the extract by ammonium sulfate fractionation and column chromatographies on DEAE-cellulose, DEAE-Sephadex A-50, hydroxyapatite, and Sepharose CL-6B. The enzyme was finally crystallized. The crystalline enzyme was almost homogeneous on electrophoresis. The enzyme had a molecular weight of about 170,000 and consisted of four subunits identical in molecular weight (approximately 42,000). The enzyme hydrolyzed N-benzoylglycine most rapidly, and N-benzoyl-l-alanine and N-benzoyl-l-aminobutyric acid. The Km value for these substrates were 0.72 mm, 0.87 mm, and 0.87mm, respectively. The optimum pH of the enzyme reaction was 7.0 to 8.0 and the enzyme was stable from pH 6.0 to 8.0. 相似文献
18.
The purpose of this study was to characterize the physical properties of cytochromec oxidase from rat liver. The enzyme was extracted from isolated mitochondria with nonionic detergents and further purified by ion-exchange chromatography on DEAE Bio-Gel A. The purified enzyme contained 9.64 nmol heme a/mg protein and one iron atom plus one copper atom for each heme a. The specific activity of the final preparation was 146 µmol of ferrocytochromec oxidized/min · mg protein, measured at pH 5.7. The spectral properties of the enzyme were characteristic of purified cytochrome oxidase and indicated that the preparation was free of cytochromesb, c, andc
1. In analytical ultracentrifugation studies, the enzyme sedimented as a single component with anS
20,w
of5.35S. The Stokes radius of the enzyme was determined by gel filtration chromatography and was equal to 75 Å. The molecular weight of the oxidase calculated from its sedimentation coefficient and Stokes' radius was 180,000, indicating that the active enzyme contained two heme a groups. The purified cytochrome oxidase was also subjected to dodecyl sulfate-polyacrylamide gel electrophoresis in order to determine its components. The enzyme was resolved into five polypeptides with the molecular weights of I, 27,100; II, 15,000; III, 11,900; IV 9800; and V, 9000. 相似文献
19.
《Bioscience, biotechnology, and biochemistry》2013,77(2):214-220
PmHNL, a hydroxynitrile lyase from Japanese apricot ume (Prunus mume) seed was purified to homogeneity by ammonium sulfate fractionation and chromatographic steps. The purified enzyme was a monomer with molecular mass of 58 kDa. It was a flavoprotein similar to other hydroxynitrile lyases of the Rosaceae family. It was active over a broad temperature, and pH range. The N-terminal amino acid sequence (20 amino acids) was identical with that of the enzyme from almond (Prunus dulcis). Based on the N-terminal sequence of the purified enzyme and the conserved amino acid sequences of the enzymes from Pr. dulcis, inverse PCR method was used for cloning of a putative PmHNL (PmHNL2) gene from a Pr. mume seedling. Then the cDNA for the enzyme was cloned. The deduced amino acid sequence was found to be highly similar (95%) to that of an enzyme from Pr. serotina, isozyme 2. The recombinant Pichia pastoris transformed with the PmHNL2 gene secreted an active enzyme in glycosylated form. 相似文献
20.
Immobilization of pectinase fromS. rolfsii was studied on different matrices of which Amberlite XAD-7 showed maximum adsorption and expression of the enzyme. The most
active preparation was obtained when XAD-7 was activated with 2% glutaraldehyde and 1.7 μkat of enzyme per g resin was used
for immobilization at pH 5.5 and 28°C. Optimum pH and temperature of theS. rolfsii pectinase remain unaltered, 3.5 and 55°C, respectively, after immobilization. However, the apparentK
M value of the enzyme decreased from 1.75 g/L for soluble enzyme preparation to 1.4 g/L for immobilized enzyme preparation.
Both soluble and immobilized enzyme preparations were most stable at pH 4.0. The immobilized enzyme preparation was more stable
than the soluble enzyme. 相似文献