共查询到20条相似文献,搜索用时 31 毫秒
1.
Some enzymatic properties were examined with the purified alkaline proteinase from Aspergillus candidus. The isoelectric point was determined to be 4.9 by polyacrylamide gel disc electrofocusing. The optimum pH for milk casein was around 11.0 to 11.5 at 30°C. The maximum activity was found at 47°C at pH 7.0 for 10 min. The enzyme was stable between pH 5.0 and 9.0 at 30°C and most stable at pH 6.0 at 50°C. The enzyme activity over 95% remained at 40°C, but was almost completely lost at 60°C for 10 min. Calcium ions protected the enzyme from heat denaturation to some extent. No metal ions examined showed stimulatory effect and Hg2+ ions inhibited the enzyme. The enzyme was also inhibited by potato inhibitor and diisopropylphosphorofluoridate, but not by metal chelating agent or sulfhydryl reagents. A. candidus alkaline proteinase exhibited immunological cross-reacting properties similar to those of alkaline proteinases of A. sojae and A. oryzae. 相似文献
2.
Sadaaki Iibuchi Yasuji Minoda Koichi Yamada 《Bioscience, biotechnology, and biochemistry》2013,77(7):803-809
Tannin acyl hydrolase (Tannase) from Asp. oryzae No. 7 was purified. The purified enzyme was homogenous on column chromatography (DEAE-Sephadex A50, Sephadex G100), ultra centrifugation and electrophoresis.The molecular weight of the enzyme estimated by gel filtration method was about 200,000.The enzyme was stable in the range of pH 3 to 7.5 for 12 hr at 5°C, and for 25 hr at the same temperature in the range of pH 4.5 to 6. The optimum pH for the reaction was 5.5. It was stable under 30°C (over one day, in 0.05 M-citrate buffer of pH 5.5), and the optimum temperature was 30~40°C (reaction for 20min). The activity was lost completely at 55°C in 20 min at pH 5.5, or at 85°C in 10 min at the same pH.Any metal salt tested did not activate the enzyme, Zink chloride and cupric chloride inhibited the activity or denatured the enzyme. The activity was lost completely by dialysis against EDTA-solution at pH 7.25, although it was not affected by dialysis against deionized water. 相似文献
3.
Yoshitaka Kokusho Shigeaki Kato Haruo Machida Shinjiro Iwasaki 《Bioscience, biotechnology, and biochemistry》2013,77(9):2515-2524
An extracellular phospholipase D from Actinomadura sp. Strain No. 362 was purified about 430-fold from the culture filtrate. The purified enzyme preparation was judged to be homogeneous on polyacrylamide gel electrophoresis. The molecular weight and isoelectric point of the enzyme were estimated to be about 50,000—60,000 and 6.4, respectively. The enzyme was most active at pH 5.5 and 50°C in the presence of Triton X-100, but showed the highest activity at pH 7.0 and 60 — 70°C in its absence. The enzyme was stable up to 30°C at pH 7.2 and also stable in the pH range of 4.0 to 8.0 on 2 hr incubation at 25°C. With regard to substrate specificity, this enzyme hydrolysed lecithin best among the phospholipids tested. It was activated by Fe3 +, Al3+, Mn2 +, Ca2 +, diethyl ether, sodium deoxycholate and Triton X-100, but was inhibited by cetyl pyridinium chloride and dodecylsulfate. 相似文献
4.
Nobuhiro Mori Bunsei Kawakami Kimiaki Hyakutome Yoshiki Tani Hideaki Yamada 《Bioscience, biotechnology, and biochemistry》2013,77(12):3015-3016
To obtain a lipase which effectively hydrolyzes castor oil, bacteria were isolated from 500 soil samples. The best strain was examined; its microbiological characteristics suggested that it belongs to the genus Pseudomonas. A lipase from this strain was purified by ammonium sulfate fractionation and chromatographies on DEAE-cellulose and DEAE-Toyopearl 650 M. The enzyme was purified about 400-fold with a yield of 13%. The purified enzyme was electrophoretically homogeneous and its molecular weight was 30,000. The optimum pH and temperature for the hydrolysis of olive oil emulsion were 7.0 and 60°C. The enzyme was stable up to 35°C at pH 7.0 for 30min and also stable from pH 9.0 to 10.0 at 4°C for 22 hr. The activity was inhibited by Fe3+ , Hg2+ , pCMB, and anionic surfactants, and enhanced by nonionic surfactants and bile salts. The enzyme efficiently hydrolyzed castor oil. 相似文献
5.
Purification, immobilization and characterization of linoleic acid isomerase on modified palygorskite 总被引:2,自引:0,他引:2
Linoleic acid isomerase from Lactobacillus delbrueckii subsp. bulgaricus 1.1480 was purified by DEAE ion-exchange chromatography and gel filtration chromatography. An overall 5.1% yield and purification
of 93-fold were obtained. The molecular weight of the purified protein was ~41 kDa which was analyzed by SDS-PAGE. The purified
enzyme was immobilized on palygorskite modified with 3-aminopropyltriethoxysilane. The immobilized enzyme showed an activity
of 82 U/g. The optimal temperature and pH for the activity of the free enzyme were 30 °C and pH 6.5, respectively; whereas
those for the immobilized enzyme were 35 °C and pH 7.0, respectively. The immobilized enzyme was more stable than the free
enzyme at 30–60 °C, and the operational stability result showed that more than 85% of its initial activity was retained after
incubation for 3 h. The K
m and V
max values of the immobilized enzyme were found to be 0.0619 mmol l−1 and 0.147 mmol h−1 mg−1, respectively. The immobilized enzyme had high operational stability and retained high enzymatic activity after seven cycles
of reuse at 37 °C. 相似文献
6.
《Bioscience, biotechnology, and biochemistry》2013,77(2):246-250
Protease secreted into the culture medium by alkalophilic Thermoactinomyces sp. HS682 was purified to an electrophoretically homogeneous state through only two chromatograhies using Butyl-Toyopearl 650M and SP-Toyopearl 650S columns. The purified enzyme has an apparent relative molecular mass of 25, 000 according to gel filtration on a Sephadex G-75 column and SDS-PAGE and an isoelectric point above 11.0.Its proteolytic activity was inhibited by active-site inhibitors of serine protease, DFP and PMSF, and metal ions, Cu2+ and Hg2+. The enzyme was stable toward some detergents, sodium perborate, sodium triphosphate, sodium-n-dodecylbenzenesulfonate, and sodium dodecyl sulfate, at a concentration of 0.1% and pH 11.5 and 37°C for 60 min. The optimum pH was pH 11.5–13.0 at 37°C and the optimum temperature was 70°C at pH 11.5. Calcium divalent cation raised the pH and heat stabilities of the enzyme. In the presence of 5 mM CaCl2, it showed maximum proteolytic activity at 80°C and stability from pH 4–12.5 at 60°C and below 75°C at pH 11.5. The stabilization by Ca2+ was observed in secondary conformation deduced from the circular dichroic spectrum of the enzyme. The protease hydrolyzed the ester bond of benzoyl leucine ester well. The amino acid terminal sequence of the enzyme showed high homology with those of Microbiol serine protease, although alanine of the NH2-terminal amino acid was deleted. 相似文献
7.
Kawai Fusako Yamada Hideaki Ogata Koichi 《Bioscience, biotechnology, and biochemistry》2013,77(11):1660-1667
Some properties of pyridoxine glucoside-synthesizing enzyme were studied using the partially and highly purified enzyme preparations from Micrococcus sp. No. 431.The enzyme was stable at pH 7.0 and between 0°C and 30°C. The maximal activity was obtained at pH 8.0 and 37°C. Besides sucrose, phenyl-α-d-glucoside and maltose served as glucosyl donor. Of vitamin B6 compounds tested, only pyridoxine served as glucosyl acceptor. The enzyme activity was inhibited by PCMB and heavy metal ions, and the inhibition was prevented by 2-mercaptoethanol, indicating the enzyme would be a sulfhydryl enzyme. The activity was not affected by chelating agents and not activated by metal ions. 相似文献
8.
The novel fungus Aspergillus niveus RS2 isolated from rice straw showed relatively high xylanase production after 5 days of fermentation. Of the different xylan-containing
agricultural by-products tested, rice husk was the best substrate; however, maximum xylanase production occurred when the
organism was cultured on purified xylan. Yeast extract was found to be the best nitrogen source for xylanase production, followed
by ammonium sulfate and peptone. The optimum pH for maximum enzyme production was 8 (18.2 U/ml); however, an appreciable level
of activity was obtained at pH 7 (10.9 U/ml). Temperature and pH optima for xylanase were 50°C and 7.0, respectively; however
the enzyme retained considerably high activity under high temperature (12.1 U/ml at 60°C) and high alkaline conditions (17.2 U/ml
at pH 8 and 13.9 U/ml at pH 9). The enzyme was strongly inhibited by Hg2+, while Mn2+ was slight activator. The half-life of the enzyme was 48 min at 50°C. The enzyme was purified by 5.08-fold using carboxymethyl-sephadex
chromatography. Zymogram analysis suggested the presence of a single candidate xylanase in the purified preparation. SDS-PAGE
revealed a molecular weight of approximately 22.5 kDa. The enzyme had K
m and V
max values of 2.5 and 26 μmol/mg per minute, respectively. 相似文献
9.
Recombinant exoinulinase was partially purified from the culture supernatant ofS. cerevisiae by (NH4)2SO4 precipitation and PEG treatment. The purified inulinase was immobilized onto Amino-cellulofine with glutaraldehyde as a cross-linking
agent. Immobilization yield based on the enzyme activity was about 15%. Optimal pH and temperature of immobilized enzyme were
found to be 5.0 and 60°C, respectively. The enzyme activity was stably maintained in the pH ranges of 4.5 to 6.0 at 60°C.
100% of enzyme activity was observed even after incubation for 24 hr at 60°C. In the operation of a packed-bed reactor containing
412 U inulinase, dahalia inulin of 7.5%(w/v) concentration was completely hydrolyzed at flow rate of 2.0 mL/min at 60°C, resulting
in a volumetric productivity of 693 g-reducing sugars/L/h. Under the reaction conditions of 1.0 mL/min flow rate with 2.5%
inulin at 60°C, the reactor was successfully operated over 30 days without loss of inulinase activity. 相似文献
10.
《Bioscience, biotechnology, and biochemistry》2013,77(5):835-836
Thermostable trehalose synthase, which catalyzes the conversion of maltose into trehalose by intramolecular transglucosylation, was purified from a cell-free extract of the thermophilic bacterium Thermus aquaticus ATCC 33923 to an electrophoretically homogeneity by successive column chromatographies. The purified enzyme had a molecular weight of 105,000 by SDS-polyacrylamide gel electrophoresis and a pI of 4.6 by gel isoelectrofocusing. The N-terminal amino acid of the enzyme was methionine. The optimum pH and temperature were pH 6.5 and 65°C, respectively. The enzyme was stable from pH 5.5 to 9.5 and up to 80°C for 60min. The trehalose synthase from Thermus aquaticus is more thermoactive and thermostable than that from Pimelobacter sp. R48. The yield of trehalose from maltose by the enzyme was independent of the substrate concentration, and tended to increase at lower temperatures. The maximum yield of trehalose from maltose by the enzyme reached 80–82% at 30–40°C. The activity was inhibited by Cu2+ , Hg2+, Zn2+, and Tris. 相似文献
11.
A milk-clotting enzyme from Bacillus subtilis K-26 was purified by gel filtration and ion-exchange chromatography resulting in a 24-fold increase in specific activity with an 80% yield. Polyacrylamide gel electrophoresis and ultracentrifugel analysis revealed that the purified enzyme was homogeneous and had a molecular weight of 27,000 and a Km of 2.77mg/ml for κ-casein. The enzyme was most stable at pH 7.5 and showed increasing clotting activity with decrease in milk pH up to 5.0. The maximum milk-clotting activity was obtained at 60°C, but the enzyme was inactivated by heating for 30 min at 60°C. The enzyme was irreversibly inhibited by EDTA and unaffected by DFP. Heavy-metal ions (Hg2+, Pb2+) inactivated the enzyme. 相似文献
12.
Another pectate lyase was purified to a nearly homogeneous state from the culture filtrate of Streptomyces nitrosporeus. The molecular weight was estimated to be about 41,000. Iso-electric point was pH 4.6. The enzyme was most active at pH 10.0 and 50°C, and was relatively stable at a pH range of 4–11 (at 2°C for 48 hr) and below 40°C (at pH 7.0 for 10 min). Ca2+ was required for maximum activity. The enzyme was an endo-pectate lyase which was more active on low methoxyl pectin than on polygalacturonic acid and had macerating activity on potato tissue and Ganpi bark. 相似文献
13.
《Bioscience, biotechnology, and biochemistry》2013,77(11):2360-2367
A strain of thermophilic bacterium, Bacillus sp., with pectolytic activity has been isolated. It produced an extracellular endo-polygalacturonate trans-eliminase (PL, EC 4.2.2.1) when grown at 60°C on a medium containing polygalacturonate (PGA). The PL was purified by hydrophobic, cation exchange, and size exclusion column chromatographies. The molecular mass of the enzyme was 50 kDa by SDS-PAGE. The isoelectric point of the enzyme was pH 5.3. The enzyme had a half-life of 13 and 1 h at 65 and 70°C, respectively, and showed optimal activity around at 70°C and pH 8.0. It had protopectinase activity, besides PL activity, on lemon protopectin and cotton fibers. The first 20 amino acids sequence of the enzyme had significant similarity with that of PL from methophilic Bacillus subtilis, with 50% identity. 相似文献
14.
Purification and characterization of a serine alkaline protease from Bacillus clausii GMBAE 42 总被引:1,自引:0,他引:1
Kazan D Denizci AA Oner MN Erarslan A 《Journal of industrial microbiology & biotechnology》2005,32(8):335-344
An extracellular serine alkaline protease of Bacillus clausii GMBAE 42 was produced in protein-rich medium in shake-flask cultures for 3 days at pH 10.5 and 37°C. Highest alkaline protease
activity was observed in the late stationary phase of cell cultivation. The enzyme was purified 16-fold from culture filtrate
by DEAE-cellulose chromatography followed by (NH4)2SO4 precipitation, with a yield of 58%. SDS-PAGE analysis revealed the molecular weight of the enzyme to be 26.50 kDa. The optimum
temperature for enzyme activity was 60°C; however, it is shifted to 70°C after addition of 5 mM Ca2+ ions. The enzyme was stable between 30 and 40°C for 2 h at pH 10.5; only 14% activity loss was observed at 50°C. The optimal
pH of the enzyme was 11.3. The enzyme was also stable in the pH 9.0–12.2 range for 24 h at 30°C; however, activity losses
of 38% and 76% were observed at pH values of 12.7 and 13.0, respectively. The activation energy of Hammarsten casein hydrolysis
by the purified enzyme was 10.59 kcal mol−1 (44.30 kJ mol−1). The enzyme was stable in the presence of the 1% (w/v) Tween-20, Tween-40,Tween-60, Tween-80, and 0.2% (w/v) SDS for 1 h
at 30°C and pH 10.5. Only 10% activity loss was observed with 1% sodium perborate under the same conditions. The enzyme was
not inhibited by iodoacetate, ethylacetimidate, phenylglyoxal, iodoacetimidate, n-ethylmaleimidate, n-bromosuccinimide, diethylpyrocarbonate or n-ethyl-5-phenyl-iso-xazolium-3′-sulfonate. Its complete inhibition by phenylmethanesulfonylfluoride and relatively high k
cat value for N-Suc-Ala-Ala-Pro-Phe-pNA hydrolysis indicates that the enzyme is a chymotrypsin-like serine protease. K
m and k
cat values were estimated at 0.655 μM N-Suc-Ala-Ala-Pro-Phe-pNA and 4.21×103 min−1, respectively. 相似文献
15.
An extracellular alkaline carboxymethycellulase (CMCase) from Bacillus subtilis was purified by salt precipitation followed by anion-exchange chromatography using DEAE-Sepharose. The cell-free supernatant containing crude enzyme had a CMCase activity of 0.34 U/mg. The purified enzyme gave a specific activity of 3.33 U/mg, with 10-fold purification and an overall activity yield of 5.6%. The purified enzyme displayed a protein band on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with an apparent molecular size of 30 kDa, which was also confirmed by zymogram analysis. The enzyme displayed multisubstrate specificity, showing significantly higher activity with lichenan and β-glucan as compared to carboxymethylcellulose (CMC), laminarin, hydroxyethylcellulose, and steam-exploded bagasse, and negligible activity with crystalline substrate such as Avicel and filter paper. It was optimally active at pH 9.2 and temperature 45°C. The enzyme was stable in the pH range 6–10 and retained 70% activity at pH 12. Thermal stability analysis revealed that the enzyme was stable in temperature range of 20°C to 45°C and retained more than 50% activity at 60°C for 30 min. The enzyme had a Km of 0.13 mg/ml and Vmax of 3.38 U/mg using CMC as substrate. 相似文献
16.
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
17.
《Bioscience, biotechnology, and biochemistry》2013,77(4):608-613
A levanase from Bacillus sp. was purified to a homogeneous state. The enzyme had a molecular weight of 135,000 and an isoelectric point of pH 4.7. The enzyme was most active at pH 6.0 and 40°C, stable from pH 6.0 to 10.0 for 20 hr of incubation at 4°C and up to 30°C for 30 min of incubation at pH 6.0. The enzyme activity was inhibited by Ag +, Hg2 +, Cu2 +, Fe3 +, Pb2+, and p-chloromercuribenzoic acid. The enzyme hydrolyzed levan and phlein endowise to produce levanheptaose as a main product. The limit of hydrolysis of levan and phlein were 71% and 96%, respectively. 相似文献
18.
《Bioscience, biotechnology, and biochemistry》2013,77(11):1968-1972
Cyclodextrin glucanotransferase (EC 2.4.1.19) from Brevibacterium sp. No. 9605 was purified to homogeneity by chromatography on butyl-Toyopearl 650M, γ-cyclodextrin-Sepharose 4B, and Toyopearl HW-55S. The molecular weight of the purified enzyme was estimated to be 75,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric point of the purified enzyme was 2.8. The optimum pH and temperature were pH 10 and 45°C, respectively. The enzyme was stable at the range of pH 6–8 and at temperatures 50°C or less in the presence of CaCl2. The enzyme produced mainly γ-cyclodextrin from starch in the initial stage of reaction, but later, the proportion of β-cyclodextrin was increased. 相似文献
19.
A. Mandal S. Kar P. K. Das Mohapatra C. Maity B. R. Pati K. C. Mondal 《Applied Biochemistry and Microbiology》2011,47(3):250-255
An extracellular xylanase from the fermented broth of Bacillus cereus BSA1 was purified and characterized. The enzyme was purified to 3.43 fold through ammonium sulphate precipitation, DEAE cellulose
chromatography and followed by gel filtration through Sephadex-G-100 column. The molecular mass of the purified xylanse was
about 33 kDa. The enzyme was an endoxylanase as it initially degraded xylan to xylooligomers. The purified enzyme showed optimum
activity at 55°C and at pH 7.0 and remained reasonably stable in a wide range of pH (5.0–8.0) and temperature (40–65°C). The
K
m
and V
max
values were found to be 8.2 mg/ml and 181.8 μmol/(min mg), respectively. The enzyme had no apparent requirement of cofactors,
and its activity was strongly inhibited by Cu2+, Hg2+. It was also a salt tolerant enzyme and stable upto 2.5 M of NaCl and retained its 85% activity at 3.0 M. For stability and
substrate binding, the enzyme needed hydrophobic interaction that revealed when most surfactants inhibited xylanase activity.
Since the enzyme was active over wide range of pH, temperature and remained active in higher salt concentration, it could
find potential uses in biobleaching process in paper industries. 相似文献
20.
《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