共查询到20条相似文献,搜索用时 31 毫秒
1.
A novel potent protease, Urechis unicinctus fibrinolytic enzyme (UFE), was first discovered by our laboratory. In this study, we further investigated the enzymatic properties
and dynamic parameters of UFE. As a low molecular weight protein, UFE appeared to be very stable to heat and pH. When the
temperature was <50°C, the remnant enzyme activity remained almost unchanged, but when the temperature was raised to 60#x00B0;C
the remnant enzyme activity began to decrease rapidly. UFE was quite stable in a pH range of 3.0–12.0, especially at slightly
alkaline pH values. Mn2+, Cu2+, and Fe2+ ions were activators of UFE, whereas Fe3+ and Ag+ ions were inhibitors. Fe2+ ion along with Fe3+ ion might regulate UFE activity in vivo. The optimum pH and temperature of UFE were about 8.0 and 50°C, respectively. When
using casein as substrate and a substrate concentration <0.1% casein (w/v), the reaction velocity was increased with substrate
concentration. Also when using casein as substrate, the determined K
m and V
max of UFE were 0.5298 mg/mL and 3.0845 mol of l-tyrosine equivalent, respectively. Our systematic research results are significant
when UFE is applied for medical and industrial purposes. 相似文献
2.
Liliana B. Areces Mirtha Biscoglio De Jiménez Bonino Marina A. A. Parry Elda R. Fraile Héctor M. Fernández Osvaldo Cascone 《Applied biochemistry and biotechnology》1992,37(3):283-294
An acid protease having milk clotting activity has been isolated fromMucor bacilliformis cultures. The enzyme was basically purified by ionic exchange chromatography. An average yield of 29 mg purified product
was obtained from 100 mL crude extract. As purity criteria, SDS-PAGE, reverse-phase HPLC, and N-terminal analysis were performed.
The protease is a protein composed of a single polypeptide chain with glycine at the N-terminus. The mol wt is approx 32,000,
and its amino acid composition is very similar to those of other fungal proteases. As expected, its clotting activity was
drastically inhibited by pepstatin A action. On the other hand, its instability against heat treatment and its clotting/proteolytic
activity ratio indicate that it may be considered as a potential substitute for bovine chymosin. Index Entries:Mucor bacilliformis protease; milk clotting enzyme; acid protease; fungal protease; aspartyl protease. 相似文献
3.
Purification and characterization of organic solvent stable serine alkaline protease from newly isolated Bacillus circulans M34
下载免费PDF全文
![点击此处可从《Biomedical chromatography : BMC》网站下载免费的PDF全文](/ch/ext_images/free.gif)
A protease from newly isolated Bacillus circulans M34 was purified by Q‐Sepharose anion exchange chromatography and Sepharose–bacitracin affinity chromatography followed by (NH4)2SO4 precipitation. The molecular mass of the purified enzyme was determined using SDS–PAGE. The optimum pH and temperature for protease activity were 11 and 50°C, respectively. The effect of various metal ions on protease activity was investigated. Alkaline protease from Bacillus circulans M34 wase activated by Zn2+, Cu2+ and Co2+ up to 31%. The purified protease was found to be stable in the organic solvents, surfactants and oxidizing agent. The substrate specificity of purified protease was investigated towards different substrates. The protease was almost completely inhibited by the serine protease inhibitor phenylmethanesulfonyl fluoride. The kinetic parameters of the purified protease, maximum rate (Vmax) and Michaelis constant (Km), were determined using a Lineweaver–Burk plot. Copyright © 2015 John Wiley & Sons, Ltd. 相似文献
4.
Purification and characterization of an alkaline protease prot 1 frombotrytis cinerea 总被引:1,自引:0,他引:1
Alkaline thiol protease named Prot 1 was isolated from a culture filtrate ofBotrytis cinerea. The enzyme was purified by ammonium sulfate fractionation, gel filtration, and ion-exchange chromatography. Thus, the enzyme
was purified to homogeneity with specific activity of 30-fold higher than that of the crude broth. The purified alkaline protease
has an apparent molecular mass of 43 kDa under denaturing conditions as estimated by sodium dodecyl sulfate-polyacrylamide
gel electrophoresis. The native molecular mass (45 kDa), determined by gel filtration, indicated that the alkaline protease
has a monomeric form. The purified protease was biochemically characterized. The enzyme is active at alkaline pH and has a
suitable and high thermostability. The optimal pH and temperature for activity were 9.0–10.0 and 60°C, respectively. This
protease was stable between pH 5.0 and 12.0. The enzyme retained 85% of its activity by treatment at 50°C over 120 min; it
maintained 50% of activity after 60 min of heating at 60°C. Furthermore, the protease retained almost complete activity after
4 wk storage at 25°C. The activity was significantly affected by thiol protease inhibitors, suggesting that the enzyme belongs
to the alkaline thiol protease family. With the aim on industrial applications, we focused on studying the stability of the
protease in several conditions. Prot 1 activity was not affected by ionic strength and different detergent additives, and,
thus, the protease shows remarkable properties as a biodetergent catalyst. 相似文献
5.
Natalya I. Dergousova Alexander Yu. Amerik Alla M. Volynskaya Lev D. Rumsh 《Applied biochemistry and biotechnology》1996,61(1-2):97-107
A new method for obtaining HIV-I protease was suggested. Fusion proteins composed of the N-terminal fragment of human γ-interferon
and HIV-I protease connected with (Asp)4Lys (protein I) or Asp-Pro (protein II) linkers were expressed inEscherichia coli cells. The fusion proteins were produced as insoluble inclusion bodies in the 20% yield of total cell protein. Protein I
was cleaved by enterokinase. The solubility of protein I was increased by treating with Nasulfite/Na-tetrathionate under denaturing
conditions.
Optimal conditions for efficient acidic hydrolysis of protein II at Asp-Pro bond were found. The hydrolysis products were
separated by reversed-phase FPLC. The amount of tryptophan and cysteine residues in the enzyme obtained was estimated. The
activity of HIV-I protease was determined using the chromogenic peptide AlaArgVal NleNphGluAlaNleNH2 and a high-mol-wt substrate consisting of β galactosidase and a fragment ofgag proteins, including pl7-p24 processing site. 相似文献
6.
Moradian F Khajeh K Naderi-Manesh H Ahmadvand R Sajedi RH Sadeghizadeh M 《Applied biochemistry and biotechnology》2006,134(1):77-87
Two Bacillus sp. strains, HR-08 and KR-8102, isolated from soil of the west and north parts of Iran were screened on gelatin agar medium
for their ability to produce alkaline protease. The enzymes were active in a wide pH range (6.0–11.0) and stable in the alkaline
range (7.0–12.0). The optimum temperatures for the protease from HR-08 and KR-8102 were 65 and 50°C, respectively. The irreversible
thermoinactivation of HR-08 and KR-8102 proteases showed that the stability of HR-08 enzyme was higher than that of KR-8102
and the half-lives of these enzymes were 95 and 32 min at 50°C, respectively. In the presence of 10 mM Ca2+, HR-08 retained 100, 90, and 20% of its initial activity after heating for 30 min at 50, 60, and 70°C, respectively. Enzymes
were inhibited by phenylmethylsulfonyl fluoride and iodoacetate. After inhibition by iodoacetate, both enzymes were reactivated
by dithiothreitol. These data show that the enzymes seem to be thiol-dependent serine alkaline proteases. The enzymes especially
from HR-08 were stable in the presence of H2O2, surfactants, and local detergents; their activities were enhanced in the presence of 5 mM Fe2+; and the presence of 5mM metal ions such as Mg2+, Cu2+, and Mn2+ produced almost no effect. 相似文献
7.
Ayse Gerze Didem Omay Yuksel Guvenilir 《Applied biochemistry and biotechnology》2005,121(1-3):335-345
Bacteria of genus Bacillus are active producers of extracellular proteases, and characteristics of enzyme production by Bacillus species have been well studied. The aim of this experimental study is isolation and partial purification of protease enzyme
from the Bacillus subtilis megatherium bacteria species. Protease enzyme is obtained by inducing spore genesis of bacteria from Bacillus species on suitable media. The partial purification was reali-zed by applying successively ammonium sulfate precipitation,
dialysis, DEAE-cellulose ion exchange chromatography to the supernatant. In this study, the effect of substrate concentration,
reaction time, the effect of inhibitor and activator on the optimum pH, optimum temperature, pH stability, and temperature
stability was determined. Molecular weight of the obtained enzyme was investigated by SDS-PAGE. In this study, the specific
activity of the supernatant, which was partially purified from Bacillus subtilis megatherium bacteria, was 10.4 U/mg, specific activity of supernatant was 13.5 U/mg after 80% ammonium sulfate fractionation. The final
enzyme preparation was 1.1-fold purer than the crude homogenate. Molecular weight of the protease was determined, and it was
found that the weight of enzyme was 45 kDa by using SDS-PAGE. 相似文献
8.
A statistical approach, response surface methodology (RSM), was used to study the production of extracellular protease fromBacillus sp., which has properties of immense industrial importance. The most influential parameters for protease production obtained
through the method of testing the parameters one at a time were starch, soybean meal, CaCl2, agitation rate, and inoculum density. This method resulted in the production of 2543 U/mL of protease in 48 h fromBacillus sp. Based on these results, face-centered central composite design falling under RSM was employed to further enhance protease
activity. The interactive effect of the most influential parameters resulted in a 1.50-fold increase in protease production,
yielding 3746 U/mL in 48 h. Analysis of variance showed the adequacy of the model and verification experiments confirmed its
validity. On subsequent scale-up in a 30-L bioreactor using conditions optimized through RSM, 3978 U/mL of protease was produced
in 18 h. This clearly indicated that the model remained valid even on a large scale. RSM is a quick process for optimization
of a large number of variables and provides profound insight into the interactive effect of various parameters involved in
protease production. 相似文献
9.
《Analytical letters》2012,45(12):2199-2208
Abstract A method is proposed to assay catalytically active protease. The method is based on the subsequent inter action of the enzyme with a protein inhibitor immobi lized on polysterene and with antibodies labelled with a high-activity enzyme. Optimal conditions have been chosen for assaying alkaline protease from Bacillus subtilis (subtilisin) by ELISA with peroxidase as label, employing two protein serine protease inhibitors isolated from duck egg white and maize seeds. The proposed method enables the detection of the catalyti cally active enzyme at concentrations as low as 1 ng/ml for 1.5 hr. The c. v. of the assay is less than 5%. The method can be used to determine the equilibrium con stant of the enzyme-inhibitor reaction in solution. 相似文献
10.
Muhammad Bashir Khan Hidayatullah Khan Muhammad Usman Shah 《Natural product research》2016,30(8):935-940
A low molecular weight serine protease from seeds of Citrullus colocynthis was purified to electrophoretic homogeneity with high level of catalytic efficiency (22,945 M?1 S?1). The enzyme was a monomer with molecular mass of 25 kDa estimated by SDS–PAGE. The enzyme was highly active over a pH range of 6.5–9.0 and temperature range of 20–80 °C, with maximum activity at pH 7.5 and at 50 °C. The Km and Kcat were 73 μg/mL and 67/s, respectively. The enzyme was strongly inhibited by PMSF, moderately by soybean trypsin inhibitor, indicating that the enzyme was a serine protease. The enzyme retained 86 and 73% of its activity in the presence of urea and DTT, respectively, and its activity was slightly enhanced in the presence of anionic detergent (SDS). Thus, the enzyme is a novel SDS-stable protease with high catalytic efficiency over wide ranges of pH and temperature which is commercially promising for various industrial applications. 相似文献
11.
A series of novel cyclic urea molecules 5,6-dihydroxy-1,3-diazepane-2,4,7-trione as HIV-1 protease inhibitors were designed
using computational techniques. The designed molecules were compared with the known cyclic urea molecules by performing docking
studies, calculating their ADME (Absorption, Distribution, Metabolism, and Excretion) properties and protein ligand interaction
energy. These novel molecules were designed by substituting the P
1/P′
1 positions (4
th
and 7
th
position of 1, 3-diazepan-2-one) with double bonded oxygens. This reduces the molecular weight and increases the bioavailability,
indicating better ADME properties. The docking studies showed good binding affinity towards HIV-1 protease. The biological
activity of these inhibitors were predicted by a model equation generated by the regression analysis between biological activity
(log 1/K
i
) of known inhibitors and their protein ligand interaction energy. The synthetic studies are in progress.
相似文献
12.
An extracellular alkaline protease from an alkalophilic bacterium, Bacillus cereus, was produced in a large amount by the method of extractive fermentation. The protease is thermostable, pH tolerant, and
compatible with commercial laundry detergerts. The protease purified and characterized in this study was found to be saperior
to endogenous protease already present in commercial laundry detergents. The enzyme was purified to homogeneity by ammonium
sulfate precipitation, concentration by ultrafiltration, anionexchange chromatography, and gel filtration. The purified enzyme
had a specific activity of 3256.05 U/mg and was found to be amonomeric protein with a molecular mass of 28 and 31 kDa, as
estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and nondenaturing PAGE, respectively. Its
maximum protease activity against casein was found to be at pH 10.5 and 50°C. Proteolytic activity of the enzyme was detected
by casein and gelatin zymography, which gave a very clear protease activity zone on gel that corresponded to the band obtained
on SDS-PAGE and nondenaturing PAGE with a molecular mass of nearly 31 kDa. The purified enzyme was analyzed through matrix-assisted
laser desorption ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS) and identified as a subtilisin class of protease.
Specific serine protease inhibitors, suggesting the presence of serine residues at the active site, inhibited the enzme significantly. 相似文献
13.
Palma Marcia B. Pinto Annette L. Gombert Andreas K. Seitz Karina H. Kivatinitz Silvia C. Castilho Leda R. Freire Denise M. G. 《Applied biochemistry and biotechnology》2000,84(1-9):1137-1145
Lipase, protease, and amylase production by Penicillium restrictum in solid-state fermentation was investigated. The basal medium was an industrial waste of babassu oil (Orbignya oleifera) production. It was enriched with peptone, oliveoil, and Tween-80. The supplementation positively influenced both enzyme
production and fungal growth. Media enriched with Tween-80 provided the highest protease activity (8.6 U/g), whereas those
enriched with peptone and olive oil led to the highest lipase (27.8 U/g) and amylase (31.8 U/g) activities, respectively. 相似文献
14.
Luciana A. I. de Azeredo Leda R. Castilho Selma G. F. Leite Rosalie R. R. Coelho Denise M. G. Freire 《Applied biochemistry and biotechnology》2003,108(1-3):749-755
Streptomyces are important microorganisms because of their capacity to produce numerous bioactive molecules. In the present work protease
production, by Streptomyces sp. 594 isolated from a Brazilian Cerrado soil, was maximized by optimizing a low-cost culture medium composition (casitone
and sugarcane molasses) using statistical experimental design. The final protease activity (56 U/mL) was 2.8-fold and 58-fold
higher than that obtained in the beginning of this study, and in a previous work, using an actinomycete selection medium,
respectively. Protease production, not growth associated, appeared to be modulated by an inducer system, whereby the C/N ratio
seemed to play a significant role. 相似文献
15.
A cassava flour-processing effluent (manipueira) was evaluated as a substrate for surfactant production by two Bacillus subtilis strains. B. subtilis ATCC 21332 reduced the surface tension of the medium to 25.9 mN/m, producing a crude biosurfactant concentration of 2.2 g/L.
The wild-type strain, B. subtilis LB5a, reduced the surface tension of the medium to 26.6 mN/m, giving a crude biosurfactant concentration of 3.0 g/L. A decrease
in surfactant concentration observed for B. subtilis ATCC 21332 seemed to be related to an increase in protease activity. The biosurfactant produced on cassava effluent medium
by B. subtilis LB5a was similar to surfactin. 相似文献
16.
In this paper, two 3‐dimensional quantitative structure‐activity relationship models for 60 human immunodeficiency virus (HIV)‐1 protease inhibitors were established using random sampling analysis on molecular surface and translocation comparative molecular field vector analysis (Topomer CoMFA). The non–cross‐validation (r2), cross‐validation (q2), correlation coefficient of external validation (Q2ext), and F of 2 models were 0.94, 0.80, 0.79, and 198.84 and 0.94, 0.72, 0.75, and 208.53, respectively. The results indicated that 2 models were reasonable and had good prediction ability. Topomer Search was used to search R groups in the ZINC database, 20 new compounds were designed, and the Topomer CoMFA model was used to predicate the biological activity. The results showed that 18 new compounds were more active than the template molecule. So the Topomer Search is effective in screening and can guide the design of new HIV/AIDS drugs. The mechanism of action was studied by molecular docking, and it showed that the protease inhibitors and Ile50, Asp25, and Arg8 sites of HIV‐1 protease have interactions. These results have provided an insight for the design of new potent inhibitors of HIV‐1 protease. 相似文献
17.
Valeria F. Soares Leda R. Castilho Elba P. S. Bon Denise M. G. Freire 《Applied biochemistry and biotechnology》2005,121(1-3):311-319
A Bacillus subtilis isolate was shown to be able to produce extracellular protease in solid-state fermentations (SSF) using soy cake as culture
medium. A significant effect of inoculum concentration and physiological age on protease production was observed. Maximum
activities were obtained for inocula consisting of exponentially growing cells at inoculum concentrations in the range of
0.7–2.0 mg g−1. A comparative study on the influence of cultivation temperature and initial medium pH on protease production in SSF and
in submerged fermentation (SF) revealed that in SSF a broader pH range (5–10), but the same optimum temperature (37°C), is
obtained when compared to SF. A kinetic study showed that enzyme production is associated with bacterial growth and that enzyme
inactivation begins before biomass reaches a maximum level for both SF and SSF. Maximum protease activity and productivity
were 960 U g−1 and 15.4 U g−1 h−1 for SSF, and 12 U mL−1 and 1.3 U mL−1 h−1 for SF. When SSF protease activity was expressed by volume of enzyme extract, the enzyme level was 10-fold higher and the
enzyme productivity 45% higher than in SF. These results indicate that this bacterial strain shows a high biotechnological
potential for protease production in solid-state fermentation. 相似文献
18.
Wei Liu Wenning Yang Xueyan Li Dongying Qi Hongjiao Chen Huining Liu Shuang Yu Guopeng Wang Yang Liu 《Molecules (Basel, Switzerland)》2022,27(15)
(1) Methods: An integrated strategy, including in vitro study (degree of hydrolysis (DH) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity) and in vivo study (absorption after oral administration in rats), was developed to evaluate the properties of the fish skin gelatin hydrolysates prepared using different proteases (pepsin, alkaline protease, bromelain, and ginger protease). Meanwhile, in order to identify the hydrolysis site of ginger protease, the peptides in the ginger protease-degraded collagen hydrolysate (GDCH) were comprehensively characterized by liquid chromatography/tandem mass spectrometry (LC-MS) method. (2) Results: The GDCH exhibited the highest DH (20.37%) and DPPH radical scavenging activity (77.73%), and in vivo experiments showed that the GDCH was more efficiently absorbed by the gastrointestinal tract. Further oral administration experiments revealed that GDCH was not entirely degraded to free amino acids and can be partially absorbed as dipeptides and tripeptides in intact forms, including Pro-Hyp, Gly-Pro-Hyp, and X-Hyp-Gly tripeptides. LC-MS results determined the unique substrate specificity of ginger protease recognizing Pro and Hyp at the P2 position based on the amino acids at the P2 position from the three types of tripeptides (Gly-Pro-Y, X-Hyp-Gly, and Z-Pro-Gly) and 136 identified peptides (>4 amino acids). Interestingly, it suggested that ginger protease can also recognize Ala in the P2 position. (3) Conclusions: This study comprehensively evaluated the properties of GDCH by combining in vitro and in vivo strategies, and is the first to identify the cleavage site of ginger protease by LC-MS technique. It provides support for the follow-up study on the commercial applications of ginger protease and bioactivities of the hydrolysate produced by ginger protease. 相似文献
19.
20.
Hoang Thi Hong Anh Esmaeil Shahsavari Nathan J. Bott Andrew S. Ball 《Molecules (Basel, Switzerland)》2021,26(11)
Although axenic microbial cultures form the basis of many large successful industrial biotechnologies, the production of single commercial microbial strains for use in large environmental biotechnologies such as wastewater treatment has proved less successful. This study aimed to evaluate the potential of the co-culture of two halophilic bacteria, Marinirhabdus sp. and Marinobacter hydrocarbonoclasticus for enhanced protease activity. The co-culture was significantly more productive than monoculture (1.6–2.0 times more growth), with Marinobacter hydrocarbonoclasticus being predominant (64%). In terms of protease activity, enhanced total activity (1.8–2.4 times) was observed in the co-culture. Importantly, protease activity in the co-culture was found to remain active over a much broader range of environmental conditions (temperature 25 °C to 60 °C, pH 4–12, and 10–30% salinity, respectively). This study confirms that the co-culturing of halophilic bacteria represents an economical approach as it resulted in both increased biomass and protease production, the latter which showed activity over arange of environmental conditions. 相似文献