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1.
Daisuke Tsuru Heizo Kira Takehiko Yamamoto Juichiro Fukumoto 《Bioscience, biotechnology, and biochemistry》2013,77(12):1261-1268
A crystalline alkaline protease was prepared from B. amylosacchariticus, which was isolated as a strain of saccharogenic α-amylase-producing Bacillus subtilis. The enzyme was most active at pH values between 10.3 and 10.7 towards casein and was stable at pH values from 6 to 11 on twenty hour incubation at 30°C. Calcium ions were effective to stabilize the enzyme especially at higher temperatures. The enzyme was markedly inactivated by DFP as well as protease inhibitor from potato and slightly by surface active agents, but not affected by sulfhydryl reagents and divalent metal ions except Hg++ .Hemoglobin was the best substrate for the enzyme and more than 20% of the peptide bonds were hydrolyzed. Of numerous synthetic peptides tested, only the two compounds, and , were found to be hydrolyzed. A cyclic peptide, gramicidin S, was split by the enzyme only at the peptide bond of -l-valyl-l-ornithyl-. Methyl n-butyrate and tributyrin were also good substrates for the alkaline protease obtained here. 相似文献
2.
Daisuke Tsuru Heizo Kira Takehiko Yamamoto Juichiro Fukumoto 《Bioscience, biotechnology, and biochemistry》2013,77(6):718-723
Substrate specificity of the crystalline neutral protease of B. amylosacchariticus was investigated using the B-chain of oxidized beef insulin as the substrate, and the results were compared with those of proteases obtained from other strains of Bacillus subtilis. The neutral protease split the B-chain at eleven sites of the peptide linkages, indicating the narrow specificity as compared with subtilopeptidase A, The results also indicated that the peptide bonds susceptible to the action of the neutral protease were mainly those involving amino group of hydrophobic amino acids and tyrosine, with a few exception. The enzyme showed potent activities in casein digestion at near neutrality and in milk clotting at pH 5.6, whereas it was completely inert on esters and keratin, and only slightly active toward elastin. 相似文献
3.
Daisuke Tsuru Takehiko Yamamoto Juichiro Fukumoto 《Bioscience, biotechnology, and biochemistry》2013,77(7):651-658
A neutral protease of Bacillus subtilis var. amylosacchariticus was purified and crystallized by sequential chromatography on columns of Duolite A-2 anion-exchange resin, CM-cellulose and DEAE-sephadex A-50. The crystalline preparation was chromatographically homogeneous and confirmed to be monodispersive by physicochemical criteria. The enzyme was most active at near pH 7 against casein and stabilized by calcium salts. Some metalchelating agents and metal ions such as Hg?, Pb?, Cu? and Fe? markedly inactivated the enzyme, whereas diisopropyl phosphorofluoridate, sulfhydryl reagents and protease inhibitor of potato did not affect the activity. The neutral protease obtained here was rather stable as compared with the neutral protease ever reported and was able to be freeze-dried without any appreciable lose in enzyme activity. 相似文献
4.
Daisuke Tsuru Heizo Kira Takehiko Yamamoto Juichiro Fukumoto 《Bioscience, biotechnology, and biochemistry》2013,77(11):1164-1169
Some physical and chemical properties and substrate specificity were investigated of the neutral protease obtained from B. amylosacchariticus, a strain of saccharogenic α-amylase producing Bacillus subtilis. The molecular weight and sedimentation coefficient of the protease were estimated to be 33,800 and 3.02, respectively, by ultracentrifugal analyses, and alanine was identified as an amino-terminal amino acid of the enzyme by the Sanger’s method. The enzyme showed more broad specificity than the neutral protease of liquefying α-amylase-producing B. subtilis, when tested with synthetic peptides, and hippuryl-l-leucinamide was the best substrate among 42 compounds tested. On a long incubation, the enzyme hydrolyzed several proteins in a degree of 10 to 25% as peptide bond cleavage. 相似文献
5.
Daisuke Tsuru Heizo Kira Takehiko Yamamoto Juichiro Fukumoto 《Bioscience, biotechnology, and biochemistry》2013,77(9):856-862
The neutral protease of Bacillus amylosacchariticus was inactivated by low concentrations of several metal-chelating agents and the inactivated enzyme with EDTA restored its activity almost completely by the addition of Zn++ or Co++ and partially by Fe++ or Mn++, if these metal ions were added shortly after the EDTA-treatment. The native enzyme was found to contain 0.19% of zinc together with a significant amount of calcium. Parallel increase in specific activity and zinc content of enzyme preparation was observed throughout the purification procedure. The elution pattern of enzyme activity on a CM-cellulose column chromatography also completely coincided with that of protein-bound zinc. A zinc-free inactive enzyme was also reactivated by the addition of zinc or cobalt ions, clearly showing that the neutral protease of B. amylosacchariticus is a zinc mctalloenzyme. 相似文献
6.
Juichiro Fukumoto Daisuke Tsuru Takehiko Yamamoto 《Bioscience, biotechnology, and biochemistry》2013,77(6):710-717
An acid protease of Rhizopus chinensis was purified by sequential chromatographies on columns of Duolite A-2, Sephadex G-100 and CM-cellulose, and crystallized from aqueous acetone solution. The preparation was shown to be monodisperse on column chromatography of ion-exchange sephadex and on ultracentrifugal analysis. The enzyme was most active at pH values between 2.9 and 3.3 and was stable over the range of pH 2.8 to 6.5. The protease was markedly inactivated by ferric ions and sodium lauryl sulfate, whereas it was affected by neither sulfhydryl reagents nor metal-chelating agents. In milk-clotting activity, the acid protease was shown to be one of the most potent enzymes among those of fungal origin. Substrate specificity experiments on several synthetic peptides indicated that the peptide bonds susceptible to the action of the enzyme were mainly those involving amino group of aromatic amino acids. 相似文献
7.
A protease from the lotus seed (Nelumbo nucifera Gaertn) was purified by acid-treatment, ammonium sulfate-fractionation, ethylalcohol-fractionation, TEAE-cellulose-treatment and Sephadex G-100 gel-filtration.The enzyme was purified about 870-fold and was homogeneous in electrophoretic and ultracentrifugal analyses.Purified lotus seed protease is an acid protease with a pH optimum at 3.8 toward urea-denatured casein. It is active for casein and hemoglobin. But other proteins such as edestin, zein, lotus seed globulin and soybean casein are slightly hydrolyzed and egg albumin is hardly hydrolyzed. This enzyme is most stable at pH 4.0 below 40°C. The enzyme is not a thiol protease, and its activity was completely inhibited by potassium permanganate, remarkably inhibited by sodium dodecylsulfate and accelerated by hydrogen peroxide. 相似文献
8.
Some physicochemical and enzymic properties of the purified lotus seed protease were investigated. The molecular weight determined from sedimentation-diffusion studies and by Sephadex G-100 gel-filtration was 36,800 and 35,500, respectively. The enzyme gave a typical ultraviolet spectrum of protein and its isoelectric point was found to be in a range of pH 3~4. The enzyme was stabilized at pH 6 in Tris-hydrochloric acid buffer by the addition of cupric ion. The inhibitory function of some reagents, especially that of potassium permanganate, and acetylation of the enzyme revealed that the tyosine residue in this enzyme protein might play some important role in the enzyme activity. Lotus seed protease was found to be fairly similar to pepsin in some properties. 相似文献
9.
Kazuyuki Morihara 《Bioscience, biotechnology, and biochemistry》2013,77(5):464-473
A discussion was made on the step of the incorporation of Ca in the synthesizing reaction of the proteinase of Ps. myxogenes sp.When the cell suspensions containing a carbon source without addition of Ca were shaken aerobically, the secreted protein content was found to be remarkably lower than that of the suspensions containing Ca, while total amounts of secreted organic nitrogen materials were almost the same despite of the presence or absence of Ca. From the experimental result of an auto-splitting phenomenon of the proteinase by Ca-removal treatment, it was assumed that the above result occurs by auto-digestion of the proteinase secreted in the medium where Ca is absent. Accordingly, the step of incorporation of Ca would be considered to occur in the synthesized Ca-free proteinase. 相似文献
10.
Kazuyuki Morihara 《Bioscience, biotechnology, and biochemistry》2013,77(12):842-847
Proteinase productions of various species of the Pseudomonas genus were comparatively examined. Among them, Ps. aeruginosa was found to be the most suitable species for obtaining a proteinase in a high yield. The proteinase production of the organism was slight in natural medium, but remarkably high in shaking culture of synthetic medium containing glucose in a high concentration and a limited content of organic nitrogen. This medium found to be suitable for the species Ps. aeruginosa, also was remarkably similar with the medium suitable for Ps. myxogenes sp., which had been identified by us and used for this series of studies on the protease of Pseudomonas. 相似文献
11.
A neutral protease, which was prepared from Bacillus polymixa, was used on the digestion of myosin. Myosin was split to HMM and LMM, The HMM fraction was further digested with the protease and a subfragment-1 was prepared.The sedimentation coefficient, , and the intrinsic viscosity of this subfragment-1 were determined as 5.6S and 0.08dl/g, respectively. The molecular weight was estimated to be about 120,000 by the sedimentation equilibrium method. This subfragment showed the characteristic myosin-type ATPase; the ATPase was activated by Ca2+ ion or EDTA and inhibited by Mg2+ ion, the maximum activation of ATPase was obtained when 3.5 SH groups per 105 g of subfragment were titrated with PCMB.The subfragment-1 possessed the ability to interact with F-actin and to accelerate G-actin polymerization. 相似文献
12.
Kishirō Yamamoto 《Bioscience, biotechnology, and biochemistry》2013,77(7):519-526
The activating factors of the inactive protease in cell-free extracts obtained from growing mycelia of Aspergillus sojae KS were studied. It was found that the several kinds of metals were involved in activation, and the role of these metals on the activation was investigated. The velocity of the activation was maximal around pH 10 as well as around pH 5. It was proved that a kinase (enzyme) capable of activating the inactive protease in alkaline solution does exist in the cell-free extract. 相似文献
13.
Chemical composition of the filamentous cells of Lactobacillus delbrueckii produced by vitamin B12 deficient culture was studied. Protein and RNA contents per unit cell volume of the filamentous cells were nearly equal to those of normal cells, but the DNA content was much reduced. A cytoplast of the filamentous cell possessed about twice as large volume as that of the normal cell. A cytoplast of either filamentous or normal cell seems to contain the same amount of DNA. DNA level and membrane formation necessary for cell division remained as future problems. 相似文献
14.
15.
Tetsuo Misaki Masao Yamada Tadayasu Okazaki Jiro Sawada 《Bioscience, biotechnology, and biochemistry》2013,77(9):1383-1392
In order to elucidate the protease constitution of Aspergillus oryzae, systematic separation of proteases was elaborated by sequential chromatography on Amberlite CG–50, DEAE-Sephadex A–50 and CM-cellulose. As the results, three kinds of proteases, that is, acid-, neutral- and alkaline proteases were isolated and purified in crystalline form except neutral one. Purified neutral protease could not be crystallized, but was confirmed to be homogeneous by ultracentrifugal analysis. Besides these proteases, a new protease which was unknown up to the present in the constitution of Asp. oryzae proteases, was first isolated and designated as “semi-alkaline protease” according to its optimal pH. 相似文献
16.
Kishirō Yamamoto 《Bioscience, biotechnology, and biochemistry》2013,77(6):453-460
Inactive protease in the cell free extracts obtained from growing cells of Aspergillus sojae KS was collected in a supernatant of ultracentrifugation at 14×l04 g, and in fractions obtained by acetone of 35~50 per cent and by ammonium sulfate of 0.5~0.6 saturation.The inactive protease has the same resistance against pH or heat treatments as active protease has. The activation of the inactive protease was maximal between pH 5~6, and was accelerated by several kinds of protease, and was not affected by thioglycollate and KCN. 相似文献
17.
Tsutomu Nishihara Masafumi Tomita Noriyasu Yamanaka Tomio Ichikawa Masaomi Kondo 《Microbiology and immunology》1980,24(2):105-112
Properties of alkali-soluble components from the spore coat of Bacillus megaterium were examined by physicochemical methods. They were composed of acidic polypeptides of various molecular weights with small amounts of phosphorus and sugar. They were allowed to dissociate to unit components by incubation with SDS. The major component was partially purified by gel filtration, and shown to have a mean molecular weight of about 11,000. 相似文献
18.
N. Hajela A. H. Pande S. Sharma D. N. Rao K. Hajela 《Journal of plant biochemistry and biotechnology.》1999,8(1):57-60
A doubleheaded protease inhibitor showing inhibition of bovine pancreatic trypsin and α-chymotrypsin was isolated and purified from the seeds of Phaseolus mungo. The molecular weight of the protease inhibitor was found to be 14.2 kD by SDS-PAGE analysis and gel filtration. The native inhibitor inhibited trypsin and α-chymotrypsin stoichiometrically at the molar ratio 1:1 and 2:1 respectively. The Ki app for trypsin was found to be 0.35 nM and for α-chymotrypsin to be 2.4 nM. Bovine pepsin was not inhibited by the inhibitor. However, the pepsin treated inhibitor was still able to inhibit trypsin and α-chymotrypsin. The inhibitor was stable in 8M urea. Addition of 0.2 M mercaptoethanol resulted in significant loss of inhibitory activity. The inhibitor was extremely heat stable with only 50% loss of inhibitory activity after heating for 100°C for 20 min. Thus, the Phaseolus mungo trypsin/chymotrypsin inhibitor resembles other Bowman-Birk protease inhibitors. 相似文献
19.
Jorge Luis Ayala-Lujan Vidhya Vijayakumar Mei Gong Rachel Smith Araceli E. Santiago Fernando Ruiz-Perez 《PloS one》2014,9(9)
The serine protease autotransporter from Enterobacteriaceae (SPATE) family, which number more than 25 proteases with apparent diverse functions, have been phylogenetically divided into two distinct classes, designated 1 and 2. We recently demonstrated that Pic and Tsh, two members of the class-2 SPATE family produced by intestinal and extraintestinal pathogenic E. coli, were able to cleave a number of O-glycosylated proteins on neutrophils and lymphocytes resulting in impaired leukocyte functions. Here we show that most members of the class-2 SPATE family have lectin-like properties and exhibit differential protease activity reliant on glycoprotein type and cell lineage. Protease activity was seen in virtually all tested O-glycosylated proteins including CD34, CD55, CD164, TIM1, TIM3, TIM4 and C1-INH. We also show that although SPATE proteins bound and cleaved glycoproteins more efficiently on granulocytes and monocytes, they also targeted glycoproteins on B, T and natural killer lymphocytes. Finally, we found that the characteristic domain-2 of class-2 SPATEs is not required for glycoprotease activity, but single amino acid mutations in Pic domain-1 to those residues naturally occurring in domain-1 of SepA, were sufficient to hamper Pic glycoprotease activity. This study shows that most class-2 SPATEs have redundant activities and suggest that they may function as immunomodulators at several levels of the immune system. 相似文献
20.
Martine Ruer Georg Krainer Philip Gröger Michael Schlierf 《Journal of molecular biology》2018,430(22):4592-4602
AAA+ proteases are essential players in cellular pathways of protein degradation. Elucidating their conformational behavior is key for understanding their reaction mechanism and, importantly, for elaborating our understanding of mutation-induced protease deficiencies. Here, we study the structural dynamics of the Thermotoga maritima AAA+ hexameric ring metalloprotease FtsH (TmFtsH). Using a single-molecule Förster resonance energy transfer approach to monitor ATPase and protease inter-domain conformational changes in real time, we show that TmFtsH—even in the absence of nucleotide—is a highly dynamic protease undergoing sequential transitions between five states on the second timescale. Addition of ATP does not influence the number of states or change the timescale of domain motions but affects the state occupancy distribution leading to an inter-domain compaction. These findings suggest that thermal energy, but not chemical energy, provides the major driving force for conformational switching, while ATP, through a state reequilibration, introduces directionality into this process. The TmFtsH A359V mutation, a homolog of the human pathogenic A510V mutation of paraplegin (SPG7) causing hereditary spastic paraplegia, does not affect the dynamic behavior of the protease but impairs the ATP-coupled domain compaction and, thus, may account for protease malfunctioning and pathogenesis in hereditary spastic paraplegia. 相似文献