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Kuniyo Inouye - One of the best experts on this subject based on the ideXlab platform.
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effects of site directed mutagenesis in the n terminal domain of thermolysin on its stabilization
Journal of Biochemistry, 2013Co-Authors: Yuichi Kawasaki, Kiyoshi Yasukawa, Kuniyo InouyeAbstract:Thermolysin [EC 3.4.24.27] is a thermostable neutral metalloproteinase produced in the culture broth of Bacillus Thermoproteolyticus (1, 2). It consists of 316 amino acid residues with one zinc ion essential for enzyme activity and four calcium ions required for structural stability (3–6). Based on the structural data (7, 8), it consists of a β-rich N-terminal domain and an α-helical C-terminal domain. The Ca2+-binding sites I, II and IV are located in the C-terminal domain, and the Ca2+-binding site III is located in the N-terminal domain (7, 8) (Fig. 1A). When thermolysin is incubated with ethylenediamine-N,N,N′,N′-tetraacetic acid (EDTA), all calcium ions are removed from the thermolysin molecule and autolysis occurs (9–11). Thermolysin catalyses specifically the hydrolysis of peptide bonds containing hydrophobic amino acid residues (12, 13). It is widely used for the peptide bond formation through reverse reaction of hydrolysis, in particular, N-carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester (ZDFM), the precursor of an artificial sweetener aspartame, from N-carbobenzoxy-L-aspartic acid (ZD) and L-phenylalanine methyl ester (FM) (2, 14). Improvement of its activity and stability and modification of its pH-activity profile are thus still important goals (15). We have reported that high concentrations (1–5 M) of neutral salts increase the activity and stability of thermolysin (13, 14, 16, 17). Fig. 1 Structure of thermolysin. (A) Whole structure of the WT. The overall protein structure (ribbon model), the mutated residues (ball and stick model) and zinc and calcium ions (sphere) are shown. (B) Close-up view of the Ca2+-binding site III of WT. The ... Site-directed mutagenesis experiments of thermolysin (18–26) and thermolysin-like protease (TLP-ste) [EC 3.4.24.4] (27, 28) have generated a number of variant enzymes with improved activity and/or stability. TLP-ste is a neutral metalloproteinase from Bacillus stearothermophilus. It consists of 319 amino acid residues and differs from thermolysin at 44 out of 319 residues. (In this study, the amino acid sequence of thermolysin is adopted for numbering that of TLP-ste.) In TLP-ste, a single mutation Ser65→Pro was first reported to increase stability (27). (In this study, the mutation of a residue, e.g. Ser65 to Pro is designated as Ser65→Pro, and the variant enzyme bearing Ser65→Pro is designated as S65P.) After that, a double mutation Gly8→Cys/Asn60→Cys, designed to introduce a disulphide bridge between the amino acid residues 8 and 60, was reported to increase stability (28). In thermolysin, we reported that the mutational combination of Gly8→Cys/Asn60→Cys and Ser65→Pro increases stability without affecting activity (20). These results suggest that the N-terminal domain is important for stability in thermolysin and TLP-ste. However, little is known about the mechanism of stabilization by these mutations. In this study, we examined the effects of the mutations Gly8→Cys/Asn60→Cys and Ser65→Pro on the thermal denaturation and inactivation of thermolysin. Our results suggest that Gly8→Cys/Asn60→Cys and Ser65→Pro stabilize thermolysin by improving its affinity to calcium ions.
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a new method for the extracellular production of recombinant thermolysin by co expressing the mature sequence and pro sequence in escherichia coli
Protein Engineering Design & Selection, 2007Co-Authors: Kiyoshi Yasukawa, Masayuki Kusano, Kuniyo InouyeAbstract:: Thermolysin, a representative zinc metalloproteinase from Bacillus Thermoproteolyticus, is synthesized as inactive pre-proenzyme and receives autocatalytic cleavage of the peptide bond linking the pro- and mature sequences. The conventional expression method for recombinant thermolysin requires the autocatalytic cleavage, so that production of a mutant thermolysin is affected by its autocatalytic digestion activity. In this study, we have established a new expression method that does not require the autocatalytic cleavage. The mature sequence of thermolysin containing an NH(2)-terminal pelB leader sequence and the pre-prosequence of thermolysin were co-expressed constitutively in Escherichia coli as independent polypeptides under the original promoter sequences in the npr gene which encodes thermolysin. Unlike the conventional expression method, not only the wild-type thermolysin but also mutant thermolysins [E143A (Glu143 is replaced with Ala), N112A, N112D, N112E, N112H, N112K and N112R] were produced into the culture medium. The wild-type enzyme expressed in the present method was indistinguishable from that expressed in the conventional method based on autocatalytic cleavage, as assessed by hydrolysis of N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide and N-carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester. The present method should be useful especially for preparation of active-site mutants of thermolysin, which might have suppressed autocatalytic digestion activity. The results also demonstrate clearly that the covalent linking between the pro- and mature sequences is not necessary for the proper folding of the mature sequence by the propeptide in thermolysin.
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engineering expression purification and production of recombinant thermolysin
Biotechnology annual review, 2007Co-Authors: Kuniyo Inouye, Masayuki Kusano, Yasuhiko Hashida, Masashi Minoda, Kiyoshi YasukawaAbstract:Abstract Thermolysin [EC 3.4.24.27] is a thermostable neutral zinc metalloproteinase originally identified in the culture broth of Bacillus Thermoproteolyticus Rokko. Since the discovery in 1962, the enzyme has been extensively studied regarding its structure and catalytic mechanism. Today, thermolysin is a representative of zinc metalloproteinase and an attractive target in protein engineering to understand the catalytic mechanism, thermostability, and halophilicity. Thermolysin is used in industry, especially for the enzymatic synthesis of N -carbobenzoxy l -Asp- l -Phe methyl ester (ZDFM), a precursor of an artificial sweetener, aspartame. Generation of genetically engineered thermolysin with higher activity in the synthesis of ZDFM has been highly desired. In accordance with the expansion of studies on thermolysin, various strategies for its expression and purification have been devised and successfully used. In this review, we aim to outline recombinant thermolysins associated with their engineering, expression, purification, and production.
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extracellular production of recombinant thermolysin expressed in escherichia coli and its purification and enzymatic characterization
Protein Expression and Purification, 2006Co-Authors: Kuniyo Inouye, Masayuki Kusano, Yasuhiko Hashida, Masashi Minoda, Teisuke Takita, Haruko Sakurama, Kiyoshi YasukawaAbstract:Thermolysin is a representative zinc metalloproteinase derived from Bacillus Thermoproteolyticus and a target in protein engineering to understand the catalytic mechanism and thermostability. Extracellular production of thermolysin has been achieved in Bacillus, but not in Escherichia coli, although it is the most widely used as a host for the production of recombinant proteins. In this study, we expressed thermolysin as a single polypeptide pre-proenzyme in E. coli under the original promoter sequences in the npr gene, the gene from B. Thermoproteolyticus, which encodes thermolysin. Active mature thermolysin (34.6 kDa) was secreted into the culture medium. The recombinant thermolysin was purified to homogeneity by sequential column chromatography procedures of the supernatant with hydrophobic-interaction chromatography followed by affinity chromatography. The purified recombinant product is indistinguishable from natural thermolysin from B. Thermoproteolyticus as assessed by hydrolysis of N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide and N-carbobenzoxy-L-asparatyl-L-phenylalanine methyl ester. The results demonstrate that our expression system should be useful for structural and functional analysis of thermolysin.
A. Beaumont - One of the best experts on this subject based on the ideXlab platform.
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Cloning and expression in Bacillus subtilis of the npr gene from Bacillus Thermoproteolyticus Rokko coding for the thermostable metalloprotease thermolysin
Biochemical Journal, 1994Co-Authors: Michael O'donohue, B. Roques, A. BeaumontAbstract:We report the isolation, cloning and expression, in Bacillus subtilis, of the gene coding for thermolysin, a thermostable metalloprotease which is produced by Bacillus Thermoproteolyticus Rokko. The nucleotide sequence has revealed that, like neutral proteases produced by other members of the Bacillus species, thermolysin is probably produced as a preproenzyme carrying a typical N-terminal membrane signal sequence. Further, the thermolysin gene shares a strong homology with two other previously cloned genes from two different strains of Bacillus stearothermophilus. The sequence of the mature secreted protease, inferred from the DNA sequence, is, with two exceptions, identical with the previously published protein sequence of thermolysin [Titani, Hermodson, Ericsson, Walsh and Neurath (1972) Nature (London) 238, 35-37]. The exceptions are Asn37 and Gln119, originally reported to be Asp and Glu respectively. The biochemical characterization of the secreted recombinant protein shows that it is indistinguishable from the wild-type thermolysin.
Alberto Saiani - One of the best experts on this subject based on the ideXlab platform.
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Enzymatically triggered peptide hydrogels for 3D cell encapsulation and culture
Journal of peptide science : an official publication of the European Peptide Society, 2014Co-Authors: Laura Szkolar, Jean-baptiste Guilbaud, Aline F. Miller, Julie E. Gough, Alberto SaianiAbstract:We have investigated the possibility of using enzymatically triggered peptide hydrogels for the encapsulation and culture of cells. Based on recent work done on the enzymatically triggered gelation of FEFK (F, phenylalanine; E, glutamic acid; K, lysine) using thermolysin, a protease enzyme from Bacillus Thermoproteolyticus Rokko, we have investigated the possibility of using this gelation triggering mechanism to encapsulate cells within a 3D hydrogel matrix. First, the properties of enzymatically triggered hydrogels prepared in phosphate buffer solution were investigated and compared with the properties of hydrogels prepared in HPLC grade water from our previous work. We showed that the use of phosphate buffer solution allowed the production of hydrogels with very high shear moduli (>1 MPa). The gelation kinetics was also investigated, and the mechanical properties of the system were shown to closely follow the synthesis of the octapeptide by the enzyme through reverse hydrolysis. In a second phase, we developed, on the basis of information acquired, a facile protocol for the encapsulation of cells and plating of the hydrogel. Human dermal fibroblasts were then used to exemplify the use of these materials. FEFEFKFK octapeptide hydrogels prepared under the same conditions and with the same mechanical properties were used as a control. We showed that no significant differences were observed between the two systems and that after a decrease in cell number on day 1, cells start to proliferate. After 5 days of culture, the cells can be seen to start to adopt a stretched morphology typical of fibroblasts. The results clearly show that the protocol developed minimises the potential detrimental effect that thermolysin can have on the cells and that these enzymatically triggered hydrogels can be used for the 3D encapsulation and culture of cells.
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Effect of Enzyme Concentration of the Morphology and Properties of Enzymatically Triggered Peptide Hydrogels
Biomacromolecules, 2013Co-Authors: Jean-baptiste Guilbaud, Cyrille Rochas, Aline F. Miller, Alberto SaianiAbstract:We have recently shown that thermolysine, a protease enzyme obtained from Bacillus Thermoproteolyticus rokko, can be used to trigger the gelation of FEFK (F, phenylalanine; E, glutamic acid; K, lysine) tetrapeptides through reverse hydrolysis and formation of longer peptide sequences, mainly octapeptides, that self-assemble readily. In this article we investigate the effect of enzyme concentration on the morphology and properties of enzymatically triggered peptide hydrogels using HPLC, FTIR, real-time SAXS, TEM, and shear rheology. We have shown that the enzyme concentration, Cenz, does not affect the final composition of the samples. Instead, this is dictated by the initial tetrapeptide concentration, C0, suggesting the existence of a chemical equilibrium. We went on to show that Cenz does not affect the self-assembly of these peptides at a molecular level either nor the structure of the fibrillar network formed at the nanometer scale. Interestingly, the mechanical properties were found to be affected by...
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Effect of Enzyme Concentration of the Morphology and Properties of Enzymatically Triggered Peptide Hydrogels
2013Co-Authors: Jean-baptiste Guilbaud, Cyrille Rochas, Aline F. Miller, Alberto SaianiAbstract:We have recently shown that thermolysine, a protease enzyme obtained from Bacillus Thermoproteolyticus rokko, can be used to trigger the gelation of FEFK (F, phenylalanine; E, glutamic acid; K, lysine) tetrapeptides through reverse hydrolysis and formation of longer peptide sequences, mainly octapeptides, that self-assemble readily. In this article we investigate the effect of enzyme concentration on the morphology and properties of enzymatically triggered peptide hydrogels using HPLC, FTIR, real-time SAXS, TEM, and shear rheology. We have shown that the enzyme concentration, Cenz, does not affect the final composition of the samples. Instead, this is dictated by the initial tetrapeptide concentration, C0, suggesting the existence of a chemical equilibrium. We went on to show that Cenz does not affect the self-assembly of these peptides at a molecular level either nor the structure of the fibrillar network formed at the nanometer scale. Interestingly, the mechanical properties were found to be affected by Cenz, where the shear moduli of the hydrogels were found to increase with increasing Cenz. These results suggest that morphological differences between the hydrogels at the microscale are at the origin of their difference in mechanical properties. In this paper, we propose a morphological model in which denser network regions are found around the enzymes, resulting in the creation of heterogeneous networks. These were confirmed by TEM measurements. The existence of these denser network regions will result in the reinforcement of the hydrogels, thus, explaining the high shear moduli obtained increasing Cenz
Kohei Oda - One of the best experts on this subject based on the ideXlab platform.
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Molecular cloning of the gene encoding Vibrio metalloproteinase vimelysin and isolation of a mutant with high stability in organic solvents.
Journal of biochemistry, 2005Co-Authors: Toshihiro Takahashi, Hiroshi Oyama, Kohei OdaAbstract:VimelysinisauniquemetalloproteinasefromVibriosp.T1800exhibitinghighactivityatlow temperature and high stability in organic solvents such as ethanol. A 1,821 bp openreading frame of the vimelysin gene encoded 607 amino acid residues consisting of anN-terminal pro-region, a mature enzyme, and a C-terminal pro-region. The matureenzyme region showed 80%, 57% and 35% sequence identity with the mature forms ofvibriolysin from V. vulnificus, pseudolysin from Pseudomonas aeruginosa, and thermo-lysin from Bacillus Thermoproteolyticus, respectively. The catalytic residues and zinc-bindingmotifsofmetalloproteinasesarewellconservedinvimelysin.ThevimelysingenewasexpressedinE.coliJM109cellsandtherecombinantenzymewaspurifiedasa38-kDamature form from cell-free extracts. The purified recombinant enzyme is indistinguish-ablefromtheenzymepurifieddirectlyfromVibrio.Toobtainmutantsexhibitinghigherstabilityinorganicsolvents,randommutationswereintroducedbyerror-pronePCRand600transformantswerescreened.TheN123Dmutantexhibitstwotimeshigherstabilityin organic solvents than the wild-type enzyme. A plausible mechanism for the stabilityof the N123D mutant in organic solvents was discussed based on homology models ofvimelysin and the N123D mutant.Key words: alcohol resistance, metalloproteinase, pseudolysin, random mutagenesis,vimelysin.Microbial proteinases have many unique characteristics.For instance, Achromobactor I proteinase (1, 2) andStaphylococcus aureus V8 proteinase (3, 4) are unique intheir strict substrate specificities. Thermolysin fromBacillus Thermoproteolyticus (5) exhibits high thermal sta-bility and proteinase from alkalophilic Bacillus (6) exhibitsmaximum activity under extremely alkaline conditions.In order to utilize it as an additive in detergents or forpeptide synthesis, we succeeded in isolating Vibrio sp.T1800 from marine bacteria, which produce a proteinaseexhibiting high stability in organic solvents such asethanol (7, 8). The purified metalloproteinase, namedvimelysin, has a molecular mass of 38 kDa and exhibitshigher stability in organic solvents than thermolysin, arepresentative metalloproteinase from microorganisms.Vimelysin showed 40% activity compared to that of a con-trol in the presence of 10% ethanol, whereas thermolysinshowed only 5% activity under the same conditions (7, 8).As for substrate specificity, vimelysin preferred Phe atthe P1
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Activity and stability of a neutral protease from Vibrio sp. (vimelysin) in a pressure-temperature gradient.
European journal of biochemistry, 2000Co-Authors: Hidekazu Ikeuchi, Shigeru Kunugi, Kohei OdaAbstract:The apparent second-order rate constant of hydrolysis of Fua-Gly-LeuNH2 by vimelysin, a neutral protease from Vibrio sp. T1800, was measured in a variable pressure–temperature gradient (0.1–400 MPa and 5–40 °C). The apparent maximum rate was observed at approximately 15 °C and 150–200 MPa; the pressure-activation ratio (kcat/Km(max)/kcat/Km(0.1 MPa)) was reached about sevenfold. The pressure dependence of the kcat and Km parameters at constant temperature (25 °C) revealed that the pressure-activation below 200 MPa was mainly caused by a change in the kcat parameter. The change in the intrinsic fluorescence intensity of vimelysin was also measured in a pressure-temperature plane (0.1–400 MPa and −20 to +60 °C). The fluorescence intensity was found to decrease by increasing pressure and temperature, and the isointensity contours were more or less circular. The tangential lines to the contours at high temperatures and low to medium pressures seem to have slightly positive slopes, which was reflected by the higher residual activities left after incubations at higher temperatures and medium pressure (200 MPa and 50 °C) and by the almost intact secondary structure left after 1 h of incubation at 200 MPa and 40 °C, as studied by circular dichroism. These results were compared with the corresponding results for thermolysin, a moderately thermostable protease from Bacillus Thermoproteolyticus. Apparent differences that might be related to the temperature adaptations of the respective source microbes are also discussed.
Michael O'donohue - One of the best experts on this subject based on the ideXlab platform.
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Cloning and expression in Bacillus subtilis of the npr gene from Bacillus Thermoproteolyticus Rokko coding for the thermostable metalloprotease thermolysin
Biochemical Journal, 1994Co-Authors: Michael O'donohue, B. Roques, A. BeaumontAbstract:We report the isolation, cloning and expression, in Bacillus subtilis, of the gene coding for thermolysin, a thermostable metalloprotease which is produced by Bacillus Thermoproteolyticus Rokko. The nucleotide sequence has revealed that, like neutral proteases produced by other members of the Bacillus species, thermolysin is probably produced as a preproenzyme carrying a typical N-terminal membrane signal sequence. Further, the thermolysin gene shares a strong homology with two other previously cloned genes from two different strains of Bacillus stearothermophilus. The sequence of the mature secreted protease, inferred from the DNA sequence, is, with two exceptions, identical with the previously published protein sequence of thermolysin [Titani, Hermodson, Ericsson, Walsh and Neurath (1972) Nature (London) 238, 35-37]. The exceptions are Asn37 and Gln119, originally reported to be Asp and Glu respectively. The biochemical characterization of the secreted recombinant protein shows that it is indistinguishable from the wild-type thermolysin.
