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1.
The conserved histidine residues, His-191, His-227, His-345, and His-378, in Bacillus stearothermophilus leucine aminopeptidase II (LAPII) were replaced with leucine by site-directed mutagenesis. The overexpressed wild-type and mutant enzymes have been purified by nickel-chelate chromatography and their molecular masses were approximately 44.5 kDa. Under assay conditions, no LAP activity was detected in H345L and H378L. Although the Km value for H191L increased more than 30% with respect to the wild-type LAPII, alteration in this residue did not lead to a significant change on the catalytic efficiency. The 39% decrease in Kcat/Km for H227L was partly caused by a 3.9-fold increase in Km value. Based on these results, it is suggested that His-345 and His-378 play a crucial role in the catalytic reaction of B. stearothermophilus LAPII. 相似文献
2.
Ruey-Shyang Chen Shin-Ling Yang Yu-Wen Hua Meng-Chun Chi Long-Liu Lin 《World journal of microbiology & biotechnology》2005,21(8-9):1477-1482
Summary To understand the structure–function relationships of Bacillus stearothermophilus leucine aminopeptidase II (LAPII), each of the four conserved asparagine residues was replaced with leucine, aspartate, and
lysine respectively by site-directed mutagenesis. The over-expressed wild-type and mutant enzymes with an apparent molecular
mass of approximately 44.5 kDa were purified to homogeneity by nickel-chelate chromatography. Substitution of Asn-245, Asn-335,
and Asn-341 with Lys generated variants with a dramatic loss of LAP activity. Kinetic analysis of Asn-373 variants with p-leucine-nitroanilide as the substrate revealed an increase in kcat with no significant change in Km, leading to a more than 2-fold increase in the catalytic efficiency. Thermostability assays showed that replacement of Asn-335,
Asn-341, and Asn-373 by aspartic acid markedly increased the half-life of the enzyme at 70 °C, indicating that the deamination
of these residues may have a deleterious effect on LAPII. 相似文献
3.
Bacillus kaustophilus leucine aminopeptidase (bkLAP) was sensitive to oxidative damage by hydrogen peroxide. To improve its oxidative stability,
the oxidation-sensitive methionine residues in the enzyme were replaced with leucine by site-directed mutagenesis. The variants,
each with an apparent molecular mass of approximately 54 kDa, were overexpressed in recombinant Escherichia coli M15 cells and purified to homogeneity by nickel-chelate chromatography. The specific activity for M282L, M285L, M289L and
M321L decreased by more than 43%, while M400L, M426L, M445L, and M485L showed 191, 79, 313, and 103%, respectively, higher
activity than the wild-type enzyme. Although the mutations did not cause significant changes in the K
m value, more than 67.8% increase in the value of k
cat/K
m was observed in the M400L, M426L, M445L and M485L. In the presence of 50 mM H2O2, most variants were more stable with respect to the wild-type enzyme, indicating that the oxidative stability of the enzyme
can be improved by engineering the methionine residues.
This revised version was published online in June 2006 with corrections to the Cover Date. 相似文献
4.
Leucine aminopeptidase (LAP) is an exopeptidase that catalyzes the hydrolysis of amino acid residues from the amino terminus of proteins and peptides. Sequence alignment shows that the conserved Ala348 and Gly350 residues of Bacillus kaustophilus LAP (BkLAP) are located right next to a coordinated ligand. We further investigated the roles of these two residues by performing computer modeling and site-directed mutagenesis. Based on the modeling, the carbonyl group of Ala348 interacts with Asn345 and Asn435, and that of Gly350 with Ile353 and Leu354, where these interactions might maintain the zinc-coordinated residues at their correct positions. Replacement of Ala348 with Arg resulted in a dramatic reduction in LAP activity. A complete loss of the activity was also observed in A348E, A348V, and the Gly350 variants. Measurement of intrinsic tryptophan fluorescence revealed alteration of the microenvironment of aromatic amino acid residues, while circular dichroism spectra were nearly identical for wild-type and all mutant enzymes. Protein modeling and site-directed mutagenesis suggest that residues Ala348 and Gly350 are essential for BkLAP in maintaining a stable active-site environment for the catalytic reaction. 相似文献
5.
Chien-Li Wu Yi-Pu Chen Jia-Ci Yang Huei-Fen Lo Long-Liu Lin 《Journal of Molecular Catalysis .B, Enzymatic》2008,54(3-4):83-89
Bacillus stearothermophilus leucine aminopeptidase II tagged C-terminally with either tri- or nona-lysine (BsLAPII-Lys3/9) was constructed and over-expressed in Escherichia coli M15 (pRep4). The recombinant enzymes were purified to homogeneity by nickel-chelate chromatography and their molecular masses were determined to be approximately 45 kDa by SDS/PAGE. Surface modification of colloidal gold with 16-mercaptohexadecanoic acid was employed to generate the carboxylated nanoparticles. BsLAPII-Lys9 was efficiently immobilized onto the carboxylated gold nanoparticles (AuNP-COOH) and the obtained bioconjugate showed excellent biocatalytic activity in the immobilized form. Additionally, the bioconjugate material exhibited a significant enhancement in temperature stability and could be reused over 5 successive cycles. 相似文献
6.
Leucine aminopeptidases (LAPs) are exopeptidases that remove the N-terminal L-leucine from peptide substrates. Oxidative stability assay showed that the recombinant Bacillus stearothermophilus LAP II (rLAPII) was sensitive to oxidative damage by hydrogen peroxide at the elevated temperature. The H2O2-treated enzyme experienced obvious changes in the secondary structure when the oxidant concentration increased to 300 mM. To investigate the role of methionine residues on the oxidative inactivation, each of the five methionine residues in the rLAPII was replaced with leucine by site-directed mutagenesis. The mutant enzymes with an apparent Mr of approximately 44.5 kDa were overexpressed in Escherichia coli and were purified to homogeneity by nickel-chelate chromatography. The specific activities for Met82Leu, Met88Leu, Met254Leu, and Met382Leu were similar to that of the wild-type enzyme, whereas a reduced activity was observed in Met136Leu. The 50% decrease in the catalytic efficiency (kcat/Km) for Met136Leu was caused by 47% decrease in kcat value. As compared with the wild-type enzyme, all mutant proteins were more sensitive to the oxidant, implying that the methionine residues of B. stearothermophilus LAP II are important for the protection of the enzyme from oxidative inactivation. 相似文献
7.
Leucine dehydrogenase (l-leucine: NAD+ oxidoreductase, deaminating, EC 1.4.1.9) has been purified to homogeneity from a moderate thermophilic bacterium, Bacillus stearothermophilus. Am improved method of preparative slab gel electrophoresis was used effectively to purify it. The enzyme has a molecular mass of about 300,000 and consists of six subunits with identical molecular mass (Mr, 49,000). The enzyme does not lose its activity by heat treatment at 70° C for 20 min, and incubation in the pH range of 5.5–10.0 at 55° C for 5 min. It is stable in 10 mM phosphate buffer (pH 7.2) containing 0.01% 2-mercaptoethanol at over 1 month, and is resistant to detergent and ethanol treatment. The enzyme catalyzes the oxidative deamination of branched-chain l-amino acids and the reductive amination of their keto analogs in the presence of NAD+ and NADH, respectively, as the coenzymes. The pH optima are 11 for the deamination of l-leucine, and 9.7 and 8.8 for the amination of -ketoisocaproate and -ketoisovalerate, respectively. The Michaelis constants were determined: 4.4 mM for l-leucine, 3.3 mM for l-valine, 1.4 mM for l-isoleucine and 0.49 mM for NAD+ in the oxidative deamination. The B. stearothermophilus enzyme shows similar catalytic properties, but higher activities than that from Bacillus sphaericus.Dedicated to Prof. Dr. G. Drews on the occasion of his 60th birthday 相似文献
8.
The importance of the conserved Tyr352 and Asp380 residues of Bacillus stearothermophilus aminopeptidase II (AP-II) was investigated by site-directed mutagenesis. The wild-type and mutant enzymes were expressed in recombinant Escherichia coli M15 cells and the 45-kD proteins were purified from the cell-free extracts by Ni(2+)-NTA resin. The specific activity for Tyr352 and Asp380 replacements was decreased by more than 3.5-fold. Detailed analysis of the kinetic consequences in the mutant proteins revealed that the K (m) values were increased 1.9- to 2.6-fold with respect to wild-type enzyme. Catalytic efficiencies (k (cat)/K (m)) of mutant proteins were between 3.5- and 31-fold lower than the corresponding value of the wild-type enzyme. Tryptophan emission fluorescence and circular dichroism spectra were nearly identical for wild-type and mutant enzymes. These results indicate that residues Tyr352 and Asp380 are essential for the proper function of AP-II. 相似文献
9.
Summary A chimericBacillus stearothermophilus leucine aminopeptidase II (LAPsbd) has been constructed by introducing the raw-starch-binding domain of Bacillus sp. strain TS-23 α-amylase into the enzyme. LAPsbd was adsorbed onto raw starch and the adsorbed enzyme could be eluted from the adsorbent by soluble starch in 20 mM Tris–HCl buffer (pH 8.0). The adsorption of LAPsbd onto raw starch was affected by raw starch concentration, pH, and temperature, while the temperature and incubation time had no obvious effects on the elution of adsorbed enzyme. The molecular weight of purified enzyme was estimated to be 61 kDa. About 84% of LAPsbd in the cell free extract was recovered through one adsorption–elution cycle with a purification of 20-fold. The high quantity and purity of the recovered enzyme coupled with the easy performance make the adsorption–elution procedure suitable for industrial applications. 相似文献
10.
Lin LL Hsu WH Wu CP Chi MC Chou WM Hu HY 《Extremophiles : life under extreme conditions》2004,8(1):79-87
Two degenerate primers established from the consensus sequences of bacterial leucine aminopeptidases (LAP) were used to amplify a 360-bp gene fragment from the chromosomal DNA of thermophilic Bacillus kaustophilus CCRC 11223 and the amplified fragment was successfully used as a probe to clone a leucine aminopeptidase (lap) gene from a genomic library of the strain. The gene consists of an open reading frame (ORF) of 1,494 bp and encodes a protein of 497 amino acid residues with a calculated molecular mass of 53.7 kDa. The complete amino acid sequence of the cloned enzyme showed greater than 30% identity with prokaryotic and eukaryotic LAPs. Phylogenetic analysis showed that B. kaustophilus LAP is closely related to the enzyme from Bacillus subtilis and is grouped with the M17 family. His6-tagged LAP was generated in Escherichia coli by cloning the coding region into pQE-30 and the recombinant enzyme was purified by nickel-chelate chromatography. The pH and temperature optima for the purified enzyme were 8 and 65°C, respectively, and 50% of its activity remained after incubation at 60°C for 32 min. The enzyme preferentially hydrolyzed l-leucine-p-nitroanilide (l-Leu-p-NA) followed by Cys derivative.Communicated by G. Antranikian 相似文献
11.
The importance of Thr-346 and Leu-352 residues in Bacillus kaustophilus leucine aminopeptidase (BkLAP) was explored by site-directed mutagenesis. The impact of substitutions at these positions was evaluated with His6-BkLAP fusion proteins expressed in Escherichia coli. Substitution of Thr-346 with Tyr, Arg, and Leu, respectively, resulted in a dramatic reduction in LAP activity. A complete loss of activity was observed in L352E and L352R variants with the exception of L352 V, which retained approximately 60% of the wild-type activity. Zinc content analysis and protein modeling suggested that Thr-346 and Leu-352 of BkLAP play a role in maintaining the coordination environment for the zinc-binding residues. 相似文献
12.
Thermostable extracellular protease of Bacillus stearothermophilus: factors affecting its production
N. A. Razak M. Y. A. Samad M. Basri W. M. Z. W. Yunus K. Ampon A. B. Salleh 《World journal of microbiology & biotechnology》1994,10(3):260-263
A strain of protease-producing Bacillus stearothermophilus has been isolated. Glycerol was the best carbon source for production whereas yeast extract was the best nitrogen source. The bacterium could grow up to 70°C but optimum protease production was at 60°C. Best initial pH for protease production was 5. Alkaline pH inhibited production. The enzyme was stable at 60°C for 18 h and was inhibited by EDTA, PMSF and HgCl2.The authors are with the Enzyme and Microbial Technology Group, Faculty of Science and Environmental Studies, Universiti Pertanian Malaysia, 43400 UPM Serdang, Selangor, Malaysia 相似文献
13.
Purification and characterization of hyperthermotolerant leucine aminopeptidase from Geobacillus thermoleovorans 47b 总被引:1,自引:0,他引:1
Deejing S Yoshimune K Lumyong S Moriguchi M 《Journal of industrial microbiology & biotechnology》2005,32(7):269-276
A thermophilic bacterium, which we designated as Geobacillus thermoleovorans 47b was isolated from a hot spring in Beppu, Oita Prefecture, Japan, on the basis of its ability to grow on bitter peptides as a sole carbon and nitrogen source. The cell-free extract from G. thermoleovorans 47b contained leucine aminopeptidase (LAP; EC 3.4.11.10), which was purified 164-fold to homogeneity in seven steps, using ammonium sulfate fractionation followed by the column chromatography using DEAE-Toyopearl, hydroxyapatite, MonoQ and Superdex 200 PC gel filtration, followed again by MonoQ and hydroxyapatite. The enzyme was a single polypeptide with a molecular mass of 42,977.2 Da, as determined by matrix-assisted laser desorption ionization and time-of-flight mass spectrometry, and was found to be thermostable at 90°C for up to 1 h. Its optimal pH and temperature were observed to be 7.6–7.8 and 60°C, respectively, and it had high activity towards the substrates Leu-p-nitroanilide (p-NA)(100%), Arg-p-NA (56.3%) and LeuGlyGly (486%). The Km and Vmax values for Leu-p-NA and LeuGlyGly were 0.658 mM and 25.0 mM and 236.2 mol min–1 mg–1 protein and 1,149 mol min–1 mg–1 protein, respectively. The turnover rate (kcat) and catalytic efficiency (kcat/ Km) for Leu-p-NA and LeuGlyGly were 10,179 s–1 and 49,543 s–1 and 15,470 mM–1 s–1 and 1981.7 mM–1 s–1, respectively. The enzyme was strongly inhibited by EDTA, 1,10-phenanthroline, dithiothreitol, -mercaptoethanol, iodoacetate and bestatin; and its apoenzyme was found to be reactivated by Co2+ . 相似文献
14.
15.
Sten Ståhl 《Archives of microbiology》1978,119(1):17-24
The calcium transport in resting vegetative cells of Bacillus stearothermophilus was studied by determining the retention of 45Ca in a membrane filter assay. The kinetics of death by vegetative cells, when suspended in buffer at 55°C, was also investigated. The calcium influx required the presence of an energy source, e.g. glucose-1-phosphate and the system exhibited saturation kinetics. The requirements for survival of the thermophilic cells reflected those of the calcium transport system. Thus, cells treated with nitrogen gas showed an increased thermal stability and a decreased efflux of calcium. The initial velocity of calcium influx correlated linearly with the survival of the cells after 1 min heating at 55° C. Lanthanum inhibited calcium influx and reduced survival. Magnesium did not inhibit calcium influx but could replace calcium as a stabilizing agent. The results suggest that the thermophilic cells are not intrinsically heat stable but survive due to a high cellular concentration of divalent ions.Abbreviations CFU
colony forming units
- CPM
counts per min
- NCA
National canners association
- CCCP
carbonyl cyanide m-chlorophenylhydrazone
- PMS
phenazine methosulfate 相似文献
16.
Genetics of leucine aminopeptidase in apple 总被引:1,自引:0,他引:1
A. G. Manganaris F. H. Alston 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1992,83(3):345-352
Summary Six zones of LAP activity were detected in apples, some of them tissue specific. Genetic studies in four of them revealed the presence of four genes LAP-1, LAP-2, LAP-3 and LAP-4 with 4, 5, 4 and 4 alleles respectively including two null alleles. There were no big differences in allelic frequency within cultivars, selections, rootstocks and Malus species. Close linkage was found between LAP-2 and resistance to mildew derived from White Angel. 相似文献
17.
Localized insertion of new S-layer during growth of Bacillus stearothermophilus strains 总被引:4,自引:0,他引:4
Bacillus stearothermophilus strains PV 72 and ATCC 12980 carry a crystalline surface layer (S-layer) with hexagonal (p6) and oblique (p2) symmetry, respectively. Sites of insertions of new subunits into the regular lattice during cell growth have been determined by the indirect fluorescent antibody technique and the protein A/colloidal gold technique.During S-layer growth on both bacillus strains the following common features were noted: 1. shedding of intact S-layer or turnover of individual subunits was not seen; 2. new S-layer was deposited in helically-arranged bands over the cylindrical surface of the cell at a pitch angle related to the orientation of the lattice vectors of the crystalline array; 3. little or no S-layer was inserted into pre-existing S-layer at the poles, and 4. septal regions and, subsequently, newly formed cell poles were covered with new S-layer protein. 相似文献
18.
Yuval Shoham Zeev Schwartz Alexander Khasin Orit Gat Zinaida Zosim Eugene Rosenberg 《Biodegradation》1992,3(2-3):207-218
During the bleaching of wood pulp for the paper industry, large amounts of chlorinated aromatic compounds are produced and released into the environment. These compounds are extremely toxic and are a major source of pollution. The paper and pulp industry is seeking for alternative methods for bleaching pulp. One such method involves the use of hemicellulases to release the colored lignohemicellulose. We have isolated and characterized several thermophilic bacteria which produce xylanases. One such strain, T-6, produced high levels of extracellular xylanase, free of cellulase and proteinase activities. Strain T-6 was classified as a strain of Bacillus stearothermophilus and was able to grow on defined medium containing xylose, methionine and asparagine at 65 °C. Xylanase activity was induced by either xylose or xylan; no activity was detected with other carbon sources, such as glycerol, acetate, lactose, glucose, maltose, fructose, mannose, galactose or sucrose. Xylanase constitutive mutants were obtained following mutagenesis and detection on p-nitrophenol -d-xylopyranoside containing agar plates. Xylanase T-6 was produced on large scale, and was purified and concentrated by a single adsorption-desorption step from a cation exchanger. The overall purification yield of a 1000 liter fermentation was 45%, resulting in a 98% pure enzyme. Xylanase T-6 was shown to partially remove lignin from unbleached pulp at 65 °C and pH 9.0, without loss in pulp viscosity. The enzyme-treated pulp was used to make handsheets that had higher brightness than untreated pulp. 相似文献
19.
The functional significance of amino acid residues Lys-265, Asp-270, Lys-277, Asp-288, Asp-347, Glu-349, and Arg-351 of Bacillus kaustophilus leucine aminopeptidase was explored by site-directed mutagenesis. Variants with an apparent molecular mass of approximately 54 kDa were overexpressed in Escherichia coli and purified to homogeneity by nickel-chelate chromatography. The purified mutant enzymes had no LAP activity, implying that these residues are important for the catalytic reaction of the enzyme. 相似文献
20.
The possibility that exopeptidases, i.e. aminopeptidases and carboxypeptidases, in addition to the previously studied endopeptidase
might also be developmentally regulated in daylily petals was examined. The level of leucine aminopeptidase and endopeptidase
activities changed after the flower was fully open while that of carboxypeptidase activity remained relatively unchanged throughout
senescence. Leucine aminopeptidase activity seemed to increase after the flower was fully open and peaked several hours earlier
than endopeptidase did. Taken together, it is postulated that leucine aminopeptidase might play a role in protein turnover
during flower opening and in the initiation of protein hydrolysis associated with petal senescence while the endopeptidase
could be responsible for the breakdown of the bulk of proteins at the later stages. The drop in leucine aminopeptidase activity
associated with the onset of daylily petal senescence was effectively halted by a cycloheximide treatment of cut daylily flowers
for 24 h which was previously shown to prolong the vase life of the flowers and prevent protein loss from the petals. Apart
from both being developmentally regulated in daylily petals, the leucine aminopeptidase activity and the previously studied
endopeptidase are different in several aspects. They appear to have different pH optima, 8 for leucine aminopeptidase and
6.2 for endopeptidase. Unlike the endopeptidase activity, no new leucine aminopeptidase isozymes appeared during petal senescence,
and the leucine aminopeptidase did not appear to belong to the cysteine class of proteolytic enzymes. 相似文献