1,721,148 research outputs found
Cometabolism of w-phenylalkanoic acids with butyric acid for efficient production of aromatic polyesters in Pseudomonas putida BM01
No full textPoly(3-hydroxy-5-phenylvalerate) [P(3HPV)] was efficiently accumulated from 5-phenylvalerate (5PV) in Pseudomonas putida BM01 in a mineral salts medium containing butyric acid (BA) as the cosubstrate. A novel aromatic copolyester, poly(5 mol% 3-hydroxy-4-phenylbutyrate co- 95 mol% 3-hydroxy-6-phenylhexanoate) [P(3HPB-co-3HPC)] was also synthesized from 6-phenylhexanoate (6PC) plus BA. The two aromatic polymers, P(3HPV) and P(3HPB-co-3HPC), were found to be amorphous and showed different glass-transition temperatures at 15°C and 10°C, respectively. When the bacterium was grown in a medium containing 20 mM 5PV as the sole carbon source for 140 h, 0.4 g/l of dry cells was obtained in a flask cultivation and 20 wt% of P(3HPV) homopolymer was accumulated in the cells. However, when it was grown with a mixture of 2 mM 5PV and 50 mM BA for 40 h, the yield of dry biomass was increased up to 25 g/l and the content of P(3HPV) in the dry cells was optimally 56 wt%. This efficient production of P(3HPV) homopolymer from the mixed substrate was feasible because BA only supported cell growth and did not induce any aliphatic PHA accumulation. The metabolites released into the PHA synthesis medium were analyzed using GC or GC/ MS. Two β-oxidation derivatives, 3-phenylpropionic acid and trans-cinnamic acid, were found in the 5PV-grown cell medium and these comprised 55-88 mol% of the 5PV consumed. In the 6PC-grown medium containing BA, seven β-oxidation and related intermediates were found, which included phenylacetic acid, 4-phenylbutyric acid, cis-4-phenyl-2-butenoic acid, trans-4-phenyl-3-butenoic acid, trans-4-phenyl-2-butenoic acid, 3-hydroxy-4-phenylbutyric acid, and 3-hydroxy-6-phenylhexanoic acid. Accordingly, based on the metabolite analysis, PHA synthesis pathways from the two aromatic carbon sources are suggested.open
Purification and characterization of a subtilisin D5, a fibrinolytic enzyme of Bacillus amyloliquefaciens DJ-5 isolated from Doenjang
No full textopen
Construction of non-invasively constitutive expression vectors using a metagenome-derived promoter for soluble expression of proteins
No full textExpression of soluble and functional proteins has been one of the critical challenges to many aspects of synthetic biology, metabolic and protein engineering. Among the current methods for expression of target proteins, constitutive expression systems offer several advantages over inducible systems, which require a chemical or physical inducer. In a previous study, a G196 DNA fragment containing constitutive promoters was mined from the soil metagenome and evaluated for the expression of target proteins in the functional and soluble state. In this study, we further improved this system by constructing a series of constitutive expression vectors, pCEM (using the CEM promoter trimmed from G196), pCEMT (incorporating rrnB T1 and T2 terminator into the downstream region of MCS in pCEM) and pRCEMT (grafting the cis-acting region of pCEMT into a low-copy-number plasmid). Subsequently, genes encoding GFPuv, esterase 1767 and β-glucosidase were subcloned into the resulting vectors, and their expression level and solubility were compared with those of IPTG-inducible vector systems pQE30 and pTrc99A. The extent of homogeneity and the ratio of the soluble fraction in the pRCEMT vector were relatively higher, without any delay of growth rate, than that of the pQE30 or pTrc99A. These results indicate that new expression vectors with moderate constitutive function could more easily lead to a homogenous population of cells expressing target proteins than those with conventionally inducible promoters.open
Preparation of soluble silk peptides by food-grade proteinases
No full textEnzymatic hydrolysis of silk fibers were investigated for the preparation of soluble silk peptides by ten food-grade proteases from Bacillus, Aspergillus, and plant sources. Silk fibers were dissolved for 1 hr in a 2:1 cosolvent (50% CaCl2: ethanol) by heating at 90°C. The silk solution was filtered to remove impurity particles and desalted for 50 hours by a dialysis process to remove the used cosolvent. When the silk hydrolysis was performed at 45°C for 2 hours, most proteases from Bacillus and Aspergillus generated large amounts of insoluble aggregates. On the contrary, proteases from plant sources produced much less aggregates during prolonged incubations and also exhibited high hydrolysis activities. In regards of the solubility and broad molecular sizes of produced silk peptides, Bromelain was finally selected and applied for the enzymatic hydrolysis of silk fibers.open
First-order kinetics analysis of monomer composition dependent polyhydroxyalkanoic acid degradation in Pseudomonas spp.
No full textThe intracellular degradation of polyhydroxyalkanoic acid (PHA) in pseudomonads was investigated by first-order kinetics analysis using the initial rate method. One type of PHA was accumulated in five Pseudomonas spp., P. oleovorans, P. aeruginosa, P. fluorescens, P. citronellolis, and P. putida, by growing them on octanoic acid. The monomer compositions of the five PHA were not significantly different from one another: 85-90 mol % 3-hydroxyoctanoic acid (3HO), 7-12 mol % 3-hydorxycaproic acid (3HC), and 3-6 mol % 3-hydroxydecanoic acid (3HD). The first-order degradation rate constants (k1) for the octanoate-derived PHA (designated P(3HO)) in the five species were in a similar range between 0.060 and 0.088 h-1. This may indicate the similar specificities of the five intracellular depolymerases. In addition, the similar k1 among the different species may correlate with the high degree of amino acid sequence identities (over 85 %) among the intracellular PHA depolymerase phaZ genes. Six other chemically different types of PHA were accumulated in P. putida from n-nonanoic acid, n-decanoic acid, 5-phenyvaleric acid, or 11-phenoxyundecanoic acid as a single or a mixed carbon source. The calculated k1 values were characteristic to each PHA, reflecting their chemical structures. In comparison with P(3HO), an increase in the levels of the two minor monomers 3HC and 3HD as in P(21 mol % 3HC-co-56 mol % 3HO-co-23 mol % 3HD) significantly slowed the rate of intracellular degradation. From the comparison of k1 values, it is suggested that the P. putida intracellular depolymerase is most active against P(3HO).open
Purification and characterization of a novel thermoacid-stable fibrinolytic enzyme from Staphylococcus sp. strain AJ isolated from Korean salt-fermented Anchovy-joet
No full textA novel fibrinolytic enzyme (AJ) was purified from Staphylococcus sp. strain AJ screened from Korean salt-fermented Anchovy-jeot. Relative molecular weight of AJ was determined as 26 kDa by using SDS-PAGE and fibrin zymography. Based on a 2D gel, AJ was found to consist of three active isoforms (pI 5.5-6.0) with the same N-terminal amino acid sequence. AJ exhibited optimum pH and temperature at 2.5-3.0 and 85°C, respectively. AJ kept 85% of the initial activity after heating at 100°C for 20 min on the zymogram gel. The Michaelis constant (K m) and K cat values of AJ towards α-casein were 0.38 mM and 19.73 s-1, respectively. AJ cleaved the Aα-chain of fibrinogen but did not affect the Bβ- and γ-chains, indicating that it is an α-fibrinogenase. The fibrinolytic activity was inhibited by diisopropyl fluorophosphate, indicating AJ is a serine protease. Interestingly, AJ was very stable at acidic condition, SDS, and heat (100°C), whereas it was easily degraded at neutral and alkaline conditions. In particular, AJ formed an active homo-dimer in the pH range from 7.0 to 8.0. To our knowledge, a similar combination of acid and heat stability has not yet been reported for other fibrinolytic enzymes.open
Characteristics of a new enantioselective thermostable dipeptidase from Brevibacillus borstelensis BCS-1 and its application to synthesis of a D-amino-acid-containing dipeptide
No full textA new thermostable dipeptidase gene was cloned from the thermophile Brevibacillus borstelensis BCS-1 by genetic complementation of the D-Glu auxotroph Escherichia coli WM335 on a plate containing D-Ala-D-Glu. Nucleotide sequence analysis revealed that the gene included an open reading frame coding for a 307-amino-acid sequence with an Mr of 35,000. The deduced amino acid sequence of the dipeptidase exhibited 52% similarity with the dipeptidase from Listeria monocytogenes. The enzyme was purified to homogeneity from recombinant E. coli WM335 harboring the dipeptidase gene from B. borstelensis BCS-1. Investigation of the enantioselectivity (E) to the P 1 and P1′ site of Ala-Ala revealed that the ratio of the specificity constant (kcat/Km) for L-enantioselectivity to the P1 site of Ala-Ala was 23.4 ± 2.2 [E = (kcat/Km)L,D/(kcat/K m)D,D], while the D-enantioselectivity to the P 1′ site of Ala-Ala was 16.4 ± 0.5 [E = (k cat/Km)L,D/(kcat/Km) L.L] at 55°C. The enzyme was stable up to 55°C, and the optimal pH and temperature were 8.5 and 65°C, respectively. The enzyme was able to hydrolyze L-Asp-D-Ala, L-Asp-D-AlaOMe, Z-D-Ala-D-AlaOBzl, and Z-L-Asp-D-AlaOBzl, yet it could not hydrolyze D-Ala-L-Asp, D-Ala-L-Ala, D-AlaNH2, and L-AlaNH2. The enzyme also exhibited >-lactamase activity similar to that of a human renal dipeptidase. The dipeptidase successfully synthesized the precursor of the dipeptide sweetener Z-L-Asp-D-AlaOBzl.open
Analysis of the fine-scale population structure of "candidatus accumulibacter phosphatis" in enhanced biological phosphorus removal sludge, using fluorescence in situ hybridization and flow cytometric sorting
No full textTo investigate the fine-scale diversity of the polyphosphate-accumulating organisms (PAO) "Candidatus Accumulibacter phosphatis" (henceforth referred to as "Ca. Accumulibacter"), two laboratory-scale sequencing batch reactors (SBRs) for enhanced biological phosphorus removal (EBPR) were operated with sodium acetate as the sole carbon source. During SBR operations, activated sludge always contained morphologically different "Ca. Accumulibacter" strains showing typical EBPR performances, as confirmed by the combined technique of fluorescence in situ hybridization (FISH) and microautoradiography (MAR). Fragments of "Ca. Accumulibacter" 16S rRNA genes were retrieved from the sludge. Phylogenetic analyses together with sequences from the GenBank database showed that "Ca. Accumulibacter" 16S rRNA genes of the EBPR sludge were clearly differentiated into four "Ca. Accumulibacter" clades, Acc-SG1, Acc-SG2, Acc-SG3, and Acc-SG4. The specific FISH probes Acc444, Acc184, Acc72, and Acc119 targeting these clades and some helpers and competitors were designed by using the ARB program. Microbial characterization by FISH analysis using specific FISH probes also clearly indicated the presence of different "Ca. Accumulibacter" cell morphotypes. Especially, members of Acc-SG3, targeted by probe Acc72, were coccobacillus-shaped cells with a size of approximately 2 to 3 μm, while members of Acc-SG1, Acc-SG2, and Acc-SG4, targeted by Acc444, Acc184, and Acc119, respectively, were coccus-shaped cells approximately 1 μm in size. Subsequently, cells targeted by each FISH probe were sorted by use of a flow cytometer, and their polyphosphate kinase 1 (ppkI) gene homologs were amplified by using a ppkl-specific PCR primer set for "Ca. Accumulibacter." The phylogenetic tree based on sequences of the ppkl gene homologs was basically congruent with that of the 16S rRNA genes, but members of Acc-SG3 with a distinct morphology comprised two different ppkl genes. These results suggest that "Ca. Accumulibacter" strains may be diverse physiologically and ecologically and represent distinct populations with genetically determined adaptations in EBPR systems.open
Identification of fibrinogen-induced nattokinase WRL101 from Bacillus subtilis WRL101 isolated from Doenjang
No full textTo increase the fibrinolytic enzyme (nattokinase WRL101) in Bacillus subtilis WRL101, bovine fibrin or
fibrinogen (1.0%, w/v) were added in the tryptic soy broth as a substrate. The fibrinolytic activity
cultured in the fibrinogen-contained medium was increased. On the other hand, fibrin decreased the
activity. Using the medium condition, nattokinase WRL101 (fibrinogen-induced nattokinase WRL101,
FIN-WRL101) was isolated by commercial chromatographic techniques and its biochemical
characteristics were investigated. The molecular weight of FIN-WRL101 was estimated to be 29 kDa.
FIN-WRL101 was optimally active at pH 11.0 and 47°C. It had high degrading activity for the Aα-chain
and B？-chain of human fibrinogen, but did not affect the ？-chain, indicating that it is an ？-fibrinogenase.
FIN-WRL101 was completely inhibited by phenylmethylsulfonyl fluoride, indicating that it belongs to the
serine protease. FIN-WRL101 exhibited high specificity for Meo-Suc-Arg-Pro-Tyr-pNA (S-2586), a
synthetic chromogenic substrate for chymotrypsin. Its nucleotide and amino acid sequences were
determined.open
A novel multifunctional cellulolytic enzyme screened from metagenomic resources representing ruminal bacteria
No full textMetagenomic resources representing ruminal bacteria were screened for novel exocellulases using a robotic, high-throughput screening system, the novel CelEx-BR12 gene was identified and the predicted CelEx-BR12 protein was characterized. The CelEx-BR12 gene had an open reading frame (ORF) of 1140 base pairs that encoded a 380-amino-acid-protein with a predicted molecular mass of 41.8kDa. The amino acid sequence was 83% identical to that of a family 5 glycosyl hydrolase from Prevotella ruminicola 23. Codon-optimized CelEx-BR12 was overexpressed in Escherichia coli and purified using Ni-NTA affinity chromatography. The Michaelis-Menten constant (Km value) and maximal reaction velocity (Vmax values) for exocellulase activity were 12.92μM and 1.55×10-4μmolmin-1, respectively, and the enzyme was optimally active at pH 5.0 and 37°C. Multifunctional activities were observed against fluorogenic and natural glycosides, such as 4-methylumbelliferyl-β-d-cellobioside (0.3Umg-1), CMC (105.9Umg-1), birch wood xylan (132.3Umg-1), oat spelt xylan (67.9Umg-1), and 2-hydroxyethyl-cellulose (26.3Umg-1). Based on these findings, we believe that CelEx-BR12 is an efficient multifunctional enzyme as endocellulase/exocellulase/xylanase activities that may prove useful for biotechnological applications.open
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