1,720,972 research outputs found
A phosphorylation assay using [γ-32P]ATP: A highly sensitive detection of protein kinase C
No full textThe involvement of protein kinase C (PKC) in many biological processes such as development, memory, cell differentiation and proliferation, and carcinogenesis has been demonstrated. Using the mep45 gene encoding the 45-kDa major envelope protein (Mep45) of Selenomonas ruminantium, a protein-fused substrate (neurogranin-Mep45, MFS-PKC) was cloned, which is a highly selective substrate for PKC. The recombinant protein-fused substrate can be constantly produced in reasonable quantities with a small outlay. In this study, a suitable strategy for the detection of the phosphorylation of a peptide-type substrate and a Mep45-fused substrate catalyzed by PKC by using a sensitive radiodetection is described. This strategy can be applicable to the development of protein microarray, which can be a useful tool for high-throughput screening in biological and medical research.open
Protective effect of hesperidin, a citrus flavanoglycone, against gamma-radiation-induced tissue damage in Sprague-Dawley rats
No full textThe present study was designed to evaluate the radioprotective effect of hesperidin, a citrus flavanoglycone, against γ-radiation-induced cellular damage in the liver, heart, and kidney of rats. Whole-body γ-radiation exposure (5 Gy) of healthy adult rats resulted in cellular damage and oxidative stress manifested as increased levels of serum marker enzymes, lipid peroxidation, and fibrosis in the tissues, accompanied by depletion of cellular glutathione and abnormal alteration in the levels of lysosomal enzymes. Treatment with hesperidin (50 and 100 mg/kg, p.o.) for 7 days was found to offer significant protection against γ-radiation-induced toxicity in the tissues, which was evident by the improved status of most of the parameters investigated. Further, the histological examination of periodic acid-Schiff-stained tissue sections of animals treated with hesperidin following radiation exposure showed minimal necrotic damage with a recovery pattern in a dose-dependent manner compared with radiation-exposed animals. The results of our study show that administration of hesperidin offers effective protection against γ-radiation-induced cellular damage and oxidative stress in rats.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
A rapid and simple method for preparing an insoluble substrate for screening of microbial xylanase
No full textSeveral types of enzymes, including cellulases and xylanases, are required to degrade hemicelluloses and cellulose, which are major components of lignocellulosic biomass. Such degradative processes can be used to produce various useful industrial biomaterials. Screening methods for detecting polysaccharide-degrading microorganisms include the use of dye-labeled substrates in growth medium and culture plate staining techniques. However, the preparation of screening plates, which typically involves chemical cross-linking to synthesize a dye-labeled substrate, is a complicated and time-consuming process. Moreover, such commercial substrates are very expensive, costing tenfold more than the natural xylan. Staining methods are also problematic because they may damage relevant microorganisms and are associated with contamination of colonies of desirable organisms with adjacent unwanted bacteria. In the present study, we describe a sonication method for the simple and rapid preparation of an insoluble substrate that can be used to screen for xylanase-expressing bacteria in microbial populations. Using this new method, we have successfully isolated a novel xylanase gene from a xylolytic microorganism termed Xyl02-KBRB and Xyl14-KBRB in the bovine rumen.open
Strategy for screening metagenomic resources for exocellulase activity using a robotic, high-throughput screening system
No full textExocellulases play a key role in cleaving the accessible ends of cellulose molecules to release soluble glucose and cellobiose. To date, there have been no screens for exocellulase owing to assay protocol limitations, the high cost of substrates, and low activity of exocellulases compared with endocellulases. This study is the first to demonstrate direct screening for exocellulase activity using a robotic, high-throughput screening (HTS) system. Cell growth in 96-well plates was measured by monitoring optical density over 11-14h at 37°C with agitation. Fluorescence of methylumbelliferyl groups released from 4-methylumbelliferyl-β-D-cellobioside was determined using a VICTOR3 microplate reader. This new HTS system enabled activity verification of more than 104 clones per day. As a result, we obtained four exocellulases clones (CelEx-SF301, CelEx-SF309, CelEx-BR12 and CelEx-BR15) from 29,006 metagenomic fosmid clones that had previously been prepared from sweet potato field soil microbes and rumen fluid. This powerful approach could be effectively applied to screen various metagenomic resources for new enzymes.open
Enhanced kinase sensing by radio-phosphorylation and oriented immobilization of a GBP-linked kinase substrate on a gold surface
No full textA new strategy for enhanced sensing of kinases was developed using radio-phosphorylation and a polypeptide with an affinity for gold that was linked directly to kinase substrates. Gold-binding polypeptide (GBP)-linked GST-fusion substrates were efficiently immobilized on gold-coated slides, which is highly desirable for protein chips. The novel fusion substrates permitted oriented substrate immobilization on gold-coated slides, thereby enhancing the sensitivity of kinase assays. Thus, the proposed strategy is applicable to the development of protein microarrays and microfluidic lab-on-a-chip (LOC) sensing the activities of protein kinases.open
Bacterial cell surface display of a multifunctional cellulolytic enzyme screened from a bovine rumen metagenomic resource
No full textA cell surface display system for heterologous expression of the multifunctional cellulase, CelEx-BR12, in Escherichia coli was developed using truncated E. coli outer membrane protein C (OmpC) as an anchor motif. Cell surface expression of CelEx-BR12 cellulase in E. coli harboring OmpC-fused CelEx-BR12, designated MC4100 (pTOCBR12), was confirmed by fluorescence-activated cell sorting and analysis of outer membrane fractions by western blotting, which verified the expected molecular mass of OmpC-fused CelEx-BR12 (~72 kDa). Functional evidence for exocellulase activity was provided by enzymatic assays of whole cells and outer membrane protein fractions from E. coli MC4100 (pTOCBR12). The stability of E. coli MC4100 (pTOCBR12) cellulase activity was tested by carrying out repeated reaction cycles, which demonstrated the reusability of recombinant cells. Finally, we showed that recombinant E. coli cells displaying the CelEx-BR12 enzyme on the cell surface were capable of growth using carboxymethyl cellulose as the sole carbon source.open
Identification and characterization of a novel cold-adapted esterase from a metagenomic library of mountain soil
No full textA novel lipolytic enzyme was isolated from a metagenomic library after demonstration of lipolytic activity on an LB agar plate containing 1% (w/v) tributyrin. A novel esterase gene (estIM1), encoding a lipolytic enzyme (EstIM1), was cloned using a shotgun method from a pFos- EstIM1 clone of the metagenomic library, and the enzyme was characterized. The estIM1 gene had an open reading frame (ORF) of 936 base pairs and encoded a protein of 311 amino acids with a molecular mass 34 kDa and a pI value of 4.32. The deduced amino acid sequence was 62% identical to that of an esterase from an uncultured bacterium (ABQ11271). The amino acid sequence indicated that EstIM1 was a member of the family IV of lipolytic enzymes, all of which contain a GDSAG motif shared with similar enzymes of lactic acid microorganisms. EstIM1 was active over atemperature range of 1-50C°, at alkaline pH. The activation energy for hydrolysis of p-nitrophenyl propionate was 1.04 kcal/mol, within a temperature range of 1-40C°. The activity of EstIM1 was about 60% of maximal even at 1C°, suggesting that EstIM1 is eYciently coldadapted. Further characterization of this cold-adapted enzyme indicated that the esterase may be very valuable in industrial applications.open
A novel bifunctional endo-/exo-type cellulase from an anaerobic ruminal bacterium
No full textAn anaerobic microorganism termed AN-C16-KBRB was isolated from the bovine rumen and demonstrated cellulolytic activity on a NB agar plate containing azo-carboxymethyl cellulose. The 16S rRNA gene of the strain was 98% similar to that of Clostridiaceae bacterium SK082 (AB298754) as the highest homology. A novel celEdx16 gene encoding a bifunctional endo-/exocellulase (CelEdx16) was cloned by the shotgun method from AN-C16-KBRB, and the enzyme was characterized. The celEdx16 gene had an open reading frame of 1,104-base pairs, which encoded 367 amino acids to yield a protein of molecular mass 40.4 kDa. The amino acid sequence was 53% identical to that of an endoglucanase from Clostridium thermocellum. CelEdx16 was overexpressed in Escherichia coli and purified using Ni-NTA affinity chromatography. The specific endocellulase and exocellulase activities of CelEdx16 were 15.9 and 3.6 × 10-2 U mg -1, respectively. The Michaelis-Menten constant (K m values) and the maximal reaction velocities (V max values) of CelEdx16 were 47.1 μM and 9.6 × 10-3 μmole min -1 when endocellulase activity was measured and 106.3 μM and 2.1 × 10-5 μmole min-1 when exocellulase activity was assessed. CelEdx16 was optimally active at pH 5.0 and 40°C.open
Enhancing functional expression of heterologous proteins through random substitution of genetic codes in the 5' coding region
No full textRecent studies using heterologous protein expression systems suggest that synonymous codons affect not only the expression but also the properties of the expressed protein. However, practical application of this information is challenging, and to date, efforts to employ bioinformatics tools to design synonymous codon mixes have been only marginally successful. Here, we sought to enhance the functional expression of heterologous protein in Escherichia coli through completely random substitution of the first ten codons with synonymous codons, using a previously isolated exocellulase CelEdx-SF301 as the model protein. Synonymous codon variants were generated by PCR using forward primers with mixed nucleotides at the third position in each codon and a conventional reverse primer. The resulting PCR products were inserted upstream of the fluorescent protein mCherry without linkers. After transformation and cultivation, colonies exhibiting red fluorescence were selected, and the activity of SF301-mCherry fusion proteins was tested. Synonymous codon variant fusion proteins exhibited 35- to 530-fold increases in functional expression compared with wild-type controls. Unlike results from other reports, we found that the stability of mRNA secondary structure in the 5' untranslated region and codon rarity were not correlated with functional expression level. Our work demonstrates that a completely random mixed of synonymous codons effectively enhances functional expression levels without the need for amino acid substitutions.open
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