1,720,963 research outputs found
Purification and characterization of a subtilisin D5, a fibrinolytic enzyme of Bacillus amyloliquefaciens DJ-5 isolated from Doenjang
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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
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 gene
No full textAspergillus terreus cadA, encoding cis-aconitate decarboxylase, is an essential gene for itaconic acid (IA) biosynthesis, but it is primarily expressed as insoluble aggregates in most industrial hosts. This has been a hurdle for the development of recombinant strategies for IA production. Here, we created a library of synonymous codon variants (scv) of the cadA gene containing synonymous codons in the first 10 codons (except ATG) and screened it in Escherichia coli. Among positive clones, E. coli scvCadA_No8 showed more than 95% of expressed CadA in the soluble fraction, and in production runs, produced threefold more IA than wild-type E. coli in Luria-Bertani broth supplemented with 0.5% glucose. In M9 minimal media containing 0.85g/L citrate and 1% glycerol, E. coli scvCadA_No8 produced 985.6±33.4mg/L IA during a 72-h culture after induction with isopropyl β-D-1-thiogalactopyranoside. In a 2-L fed-batch fermentation consisting of two stages (growth and nitrogen limitation conditions), we obtained 7.2g/L IA by using E. coli by introducing only the scv_cadA gene and optimizing culture conditions for IA production. These results could be combined with metabolic engineering and generate an E. coli strain as an industrial IA producer. Biotechnol. Bioeng. 2016;9999: 1-7.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
Multiple-layer substrate zymography for detection of several enzymes in a single sodium dodecyl sulfate gel
No full textWe have developed a system to detect three hydrolytic enzymes (cellulase, lipase, and protease) using a single sodium dodecyl sulfate (SDS) gel and an electrotransfer system. After electrophoresis, proteins in the gel were transferred to three sandwiched substrate gels containing glycerol tributyrate, azo-carboxymethyl cellulose (Azo-CMC), and fibrin for detection of cellulase, lipase, and protease, respectively. We show that three cellulases (from a Paenibacillus sp. and two Bacillus sp. strains), one lipase (from a Staphylococcus sp.), and two proteases (from two Bacillus sp. strains) can be detected simultaneously with our zymogram system.open
Mixed-substrate (glycerol tributyrate and fibrin) zymography for simultaneous detection of lipolytic and proteolytic enzymes on a single gel
No full textA new zymography method for simultaneous detection of two different enzymatic
activities (lipolytic and proteolytic) using a single SDS-containing or native-conformation
gel and a mixed-substrate (glycerol tributyrate and fibrin) (MS)1 gel was developed. After
routine electrophoresis, SDS in the gel was removed by treatment with Triton X-100. Gel
proteins were electrotransferred to the MS gel. To visualize lipolytic activity, the MS gel
was incubated at 371C (for 6 or 24 h) until clear bands against an opaque background
were observed. To detect proteolytic activity, the same MS gel was stained with
Coomassie brilliant blue. Using this method, we show that six lipolytic enzymes from
Staphylococcus pasteuri NJ-1 and four proteolytic enzymes from two Bacillus strains,
B. licheniformis DJ-2 and B. licheniformis NJ-5, isolated from soil, can be simultaneously
detected.open
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