A novel endophytic fungus producing beta-glucosidase was isolated and characterized from pigeon pea (Cajanus cajan [L.] Millsp.), which has excellent properties in converting ginsenoside Rb1 to ginsenoside Rd in Panax notoginseng. According to the 16S rDNA gene sequence, the G11-7 strain was identified as Fusarium proliferatum, and the accession number KY303906 was confirmed in GenBank. The G11-7 immobilized spores, in which the activity of beta-glucosidase could reach 0.95 U/mL, were co-cultured with P. notoginseng plant material to obtain a continuous beta-glucosidase supply for the biotransformation of ginsenoside Rb1 to Rd. Under the liquid-solid ratio (20:1), initial pH (6.0), and temperature (30 °C) constituents, the maximum ginsenoside Rd yield was obtained as 9.15 ± 0.65 mg/g, which was 3.67-fold higher than that without fungal spore co-culture (2.49 ± 0.98 mg/g). Furthermore, immobilized G11-7 spores showed significant beta-glucosidase producing ability which could be recovered and reused for 6 cycles. Overall, these results suggested that immobilized G11-7 offered a promising and effective approach to enhance the production of ginsenoside Rd for possible nutraceutical and pharmaceutical uses.
The bacterium Pseudomonas putida KT2440 is gaining considerable interest as a microbial platform for biotechnological valorization of polymeric organic materials, such as lignocellulosic residues or plastics. However, P. putida on its own cannot make much use of such complex substrates, mainly because it lacks an efficient extracellular depolymerizing apparatus. We seek to address this limitation by adopting a recombinant cellulosome strategy for this host. In this work, we report an essential step in this endeavor-a display of designer enzyme-anchoring protein "scaffoldins", encompassing cohesin binding domains from divergent cellulolytic bacterial species on the P. putida surface. Two P. putida chassis strains, EM42 and EM371, with streamlined genomes and differences in the composition of the outer membrane were employed in this study. Scaffoldin variants were optimally delivered to their surface with one of four tested autotransporter systems (Ag43 from Escherichia coli), and the efficient display was confirmed by extracellular attachment of chimeric β-glucosidase and fluorescent proteins. Our results not only highlight the value of cell surface engineering for presentation of recombinant proteins on the envelope of Gram-negative bacteria but also pave the way toward designer cellulosome strategies tailored for P. putida.
- MeSH
- beta-glukosidasa metabolismus MeSH
- celulosa metabolismus MeSH
- celulozómy metabolismus MeSH
- chromozomální proteiny, nehistonové chemie MeSH
- Escherichia coli metabolismus MeSH
- genom bakteriální * MeSH
- membránové proteiny metabolismus MeSH
- metabolické inženýrství metody MeSH
- proteinové domény MeSH
- proteiny buněčného cyklu chemie MeSH
- proteiny z Escherichia coli metabolismus MeSH
- Pseudomonas putida genetika metabolismus MeSH
- rekombinantní proteiny metabolismus MeSH
- vnější bakteriální membrána metabolismus MeSH
- zelené fluorescenční proteiny metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Bacteria represent an underexplored source of diglycosidases. Twenty-five bacterial strains from the genera Actinoplanes, Bacillus, Corynebacterium, Microbacterium, and Streptomyces were selected for their ability to grow in diglycosylated flavonoids-based media. The strains Actinoplanes missouriensis and Actinoplanes liguriae exhibited hesperidin deglycosylation activity (6-O-α-L-rhamnosyl-β-D-glucosidase activity, EC 3.2.1.168), which was 3 to 4 orders of magnitude higher than the corresponding monoglycosidase activities. The diglycosidase production was confirmed in A. missouriensis by zymographic assays and NMR analysis of the released disaccharide, rutinose. The gene encoding the 6-O-α-L-rhamnosyl-β-D-glucosidase was identified in the genome sequence of A. missouriensis 431(T) (GenBank accession number BAL86042.1) and functionally expressed in Escherichia coli. The recombinant protein hydrolyzed hesperidin and hesperidin methylchalcone, but not rutin, which indicates its specificity for 7-O-rutinosylated flavonoids. The protein was classified into the glycoside hydrolase family 55 (GH55) in contrast to the known eukaryotic diglycosidases, which belong to GH1 and GH5. These findings demonstrate that organisms other than plants and filamentous fungi can contribute to an expansion of the diglycosidase toolbox.
- MeSH
- bakteriální proteiny genetika metabolismus MeSH
- beta-glukosidasa genetika metabolismus MeSH
- chalkonoidy chemie metabolismus MeSH
- disacharidy chemie metabolismus MeSH
- Escherichia coli genetika metabolismus MeSH
- exprese genu MeSH
- flavonoidy chemie metabolismus MeSH
- fylogeneze MeSH
- glykosidy chemie metabolismus MeSH
- hesperidin analogy a deriváty chemie metabolismus MeSH
- hydrolýza MeSH
- klonování DNA MeSH
- Micromonosporaceae klasifikace genetika metabolismus MeSH
- rekombinantní proteiny genetika metabolismus MeSH
- rhamnosa chemie metabolismus MeSH
- substrátová specifita MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Enzymatic depolymerization of chitosan, a β-(1,4)-linked polycationic polysaccharide composed of d-glucosamine (GlcN) and N-acetyl-d-glucosamine (GlcNAc) provides a possible route to the exploitation of chitin-rich biomass. Complete conversion of chitosan to mono-sugars requires the synergistic action of endo- and exo- chitosanases. In the present study we have developed an efficient and cost-effective chitosan-degrading enzyme cocktail containing only two enzymes, an endo-attacking bacterial chitosanase, ScCsn46A, from Streptomyces coelicolor, and an exo-attacking glucosamine specific β-glucosaminidase, Tk-Glm, from the archaeon Thermococcus kodakarensis KOD1. Moreover, we developed a fast, reliable quantitative method for analysis of GlcN using high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The sensitivity of this method is high and less than 50 pmol was easily detected, which is about 1000-fold better than the sensitivity of more commonly used detection methods based on refractive index. We also obtained qualitative insight into product development during the enzymatic degradation reaction by means of ElectroSpray Ionization-Mass Spectrometry (ESI-MS).
- MeSH
- bakteriální proteiny metabolismus MeSH
- beta-glukosidasa metabolismus MeSH
- chitosan chemie MeSH
- chromatografie iontoměničová metody MeSH
- glukosamin analýza chemie MeSH
- glykosidhydrolasy metabolismus MeSH
- hmotnostní spektrometrie s elektrosprejovou ionizací MeSH
- Streptococcus enzymologie MeSH
- substrátová specifita MeSH
- Thermococcus enzymologie MeSH
- Publikační typ
- časopisecké články MeSH
Saturation mutagenesis is a cornerstone technique in protein engineering because of its utility (in conjunction with appropriate analytical techniques) for assessing effects of varying residues at selected positions on proteins' structures and functions. Site-directed mutagenesis with degenerate primers is the simplest and most rapid saturation mutagenesis technique. Thus, it is highly appropriate for assessing whether or not variation at certain sites is permissible, but not necessarily the most time- and cost-effective technique for detailed assessment of variations' effects. Thus, in the presented study we applied the technique to randomize position W373 in β-glucosidase Zm-p60.1, which is highly conserved among β-glucosidases. Unexpectedly, β-glucosidase activity screening of the generated variants showed that most variants were active, although they generally had significantly lower activity than the wild type enzyme. Further characterization of the library led us to conclude that a carefully selected combination of randomized codon-based saturation mutagenesis and site-directed mutagenesis may be most efficient, particularly when constructing and investigating randomized libraries with high fractions of positive hits.
- MeSH
- aktivace enzymů MeSH
- beta-glukosidasa genetika metabolismus MeSH
- databáze proteinů MeSH
- genová knihovna MeSH
- hydrolýza MeSH
- kodon MeSH
- kukuřice setá genetika metabolismus MeSH
- mutageneze MeSH
- proteinové inženýrství * metody MeSH
- rostlinné proteiny genetika metabolismus MeSH
- substrátová specifita MeSH
- výpočetní biologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Extensive screening for a robust producer of α-L-rhamnosidase activity from well-defined strains of filamentous fungi, including multifactorial optimization (inducers, cultivation conditions) was accomplished. Enzyme production of the optimal producer Aspergillus terreus (non-toxigenic) was scaled up to 50L. α-L-Rhamnosidase, which was fully characterized, proved to be thermo- and alkali-tolerant, thus enabling effective operation at 70°C and pH 8.0. These conditions allow for a very high substrate (rutin) load up to 100-300 g/L, thus enabling very high volumetric productivity of the reaction product quercetin-3-β-D-glucopyranoside (isoquercitrin). Here, a novel concept of "immobilised substrate" is used. Isoquercitrin is a highly effective and biocompatible antioxidant with strong anti-inflammatory activities. Rutin biotransformation was optimized and scaled up to ca 10 kg production and thus the robustness of the large-scale production was demonstrated. Isoquercitrin can be produced to a very high purity (98%) in multikilogram amounts, without any quercetin and directly applicable in nutraceuticals.
- MeSH
- alkálie farmakologie MeSH
- Aspergillus účinky léků enzymologie MeSH
- beta-glukosidasa metabolismus MeSH
- bioreaktory mikrobiologie MeSH
- biotechnologie metody MeSH
- biotransformace účinky léků MeSH
- fyziologická adaptace účinky léků MeSH
- glukosidy biosyntéza chemie MeSH
- glykosidhydrolasy metabolismus MeSH
- koncentrace vodíkových iontů účinky léků MeSH
- quercetin analogy a deriváty biosyntéza chemie MeSH
- rutin chemie metabolismus MeSH
- stabilita enzymů účinky léků MeSH
- teplota MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Cytokinin (CK) activity is regulated by the complex interplay of their metabolism, transport, stability and cellular/tissue localization. O-glucosides of zeatin-type CKs are postulated to be storage and/or transport forms. Active CK levels are determined in part by their differential distribution of CK metabolites across different subcellular compartments. We have previously shown that overexpressing chloroplast-localized Zm-p60.1, a maize β-glucosidase capable of releasing active cytokinins from their O- and N3-glucosides, perturbs CK homeostasis in transgenic tobacco. We obtained tobacco (Nicotiana tabacum L., cv Petit Havana SR1) plants overexpressing a recombinant Zm-p60.1 that is targeted to the vacuole. The protein is correctly processed and localized to the vacuole. When grown on medium containing exogenous zeatin, transgenic seedlings rapidly accumulate fresh weight due to ectopic growths at the base of the hypocotyl. The presence of the enzyme in these ectopic structures is shown by histochemical staining. CK quantification reveals that these transgenic seedlings are unable to accumulate zeatin-O-glucoside to levels similar to those observed in the wild type. When crossed with tobacco overexpressing the zeatin-O-glucosyltransferase gene from Phaseolus, the vacuolar variant shows an almost complete reversion in the root elongation assay. This is the first evidence from intact plants that the vacuole is the storage organelle for CK O-glucosides and that they are available to attack by Zm-p60.1. We propose the use of Zm-p60.1 as a robust molecular tool that exploits the reversibility of O-glucosylation and enables delicate manipulations of active CK content at the cellular level.
- MeSH
- beta-glukosidasa genetika metabolismus MeSH
- chloroplasty účinky léků metabolismus MeSH
- cytokininy metabolismus MeSH
- fazol genetika MeSH
- geneticky modifikované rostliny MeSH
- glukosidy metabolismus MeSH
- hybridizace genetická MeSH
- kukuřice setá enzymologie genetika MeSH
- semenáček cytologie účinky léků růst a vývoj MeSH
- tabák cytologie účinky léků genetika metabolismus MeSH
- vakuoly účinky léků genetika metabolismus MeSH
- zeatin farmakologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Moonlighting proteins have two different functions within a single polypeptide chain. Exploring moonlighting enzymes from the environment using the metagenomic approach is interesting. In the present study, a novel β-glucosidase gene, designated as bgl1D, with lipolytic activity (renamed Lip1C) was cloned through function-based screening of a metagenomic library from uncultured soil microorganisms. The deduced amino acid sequence comparison and phylogenetic analysis also indicated that Lip1C and other putative lipases are closely related. Biochemical characterization demonstrated that the maximum activity of the recombinant Lip1C protein occurs at pH 8.0 and 30°C using 4-nitrophenyl butyrate as substrate. The putative lipase had an apparent K(m) value of 0.88 mmol/L, a k(cat) value of 212/min, and a k(cat)/K(m) value of 241 L/mmol/min. Lip1C exhibited habitat-specific characteristics with 5 mmol/L AlCl(3), CuCl(2), and LiCl. The characterization of the biochemical properties of Lip1C enhances our understanding of this novel moonlighting enzyme isolated from a soil metagenome.
- MeSH
- Bacteria klasifikace enzymologie genetika izolace a purifikace MeSH
- bakteriální proteiny chemie genetika metabolismus MeSH
- beta-glukosidasa chemie genetika metabolismus MeSH
- fylogeneze MeSH
- kinetika MeSH
- lipasa chemie genetika metabolismus MeSH
- metagenom MeSH
- molekulární sekvence - údaje MeSH
- půdní mikrobiologie MeSH
- sekvence aminokyselin MeSH
- sekvenční seřazení MeSH
- stabilita enzymů MeSH
- substrátová specifita MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Saprotrophic wood-inhabiting basidiomycetes are the most important decomposers of lignin and cellulose in dead wood and as such they attracted considerable attention. The aims of this work were to quantify the activity and spatial distribution of extracellular enzymes in coarse wood colonised by the white-rot basidiomycete Fomes fomentarius and in adjacent fruitbodies of the fungus and to analyse the diversity of the fungal and bacterial community in a fungus-colonised wood and its potential effect on enzyme production by F. fomentarius. Fungus-colonised wood and fruitbodies were collected in low management intensity forests in the Czech Republic. There were significant differences in enzyme production by F. fomentarius between Betula pendula and Fagus sylvatica wood, the activity of cellulose and xylan-degrading enzymes was significantly higher in beech wood than in birch wood. Spatial analysis of a sample B. pendula log segment proved that F. fomentarius was the single fungal representative found in the log. There was a high level of spatial variability in the amount of fungal biomass detected, but no effects on enzyme activities were observed. Samples from the fruiting body showed high β-glucosidase and chitinase activities compared to wood samples. Significantly higher levels of xylanase and cellobiohydrolase were found in samples located near the fruitbody (proximal), and higher laccase and Mn-peroxidase activities were found in the distal ones. The microbial community in wood was dominated by the fungus (fungal to bacterial DNA ratio of 62-111). Bacterial abundance composition was lower in proximal than distal parts of wood by a factor of 24. These results show a significant level of spatial heterogeneity in coarse wood. One of the explanations may be the successive colonization of wood by the fungus: due to differential enzyme production, the rates of biodegradation of coarse wood are also spatially inhomogeneous.
- MeSH
- Bacteria enzymologie izolace a purifikace MeSH
- beta-glukosidasa metabolismus MeSH
- biodegradace MeSH
- bříza enzymologie mikrobiologie MeSH
- buk (rod) enzymologie mikrobiologie MeSH
- celulosa-1,4-beta-cellobiosidasa metabolismus MeSH
- chitinasy metabolismus MeSH
- Coriolaceae enzymologie izolace a purifikace MeSH
- DNA bakterií analýza MeSH
- DNA fungální analýza MeSH
- dřevo enzymologie mikrobiologie MeSH
- ekologie MeSH
- lakasa metabolismus MeSH
- lignin metabolismus MeSH
- mikrobiální společenstva MeSH
- peroxidasy metabolismus MeSH
- stromy enzymologie mikrobiologie MeSH
- xylany metabolismus MeSH
- xylosidasy metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Česká republika MeSH
A new targeting potentially intravenous conjugate Amphotericin B (AMB)-star poly(ethylene glycol) (sPEG) (M=25,160) has been synthesized and characterized. It contains a beta-d-glucopyranoside molecular switch which is sensitive to beta-glucosidases (E.C.3.2.1.21). The beta-glucosidase-catalyzed release of AMB from the polymeric carrier was proved in vitro by means of spectrophotometry and HPLC.
- MeSH
- amfotericin B analogy a deriváty farmakologie chemická syntéza MeSH
- antifungální látky farmakologie chemická syntéza MeSH
- beta-glukosidasa metabolismus MeSH
- chemické modely MeSH
- financování organizované MeSH
- katalýza MeSH
- lidé MeSH
- polyethylenglykoly farmakologie chemická syntéza MeSH
- racionální návrh léčiv MeSH
- spektrofotometrie ultrafialová MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- Check Tag
- lidé MeSH
