scholarly journals Production of endoglucanase A of Clostridium thermocellum in Bacillus subtilis

2014 ◽  
Vol 17 (4) ◽  
pp. 74-82
Author(s):  
Thang Luong Pham ◽  
Trang Thi Phuong Phan ◽  
Thuoc Linh Tran ◽  
Hoang Duc Nguyen
Keyword(s):  
Bacillus Subtilis ◽  
Catalytic Activity ◽  
Clostridium Thermocellum ◽  
Anaerobic Bacterium ◽  
Slow Growth ◽  
Bacterial Strains ◽  
Industrial Enzymes ◽  
Glucanase Activity ◽  
C Terminus ◽  
Sds Page

Endoglucanase A (CelA) is a component of thermostable complex - cellulosome which is produced by anaerobic bacterium Clostridium thermocellum. In this complex, CelA has the highest catalytic activity and it can cleave randomly β-1,4 glucosidic linkage of cellulose to smaller oligosaccharides. Bacillus subtilis a Gram positive and aerobic fast growth bacterium, is commonly used to produce industrial enzymes. This bacterium can be used to replace the anaerobic slow growth bacterium, C. thermocellum, to produce CelA. To create B. subtilis strains which can secret recombinant CelA enzyme, celA gene was amplified using C. thermocellum DNA genome by PCR and inserted in the C-terminus of signal peptide of amyQ (samyQ) in plasmid pHT43. The plasmid pHT43-celA was then transformed into B. subtilis 1012 and WB800N, an extracellular protease deficiency strain. Next, CelA production was induced by IPTG at different concentrations. Cultural supernatant was collected from 2 to 24 hours after addition of IPTG. The expression levels were evaluated by SDS-PAGE and endo-β-1,4- glucanase activity. This report demonstrates that CelA can be expressed in B. subtilis and its potentials for development of bacterial strains which can produce CelA to hydrolyze cellulose in the future.

Clostridium thermocellum Nitrilase Expression and Surface Display on Bacillus subtilis Spores

2015 ◽  
Vol 25 (6) ◽  
pp. 381-387 ◽  
Author(s):  
Huayou Chen ◽  
Tianxi Zhang ◽  
Tengyun Sun ◽  
Zhong Ni ◽  
Yilin Le ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Clostridium Thermocellum ◽  
Surface Display ◽  
Effective Means ◽  
Free Enzyme ◽  
Reaction Conditions ◽  
Sds Page ◽  
Adverse Environmental Conditions ◽  
Anchoring Motif ◽  
Spore Coat Protein

Nitrilases are an important class of industrial enzymes. They require mild reaction conditions and are highly efficient and environmentally friendly, so they are used to catalyze the synthesis of carboxylic acid from nitrile, a process considered superior to conventional chemical syntheses. Nitrilases should be immobilized to overcome difficulties in recovery after the reaction and to stabilize the free enzyme. The nitrilase from<i> Clostridium thermocellum</i> was expressed, identified and displayed on the surface of <i>Bacillus subtilis </i>spores by using the spore coat protein G of <i>B. subtilis </i>as an anchoring motif. In a free state, the recombinant nitrilase catalyzed the conversion of 3-cyanopyridine to niacin and displayed maximum catalytic activity (8.22 units/mg protein) at 40°C and pH 7.4. SDS-PAGE and Western blot were used to confirm nitrilase display. Compared with the free enzyme, the spore-immobilized nitrilase showed a higher tolerance for adverse environmental conditions. After the reaction, recombinant spores were recovered via centrifugation and reused 3 times to catalyze the conversion of 3-cyanopyridine with 75.3% nitrilase activity. This study demonstrates an effective means of nitrilase immobilization via spore surface display, which can be applied in biological processes or conversion.

scholarly journals The δ subunit and NTPase HelD institute a two-pronged mechanism for RNA polymerase recycling

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Hao-Hong Pei ◽  
Tarek Hilal ◽  
Zhuo A. Chen ◽  
Yong-Heng Huang ◽  
Yuan Gao ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Nucleic Acids ◽  
Rna Polymerase ◽  
Active Site ◽  
Genome Stability ◽  
Slow Growth ◽  
Coiled Coil ◽  
Main Channel ◽  
Dependent Manner ◽  
Secondary Channel

AbstractCellular RNA polymerases (RNAPs) can become trapped on DNA or RNA, threatening genome stability and limiting free enzyme pools, but how RNAP recycling into active states is achieved remains elusive. In Bacillus subtilis, the RNAP δ subunit and NTPase HelD have been implicated in RNAP recycling. We structurally analyzed Bacillus subtilis RNAP-δ-HelD complexes. HelD has two long arms: a Gre cleavage factor-like coiled-coil inserts deep into the RNAP secondary channel, dismantling the active site and displacing RNA, while a unique helical protrusion inserts into the main channel, prying the β and β′ subunits apart and, aided by δ, dislodging DNA. RNAP is recycled when, after releasing trapped nucleic acids, HelD dissociates from the enzyme in an ATP-dependent manner. HelD abundance during slow growth and a dimeric (RNAP-δ-HelD)2 structure that resembles hibernating eukaryotic RNAP I suggest that HelD might also modulate active enzyme pools in response to cellular cues.

scholarly journals Intracellular serine proteinase of Bacillus subtilis strain Marburg 168. Comparison with the homologous enzyme from Bacillus subtilis strain A-50

1979 ◽  
Vol 179 (2) ◽  
pp. 333-339 ◽  
Author(s):  
A Y Strongin ◽  
D I Gorodetsky ◽  
I A Kuznetsova ◽  
V V Yanonis ◽  
Z T Abramov ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Serine Proteinase ◽  
Subtilis Strain ◽  
Bacterial Strains ◽  
Terminal Sequence ◽  
Gramicidin S ◽  
Intracellular Enzymes ◽  
Sepharose 4B ◽  
Homologous Enzyme ◽  
Strict Selection

Intracellular serine proteinase was isolated from sporulating cells of Bacillus subtilis Marburg 168 by gramicidin S-Sepharose 4B affinity chromatography. The enzymological characteristics, the amino acid composition and the 19 residues of the N-terminal sequence of the enzyme are reported. The isolated proteinase was closely related to, but not completely identical with, the intracellular serine proteinase of B. subtilis A-50. The divergence between these two intracellular enzymes was less than that between the corresponding extracellular serine proteinases (subtilisins) of types Carlsberg and BPN′!, produced by these bacterial strains. This may be connected with the more strict selection constraints imposed in intracellular enzymes during evolution.

scholarly journals Cloning and Over-expression of xynB Gene of Bacillus subtilis subsp. spizizenii W23 into Escherichia coli Origami Host Cells

2017 ◽  
Vol 3 (5) ◽  
pp. 139
Author(s):  
Mariana Wahjudi ◽  
Catherina . ◽  
Nita Marcelia Wangunhardjo ◽  
Ernest Suryadjaja ◽  
Xavier Daniel
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Recombinant Plasmid ◽  
Sodium Dodecyl ◽  
Xylanase Activity ◽  
Cellular Protein ◽  
Host Cells ◽  
Chromosomal Dna ◽  
Clear Zone ◽  
Sds Page

<p class="Els-Abstract-text">The <em>xyn</em>B gene of <em>Bacillus</em><em> subtilis</em> subsp. spizizenii W23 is predicted to encode a xylan 1,4-beta-xylosidase. Application of XynB enzymes in industries is wide. Production of this enzyme in its host cells is naturally restricted by repression process. It will give certain beneficial to over-expressed the enzymes in other host-cells under inducing promoter. This study aimed to clone the <em>xyn</em>B gene from <em>Bacillus</em><em> subtilis</em> subsp. spizizenii W23, to pMMB67EH plasmid, and to over-express the <em>xyn</em>B gene in <em>Escherichia coli </em>Origami as host cells. The <em>x</em><em>yn</em>B gene was successfully amplified by polymerase chain reaction (PCR) technique using a pair of primers flanking the gene sequence and chromosomal DNA of the W23 strain as a template. The <em>xyn</em>B gene inserted in recombinant plasmid was confirmed by PCR detection using primers pair’s specific for <em>xyn</em>B gene and for the vector, then continued by restriction analyses.  The result showed that transformants clone 9 and 10 bear the recombinant pMMB-<em>xyn</em>B plasmid. The xylanase activity of <em>xyn</em>B gene in <em>Escherichia coli</em> Origami clone 10 was detected by sodium-dodecyl-sulfate polyacrylamide gel analyses and with addition of isopropyl-β-D-thio-galactoside (IPTG) as an inducer. The protein seem to be over-expressed as intra- and extra-cellular protein detected on SDS-PAGE gel. Result from xylan degrading activity on Luria-Bertani-xylan-IPTG plate with addition of Congo Red, showed that the cells with pMMB-<em>xyn</em>B recombinant plasmid have clear zone around the colonies while the transformant bearing an empty plasmid showed no clear zone. It could be concluded that the <em>xyn</em>B gene of <em>Bacillus subtilis</em> subsp.spizizenii W23 has been successfully been cloned on pMMB67EH plasmid and over-expressed in the <em>Escherichia coli</em> Origami cells as intra- and extra-cellular protein, as observed on SDS-PAGE gel analysis. The protein has activity on xylan degradation.</p>

scholarly journals MARCKS domain phosphorylation regulates the differential interaction of Diacylglycerol Kinase ζ with Rac1, RhoA and Syntrophin

2020 ◽  
Author(s):  
Ryan Ard ◽  
Jean-Christian Maillet ◽  
Elias Daher ◽  
Michael Phan ◽  
Radoslav Zinoviev ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Molecular Mechanisms ◽  
Rho Gtpase ◽  
Pdz Domain ◽  
Diacylglycerol Kinase ◽  
Binding Motif ◽  
Membrane Blebbing ◽  
Rhoa Activity ◽  
C Terminus ◽  
Mechanistic Basis

AbstractCells can switch between Rac1, lamellipodia-based and RhoA, blebbing-based migration modes but the molecular mechanisms regulating this choice are not fully understood. Diacylglycerol kinase ζ (DGKζ), which phosphorylates diacylglycerol to yield phosphatidic acid, forms independent complexes with Rac1 and RhoA, selectively dissociating each from RhoGDI. DGKζ catalytic activity is required for Rac1 dissociation but is dispensable for RhoA dissociation. Instead, DGKζ functions as a scaffold that stimulates RhoA release by enhancing RhoGDI phosphorylation by protein kinase Cα (PKCα). Here, PKCα-mediated phosphorylation of the DGKζ MARCKS domain increased DGKζ association with RhoA and decreased its interaction with Rac1. The same modification increased binding of the DGKζ C-terminus to the α1-syntrophin PDZ domain. Expression of a phosphomimetic DGKζ mutant stimulated membrane blebbing in mouse embryonic fibroblasts and C2C12 myoblasts, which was augmented by inhibition of endogenous Rac1. DGKζ expression in differentiated C2 myotubes, which have low endogenous Rac1 levels, also induced substantial membrane blebbing via the Rho-ROCK pathway. These events were independent of DGKζ catalytic activity, but dependent upon a functional C-terminal PDZ-binding motif. Rescue of RhoA activity in DGKζ-null cells required the PDZ-binding motif, suggesting syntrophin interaction is necessary for optimal RhoA activation. Collectively, our results define a switch-like mechanism involving DGKζ phosphorylation by PKCα that favours RhoA-driven blebbing over Rac1-driven lamellipodia formation and macropinocytosis. These findings provide a mechanistic basis for the effect of PKCα signaling on Rho GTPase activity and suggest PKCα activity plays a role in the interconversion between Rac1 and RhoA signaling that underlies different migration modes.

scholarly journals A new dopachrome-rearranging enzyme from the ejected ink of the cuttlefish Sepia officinalis.

1994 ◽  
Vol 299 (3) ◽  
pp. 839-844 ◽  
Author(s):  
A Palumbo ◽  
M d'Ischia ◽  
G Misuraca ◽  
L De Martino ◽  
G Prota
Keyword(s):  
Catalytic Activity ◽  
Methyl Ester ◽  
Substrate Specificity ◽  
Molecular Mass ◽  
Significant Inhibition ◽  
Single Band ◽  
Pigment Cells ◽  
Sepia Officinalis ◽  
High Substrate ◽  
Sds Page

A melanogenic enzyme catalysing the rearrangement of dopachrome has been identified in the ejected ink of the cuttlefish Sepia officinalis. This enzyme occurs as a heat-labile protein which co-migrates with tyrosinase under a variety of chromatographic and electrophoretic conditions. On SDS/PAGE it shows like a single band with an approx. molecular mass of 85 kDa. The enzyme possesses high substrate specificity, acting on L-dopachrome (Km = 1 mM at pH 6.8) and on L-alpha-methyl-dopachrome, but not on D-dopachrome, L-dopachrome methyl ester, dopaminochrome and adrenochrome. Significant inhibition of the catalytic activity was observed with tropolone and L-mimosine. H.p.1.c. analysis of the enzyme-catalysed rearrangement of L-dopachrome revealed the quantitative formation of the decarboxylated product, 5,6-dihydroxyindole. These results point to marked differences between melanogenesis in cephalopod pigment cells and in melanocytes, which may have important implications in relation to the use of sepiomelanin as a model for studies of mammalian melanins.

scholarly journals Studies on poly-3-hydroxyoctanoate biosynthesis by a consortium of microorganisms

2016 ◽  
Vol 27 (1) ◽  
pp. 44-47 ◽  
Author(s):  
Mihaela Carmen Eremia ◽  
Irina Lupescu ◽  
Mariana Vladu ◽  
Maria Petrescu ◽  
Gabriela Savoiu ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Carbon Source ◽  
Pseudomonas Putida ◽  
Sole Carbon Source ◽  
Bacterial Biomass ◽  
Fermentation Medium ◽  
Energy Reserve ◽  
Bacterial Strains ◽  
Sodium Octanoate ◽  
Intracellular Energy

Abstract Polyhydroxyalcanoates (PHAs) are specifically produced by a wide variety of bacteria, as an intracellular energy reserve in the form of homo- and copolymers of [R]-β-hydroxyalkanoic acids, depending on the C source used for microorganism growth, when the cells are grown under stressing conditions. In this paper we present microbiological accumulation of poly-3-hydroxyoctanoate (PHO) by using a consortium of bacterial strains, Pseudomonas putida and Bacillus subtilis, in a rate of 3:1, grown on a fermentation medium based on sodium octanoate as the sole carbon source. The experiments performed in the above mentioned conditions led to the following results: from 18.70 g sodium octanoate (7.72 g/L in the fermentation medium) used up during the bioprocess, 3.93-3.96 g/L dry bacterial biomass and 1.834 - 1.884 g/L PHA, containing 85.83 - 86.8% PHO, were obtained.

scholarly journals Proinflammatory cytokines induce accumulation of glypican-1-derived heparan sulfate and the C-terminal fragment of β-cleaved APP in autophagosomes of dividing neuronal cells

Glycobiology ◽  
2020 ◽  
Vol 30 (8) ◽  
pp. 539-549
Author(s):  
Fang Cheng ◽  
Lars-Åke Fransson ◽  
Katrin Mani
Keyword(s):  
Heparan Sulfate ◽  
Proinflammatory Cytokines ◽  
Polyacrylamide Gel Electrophoresis ◽  
Neuronal Cells ◽  
Sodium Dodecyl ◽  
Terminal Fragment ◽  
C Terminus ◽  
Tnf Α ◽  
Sds Page ◽  
N Terminus

Abstract Proinflammatory cytokines stimulate expression of β-secretase, which increases processing of amyloid precursor protein (APP), ultimately leading to the deposition of amyloid beta (Aβ). The N-terminal domain of β-cleaved APP supports Cu/NO-dependent release of heparan sulfate (HS) from the glypican-1 (Gpc-1) proteoglycan. HS is an inhibitor of β-secretase, thereby constituting a regulatory, negative feedback loop. Here, we have investigated the effect of the proinflammatory cytokines TNF-α, IL-1β and IL-6 on the interplay between APP processing and release of HS from Gpc-1 in neuronal cells. We have used deconvolution immunofluorescence microscopy and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and a panel of monoclonal/polyclonal antibodies recognizing the released HS, the N-terminus of Aβ, Aβ, the C-terminus of APP and the autophagosome marker LC3 as well as the chemical lysosome marker LysoTrackerRed (LTR). We repeatedly found that N2a neuroblastoma cells and human neural stem cells grown in the presence of the cytokines developed large cytoplasmic clusters, which stained positive for HS, the N-terminus of Aβ, Aβ, the C-terminus of APP, LC3 and LTR, indicating accumulation of HS and APP/APP degradation products in enlarged autophagosomes/lysosomes. The SDS-PAGE of immunoisolates obtained from TNF-α-treated N2a cells by using anti-C-terminus of APP revealed the presence of SDS-stable complexes between HS and the C-terminal fragment of β-cleaved APP (βCTF) migrating in the range 10–18 kDa. Clustered accumulation of βCTF disappeared when HS release was prevented and slightly enhanced when HS release was increased. Hence, when proinflammatory cytokines induce increased processing of APP, inhibition of β-secretase by HS is insufficient, which may lead to the impaired autophagosomal degradation.

scholarly journals Bacterial Community Members Increase Bacillus subtilis Maintenance on the Roots of Arabidopsis thaliana

2020 ◽  
Vol 4 (4) ◽  
pp. 303-313
Author(s):  
Noam Eckshtain-Levi ◽  
Susanna Leigh Harris ◽  
Reizo Quilat Roscios ◽  
Elizabeth Anne Shank
Keyword(s):  
Arabidopsis Thaliana ◽  
Bacillus Subtilis ◽  
Crop Production ◽  
Crop Yields ◽  
Plant Root ◽  
Plant Roots ◽  
Plant Growth Promoting Bacteria ◽  
Bacterial Strains ◽  
Root Microbiome ◽  
Over Time

Plant-growth-promoting bacteria (PGPB) are used to improve plant health and promote crop production. However, because some PGPB (including Bacillus subtilis) do not maintain substantial colonization on plant roots over time, it is unclear how effective PGPB are throughout the plant growing cycle. A better understanding of the dynamics of plant root community assembly is needed to develop and harness the potential of PGPB. Although B. subtilis is often a member of the root microbiome, it does not efficiently monoassociate with plant roots. We hypothesized that B. subtilis may require other primary colonizers to efficiently associate with plant roots. We utilized a previously designed hydroponic system to add bacteria to Arabidopsis thaliana roots and monitor their attachment over time. We inoculated seedlings with B. subtilis and individual bacterial isolates from the native A. thaliana root microbiome either alone or together. We then measured how the coinoculum affected the ability of B. subtilis to colonize and maintain on A. thaliana roots. We screened 96 fully genome-sequenced strains and identified five bacterial strains that were able to significantly improve the maintenance of B. subtilis. Three of these rhizobacteria also increased the maintenance of two strains of B. amyloliquefaciens commonly used in commercially available bioadditives. These results not only illustrate the utility of this model system to address questions about plant–microbe interactions and how other bacteria affect the ability of PGPB to maintain their relationships with plant roots but also may help inform future agricultural interventions to increase crop yields. [Formula: see text] Copyright © 2020 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

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