Fibrobacter succinogenes S85 possesses at least nine distinct glucanase genes

1993 ◽  
Vol 39 (9) ◽  
pp. 882-891 ◽  
Author(s):  
Laercio M. Malburg Jr. ◽  
Cecil W. Forsberg
Keyword(s):  
Escherichia Coli ◽  
Restriction Fragment ◽  
Carboxymethyl Cellulose ◽  
Southern Hybridization ◽  
Fibrobacter Succinogenes ◽  
Rumen Bacteria ◽  
Genomic Libraries ◽  
Zymogram Analysis ◽  
Sds Page ◽  
Specific Activities

The construction of genomic libraries of Fibrobacter succinogenes S85 in λ-Dash, λ-DashII, and pUC19, and the screening of recombinant clones for carboxymethylcellulose hydrolysis yielded 38 glucanase clones. These clones along with a collection of 10 glucanase clones in pUC8, were compared by restriction fragment and Southern hybridization analyses. Seven distinct glucanase clones (pCe14, pCe15, and pCe17 in pUC8, pCe16 in pUC19, and LCe18, LCel10, and LCel12 in λ-Dash) were identified, which were nonhomologous to previously studied cel3, lichenase, and cello-dextrinase genes from F. succinogenes S85. Specific activities of the encoded enzymes expressed in Escherichia coli were higher for carboxymethyl cellulose, barley β-glucan, and lichenan, and lower for acid-swollen cellulose, laminarin, and xylan. The enzymes were predominantly acidic, with pIs between 3.5 and 5.2, with the exception of the pCe16 enzyme, which was basic with a pI of about 8.4. As determined by zymogram analysis after SDS-PAGE, the LCe18 enzyme was 97 kDa, the pCel10 enzyme exhibited components of 70 and 45 kDa, and the pCel12 enzyme components were 69 and 65 kDa. These seven new glucanase clones, along with the cel3 and lichenase genes, indicate that F. succinogenes S85 possesses at least nine distinct endoglucanase genes.Key words: Fibrobacter succinogenes, rumen bacteria, cellulolytic, endoglucanases, glucanases, genes.

Cloning of a xylanase gene from the ruminal fungus Neocallimastix patriciarum 27 and its expression in Escherichia coli

1993 ◽  
Vol 39 (1) ◽  
pp. 134-139 ◽  
Author(s):  
J. M. Tamblyn Lee ◽  
Y. Hu ◽  
H. Zhu ◽  
K. J. Cheng ◽  
P. J. Krell ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Xylanase Activity ◽  
Hydrolytic Activity ◽  
Crystalline Cellulose ◽  
Xylanase Gene ◽  
Amorphous Cellulose ◽  
Zymogram Analysis ◽  
Sds Page ◽  
Neocallimastix Patriciarum ◽  
Temperature Optima

An endo-β-1,4-xylanase gene was cloned from Neocallimastix patriciarum 27 in the bacteriophage vector λgtWESλB and was subcloned into the plasmid vectors pUC18 and pUC19 in which xylanase activity was expressed in both orientations. The xylanase was located in the periplasmic space of the host, Escherichia coli HB101. The pH and temperature optima for periplasmic xylanase activity were 6.2 and 40 °C, respectively, and the Km for oat spelt xylan hydrolysis was 0.89 mg∙mL−1. It also exhibited hydrolytic activity on carboxymethyl cellulose that was equivalent to 28% of the activity exhibited by the enzyme on xylan. It bound to crystalline cellulose, but lacked hydrolytic activity on amorphous cellulose. SDS-PAGE followed by zymogram analysis showed active bands of 68, 58, and 51 kDa. Isoelectric focusing in gels combined with zymogram analysis showed one band of xylanase activity with a pI of 3.6.Key words: Neocallimastix patriciarum, xylanase, gene.

Expression of Trichoderma reesei β-Xylanase in Escherichia coli and its Function in Degradation of Juncao Holocellulose

2011 ◽  
Vol 236-238 ◽  
pp. 1058-1062
Author(s):  
Li Hua Liu ◽  
Zhi Wei Lin ◽  
Ling Lin ◽  
Yan Ling Yang ◽  
Zhan Xi Lin ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Metal Ions ◽  
Molecular Mass ◽  
Trichoderma Reesei ◽  
Optimum Activity ◽  
Zymogram Analysis ◽  
Sds Page ◽  
Ph Range

In this study, the xyn2 gene, which encodes an endo-β-1,4-xylanase, was isolated with holocellulose extracted from Juncao Miscanthus floridulu as an inducer. The xyn2 gene expressed in Escherichia coli, with the estimated yield of 349 U·mL-1. Zymogram analysis showed that the purified Xyn2 had only one band on SDS-PAGE with an estimated molecular mass of 28 kDa. Enzymology analysis demonstrated that its optimum activity was at pH 6.0 and 60°C, with stability at pH range 4.0~7.0 and temperature up to 50°C. The metal ions Cu2+ and Mg2+ showed some inhibition effects, while Fe2+ and Fe3+ had small stimulating effects. Its values of Km and Vmax are 2.85 mM and 50.2 mM/min, respectively. Based on our results, we propose a novel way to convert Juncao biomass into energy and other useful products.

scholarly journals Gut Digestive Function and Microbiome after Correction of Experimental Dysbiosis in Rats by Indigenous Bifidobacteria

2021 ◽  
Vol 9 (3) ◽  
pp. 522
Author(s):  
Lyudmila V. Gromova ◽  
Elena I. Ermolenko ◽  
Anastasiya L. Sepp ◽  
Yulia V. Dmitrieva ◽  
Anna S. Alekseeva ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Digestive Enzymes ◽  
Specific Activity ◽  
Control Group ◽  
Enteropathogenic Escherichia Coli ◽  
Beneficial Bacteria ◽  
Opportunistic Bacteria ◽  
Dyspeptic Symptoms ◽  
Specific Activities ◽  
Impaired Motor Function

In recent years, great interest has arisen in the use of autoprobiotics (indigenous bacteria isolated from the organism and introduced into the same organism after growing). This study aimed to evaluate the effects of indigenous bifidobacteria on intestinal microbiota and digestive enzymes in a rat model of antibiotic-associated dysbiosis. Our results showed that indigenous bifidobacteria (the Bf group) accelerate the disappearance of dyspeptic symptoms in rats and prevent an increase in chyme mass in the upper intestine compared to the group without autoprobiotics (the C1 group), but significantly increase the mass of chyme in the colon compared to the C1 group and the control group (healthy animals). In the Bf group in the gut microbiota, the content of opportunistic bacteria (Proteus spp., enteropathogenic Escherichia coli) decreased, and the content of some beneficial bacteria (Bifidobacterium spp., Dorea spp., Blautia spp., the genus Ruminococcus, Prevotella, Oscillospira) changed compared to the control group. Unlike the C1 group, in the Bf group there was no decrease in the specific activities of maltase and alkaline phosphatase in the mucosa of the upper intestine, but the specific activity of maltase was decreased in the colon chyme compared to the control and C1 groups. In the Bf group, the specific activity of aminopeptidase N was reduced in the duodenum mucosa and the colon chyme compared to the control group. We concluded that indigenous bifidobacteria can protect the microbiota and intestinal digestive enzymes in the intestine from disorders caused by dysbiosis; however, there may be impaired motor function of the colon.

Varying influence of the autolysin, N-acetyl muramidase, and the cell envelope proteinase on the rate of autolysis of six commercial Lactococcus lactis cheese starter bacteria grown in milk

2000 ◽  
Vol 67 (4) ◽  
pp. 585-596 ◽  
Author(s):  
SELVARANI GOVINDASAMY-LUCEY ◽  
PRAMOD K. GOPAL ◽  
PATRICK A. SULLIVAN ◽  
CHRISTOPHER J. PILLIDGE
Keyword(s):  
Lactococcus Lactis ◽  
Cell Walls ◽  
Cell Envelope ◽  
Laboratory Strain ◽  
Proteolytic Cleavage ◽  
Zymogram Analysis ◽  
Sds Page ◽  
Derivatives Of ◽  
Free Strains

The autolysin, N-acetyl muramidase (AcmA), of six commercial Lactococcus lactis subsp. cremoris starter strains and eight Lc. lactis subsp. cremoris derivatives or plasmid-free strains was shown by renaturing SDS-PAGE (zymogram analysis) to be degraded by the cell envelope proteinase (lactocepin; EC 3.4.21.96) after growth of strains in milk at 30 °C for 72 h. Degradation of AcmA was less in starter strains and derivatives producing lactocepin I/III (intermediate specificity) than in strains producing lactocepin I. This supports previous observations on AcmA degradation in derivatives of the laboratory strain Lc. lactis subsp. cremoris MG1363 (Buist et al. Journal of Bacteriology180 5947–5953 1998). In contrast to the MG1363 derivatives, however, the extent of autolysis in milk of the commercial Lc. lactis subsp. cremoris starter strains in this study did not always correlate with lactocepin specificity and AcmA degradation. The distribution of autolysins within the cell envelope of Lc. lactis subsp. cremoris starter strains and derivatives harvested during growth in milk was compared by zymogram analysis. AcmA was found associated with cell membranes as well as cell walls and some cleavage of AcmA occurred independently of lactocepin activity. An AcmA product intermediate in size between precursor (46 kDa) and mature (41 kDa) forms of AcmA was clearly visible on zymograms, even in the absence of lactocepin I activity. These results show that autolysis of commercial Lc. lactis subsp. cremoris starter strains is not primarily determined by AcmA activity in relation to lactocepin specificity and that proteolytic cleavage of AcmA in vivo is not fully defined.

scholarly journals σE Regulates and Is Regulated by a Small RNA in Escherichia coli

2007 ◽  
Vol 189 (11) ◽  
pp. 4243-4256 ◽  
Author(s):  
Karl M. Thompson ◽  
Virgil A. Rhodius ◽  
Susan Gottesman
Keyword(s):  
Escherichia Coli ◽  
Small Rnas ◽  
Noncoding Rna ◽  
Response Regulator ◽  
Heterologous Promoter ◽  
Genomic Libraries ◽  
Multicopy Suppressors ◽  
Intergenic Regions ◽  
Transposon Mutants

ABSTRACT RybB is a small, Hfq-binding noncoding RNA originally identified in a screen of conserved intergenic regions in Escherichia coli. Fusions of the rybB promoter to lacZ were used to screen plasmid genomic libraries and genomic transposon mutants for regulators of rybB expression. A number of plasmids, including some carrying rybB, negatively regulated the fusion. An insertion in the rep helicase and one upstream of dnaK decreased expression of the fusion. Multicopy suppressors of these insertions led to identification of two plasmids that stimulated the fusion. One contained the gene for the response regulator OmpR; the second contained mipA, encoding a murein hydrolase. The involvement of MipA and OmpR in cell surface synthesis suggested that the rybB promoter might be dependent on σE. The sequence upstream of the +1 of rybB contains a consensus σE promoter. The activity of rybB-lacZ was increased in cells lacking the RseA anti-sigma factor and when σE was overproduced from a heterologous promoter. The activity of rybB-lacZ and the detection of RybB were totally abolished in an rpoE-null strain. In vitro, σE efficiently transcribes from this promoter. Both a rybB mutation and an hfq mutation significantly increased expression of both rybB-lacZ and rpoE-lacZ fusions, consistent with negative regulation of the σE response by RybB and other small RNAs. Based on the plasmid screens, NsrR, a repressor sensitive to nitric oxide, was also found to negatively regulate σE-dependent promoters in an RseA-independent fashion.

scholarly journals BACTERIAL AGGLUTINATION BY A LECTIN FROM THE LEAVES OF THE MEDICINAL PLANT, PIMENTA DIOICA (L.) MERR

2018 ◽  
Vol 10 (4) ◽  
pp. 35 ◽  
Author(s):  
Surya P H ◽  
Elyas K K ◽  
Deepti Madayi
Keyword(s):  
Escherichia Coli ◽  
Medicinal Plant ◽  
Biochemical Characterization ◽  
Exchange Chromatography ◽  
Bacterial Typing ◽  
Gram Negative ◽  
Sds Page ◽  
Bacterial Filters ◽  
Pimenta Dioica ◽  
Bacterial Agglutination

Objective: The current investigation involves the purification, characterization of the lectin from the leaves of Pimenta dioica (L.) Merr. (Myrtaceae) a medicinal plant, and its application in bacterial typing.Methods: A lectin was purified from the leaves by cation exchange chromatography. SDS PAGE revealed the molecular weight of the purified lectin. Biochemical characterization was carried out by performing various tests. Hemagglutination inhibition was conducted to detect the sugar specificity. Additionally, bacterial agglutination was performed to predict whether the purified lectin was able to agglutinate the bacterial strains.Results: SDS PAGE analysis revealed the lectin to be a tetramer in the range of 43-66 kDa. The purified lectin agglutinated human, avian, and mouse erythrocytes, and was inhibited by 125 mmol of mannose and xylose. The lectin was stable at 0-60 ° C for 30 min and was unaffected by either 2-Mercaptoethanol (2-ME) or Dithiothreitol (DTT) (50-250µM). A pH of 6.0–8.0 was found optimum for its activity and was nearly independent of metal ions. The purified lectin contained about 20% carbohydrate as estimated by Anthrone method. Purified lectin agglutinated the Gram-negative Escherichia coli and Proteus vulgaris.Conclusion: The isolated lectin was found to possess significant hemagglutinating activity. Due to its ability to agglutinate Gram negative bacteria such as Escherichia coli and Proteus vulgaris, it could be used for bacterial typing and for the design of bacterial filters.

Comparative Analysis of Cytoplasmic Membrane Proteomes of Escherichia coli Using 2D Blue Native/SDS-PAGE

2010 ◽  
pp. 257-269 ◽  
Author(s):  
Susan Schlegel ◽  
Mirjam Klepsch ◽  
David Wickström ◽  
Samuel Wagner ◽  
Jan-Willem de Gier
Keyword(s):  
Escherichia Coli ◽  
Comparative Analysis ◽  
Cytoplasmic Membrane ◽  
Sds Page ◽  
Blue Native

scholarly journals Identification and Function of the pdxY Gene, Which Encodes a Novel Pyridoxal Kinase Involved in the Salvage Pathway of Pyridoxal 5′-Phosphate Biosynthesis in Escherichia coli K-12

1998 ◽  
Vol 180 (7) ◽  
pp. 1814-1821 ◽  
Author(s):  
Yong Yang ◽  
Ho-Ching Tiffany Tsui ◽  
Tsz-Kwong Man ◽  
Malcolm E. Winkler
Keyword(s):  
Escherichia Coli ◽  
De Novo ◽  
Specific Activity ◽  
Salvage Pathway ◽  
Pyridoxal Kinase ◽  
Triple Mutant ◽  
Reading Frame ◽  
E Coli ◽  
Specific Activities ◽  
K 12

ABSTRACT pdxK encodes a pyridoxine (PN)/pyridoxal (PL)/pyridoxamine (PM) kinase thought to function in the salvage pathway of pyridoxal 5′-phosphate (PLP) coenzyme biosynthesis. The observation that pdxK null mutants still contain PL kinase activity led to the hypothesis that Escherichia coli K-12 contains at least one other B6-vitamer kinase. Here we support this hypothesis by identifying the pdxY gene (formally, open reading frame f287b) at 36.92 min, which encodes a novel PL kinase. PdxY was first identified by its homology to PdxK in searches of the complete E. coli genome. Minimal clones of pdxY + overexpressed PL kinase specific activity about 10-fold. We inserted an omega cassette intopdxY and crossed the resultingpdxY::ΩKanr mutation into the bacterial chromosome of a pdxB mutant, in which de novo PLP biosynthesis is blocked. We then determined the growth characteristics and PL and PN kinase specific activities in extracts ofpdxK and pdxY single and double mutants. Significantly, the requirement of the pdxB pdxK pdxY triple mutant for PLP was not satisfied by PL and PN, and the triple mutant had negligible PL and PN kinase specific activities. Our combined results suggest that the PL kinase PdxY and the PN/PL/PM kinase PdxK are the only physiologically important B6vitamer kinases in E. coli and that their function is confined to the PLP salvage pathway. Last, we show thatpdxY is located downstream from pdxH (encoding PNP/PMP oxidase) and essential tyrS (encoding aminoacyl-tRNATyr synthetase) in a multifunctional operon.pdxY is completely cotranscribed with tyrS, but about 92% of tyrS transcripts terminate at a putative Rho-factor-dependent attenuator located in thetyrS-pdxY intercistronic region.

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>

Sign in / Sign up

Export Citation Format

Share Document