scholarly journals Spontaneous Cleavages of a Heterologous Protein, the CenA Endoglucanase of Cellulomonas fimi, in Escherichia coli

2021 ◽  
Vol 14 ◽  
pp. 117863612110246
Author(s):  
Cheuk Yin Lai ◽  
Ka Lun Ng ◽  
Hao Wang ◽  
Chui Chi Lam ◽  
Wan Keung Raymond Wong
Keyword(s):  
Escherichia Coli ◽  
Cellulolytic Bacterium ◽  
Systematic Screening ◽  
Cellulomonas Fimi ◽  
E Coli ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Glycosylation Sites ◽  
Endogenous Proteins ◽  
Cellulose Binding

CenA is an endoglucanase secreted by the Gram-positive cellulolytic bacterium, Cellulomonas fimi, to the environment as a glycosylated protein. The role of glycosylation in CenA is unclear. However, it seems not crucial for functional activity and secretion since the unglycosylated counterpart, recombinant CenA (rCenA), is both bioactive and secretable in Escherichia coli. Using a systematic screening approach, we have demonstrated that rCenA is subjected to spontaneous cleavages (SC) in both the cytoplasm and culture medium of E. coli, under the influence of different environmental factors. The cleavages were found to occur in both the cellulose-binding (CellBD) and catalytic domains, with a notably higher occurring rate detected in the former than the latter. In CellBD, the cleavages were shown to occur close to potential N-linked glycosylation sites, suggesting that these sites might serve as ‘attributive tags’ for differentiating rCenA from endogenous proteins and the points of initiation of SC. It is hypothesized that glycosylation plays a crucial role in protecting CenA from SC when interacting with cellulose in the environment. Subsequent to hydrolysis, SC would ensure the dissociation of CenA from the enzyme-substrate complex. Thus, our findings may help elucidate the mechanisms of protein turnover and enzymatic cellulolysis.

scholarly journals Intramolecular chaperone and inhibitor activities of a propeptide from a bacterial zinc aminopeptidase

1999 ◽  
Vol 341 (1) ◽  
pp. 25-31 ◽  
Author(s):  
Satoru NIRASAWA ◽  
Yoshiaki NAKAJIMA ◽  
Zhen-Zhong ZHANG ◽  
Michiteru YOSHIDA ◽  
Kiyoshi HAYASHI
Keyword(s):  
Escherichia Coli ◽  
Conformational Changes ◽  
Amino Acid Residues ◽  
E Coli ◽  
Substrate Complex ◽  
Extracellular Secretion ◽  
Enzyme Substrate ◽  
Aeromonas Caviae ◽  
Km Value

An aminopeptidase from Aeromonas caviae T-64 was translated as a preproprotein consisting of three domains; a signal peptide (19 amino acid residues), an N-terminal propeptide (101 residues) and a mature region (273 residues). We demonstrated that a proteinase, which was isolated from the culture filtrate of A. caviae T-64, activated the recombinant pro-aminopeptidase by removal of the majority of the propeptide. Using L-Leu-p-nitroanilide as a substrate, the processed aminopeptidase showed a large increase in kcat when compared with the unprocessed enzyme, whereas the Km value remained relatively unchanged. The similar Km values for the pro-aminopeptidase and the mature aminopeptidase indicated that the N-terminal propeptide of the pro-aminopeptidase did not influence the formation of the enzyme-substrate complex, suggesting the absence of marked conformational changes in the active domain. In contrast, the marked difference in kcat suggests a significant decrease in the energy of one or more of the transition states of the enzyme-substrate reaction coordinate. Moreover, we showed that the activity of the urea-denatured pro-aminopeptidase could be recovered by dialysis, whereas the activity of the urea-denatured mature aminopeptidase, which lacked the propeptide, could not. Further to this, the propeptide-deleted aminopeptidase formed an inclusion body in the cytoplasmic space in Escherichia coli and was not secreted at all. These results suggested that the propeptide of the pro-aminopeptidase acted as an intramolecular chaperone that was involved with the correct folding of the enzyme in vitro and was required for extracellular secretion in E. coli.

Glycosylation by Pichia pastoris decreases the affinity of a family 2a carbohydrate-binding module from Cellulomonas fimi: a functional and mutational analysis

2001 ◽  
Vol 358 (2) ◽  
pp. 423-430 ◽  
Author(s):  
Alisdair B. BORASTON ◽  
R. Antony J. WARREN ◽  
Douglas G. KILBURN
Keyword(s):  
Pichia Pastoris ◽  
Association Constant ◽  
Mutational Analysis ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Module ◽  
Wild Type ◽  
Cellulomonas Fimi ◽  
E Coli ◽  
Glycosylation Sites ◽  
High Mannose

When produced by Pichia pastoris, three of the five Asn-Xaa-Ser/Thr sequences (corresponding to Asn-24, Asn-73 and Asn-87) in the carbohydrate-binding module CBM2a of xylanase 10A from Cellulomonas fimi are glycosylated. The glycans are of the high-mannose type, ranging in size from GlcNAc2Man8 to GlcNAc2Man14. The N-linked glycans block the binding of CBM2a to cellulose. Analysis of mutants of CBM2a shows that glycans on Asn-24 decrease the association constant (Ka) for the binding of CBM2a to bacterial microcrystalline cellulose approx. 10-fold, whereas glycans on Asn-87 destroy binding. The Ka of a mutant of CBM2a lacking all three N-linked glycosylation sites is the same when the polypeptide is produced by either Escherichia coli or P. pastoris and is approx. half that of wild-type CBM2a produced by E. coli.

scholarly journals From reporters to endogenous genes: the impact of the first five codons on translation efficiency in Escherichia coli

2019 ◽  
Author(s):  
Mariana H. Moreira ◽  
Géssica C. Barros ◽  
Rodrigo D. Requião ◽  
Silvana Rossetto ◽  
Tatiana Domitrovic ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Protein Synthesis ◽  
Protein Expression ◽  
Nucleotide Composition ◽  
Control Mechanisms ◽  
Translation Efficiency ◽  
Coding Region ◽  
E Coli ◽  
Endogenous Proteins ◽  
The Impact

ABSTRACTTranslation initiation is a critical step in the regulation of protein synthesis, and it is subjected to different control mechanisms, such as 5’ UTR secondary structure and initiation codon context, that can influence the rates at which initiation and consequentially translation occur. For some genes, translation elongation also affects the rate of protein synthesis. With a GFP library containing nearly all possible combinations of nucleotides from the 3rd to the 5th codon positions in the protein coding region of the mRNA, it was previously demonstrated that some nucleotide combinations increased GFP expression up to four orders of magnitude. While it is clear that the codon region from positions 3 to 5 can influence protein expression levels of artificial constructs, its impact on endogenous proteins is still unknown. Through bioinformatics analysis, we identified the nucleotide combinations of the GFP library in Escherichia coli genes and examined the correlation between the expected levels of translation according to the GFP data with the experimental measures of protein expression. We observed that E. coli genes were enriched with the nucleotide compositions that enhanced protein expression in the GFP library, but surprisingly, it seemed to affect the translation efficiency only marginally. Nevertheless, our data indicate that different enterobacteria present similar nucleotide composition enrichment as E. coli, suggesting an evolutionary pressure towards the conservation of short translational enhancer sequences.

Effect of endotoxin on capillary permeability to macromolecules

1964 ◽  
Vol 207 (3) ◽  
pp. 518-522 ◽  
Author(s):  
S. Chien ◽  
D. G. Sinclair ◽  
R. J. Dellenback ◽  
C. Chang ◽  
B. Peric ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Intravenous Injection ◽  
Thoracic Duct ◽  
Capillary Permeability ◽  
Lymph Flow ◽  
Thoracic Duct Lymph ◽  
E Coli ◽  
Duct Lymph ◽  
Endogenous Proteins

The intravenous injection of Escherichia coli endotoxin (3 mg/kg) into dogs caused an increase in lymph flow from the thoracic duct. The lymph concentrations of macromolecules (dextran with mol. wt. of 250,000, albumin-I131, and endogenous proteins) increased and the lymph-to-plasma ratios approached 1. These results indicate that E. coli endotoxin causes an increase in capillary permeability to both the fluid and the macromolecules in plasma. The increase in capillary permeability for albumin-I131 was greater than that for dextran with mol. wt. of 250,000. Eighty minutes after endotoxin, the lymph flow returned to normal, but albumin-I131 and dextran injected at this time were still transferred into the thoracic duct lymph at enhanced rates.

Analysis of Molecular Size Distributions of Cellulose Molecules during Hydrolysis of Cellulose by Recombinant Cellulomonas fimiβ-1,4-Glucanases

1998 ◽  
Vol 64 (7) ◽  
pp. 2374-2379 ◽  
Author(s):  
Henrik Stålbrand ◽  
Shawn D. Mansfield ◽  
John N. Saddler ◽  
Douglas G. Kilburn ◽  
R. Antony J. Warren ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Molecular Size ◽  
Cellulose Hydrolysis ◽  
Hydrolytic Activity ◽  
Cellulolytic Bacterium ◽  
Size Distributions ◽  
Cellulomonas Fimi ◽  
Hydrolysis Of Cellulose ◽  
The Individual ◽  
Hydrolysis Of

ABSTRACT Four β-1,4-glucanases (cellulases) of the cellulolytic bacteriumCellulomonas fimi were purified from Escherichia coli cells transformed with recombinant plasmids. Previous analyses using soluble substrates had suggested that CenA and CenC were endoglucanases while CbhA and CbhB resembled the exo-acting cellobiohydrolases produced by cellulolytic fungi. Analysis of molecular size distributions during cellulose hydrolysis by the individual enzymes confirmed these preliminary findings and provided further evidence that endoglucanase CenC has a more processive hydrolytic activity than CenA. The significant differences between the size distributions obtained during hydrolysis of bacterial microcrystalline cellulose and acid-swollen cellulose can be explained in terms of the accessibility of β-1,4-glucan chains to enzyme attack. Endoglucanases and cellobiohydrolases were much more easily distinguished when the acid-swollen substrate was used.

Enzyme-Linked Immunomagnetic Electrochemical Detection of Live Escherichia coli O157:H7 in Apple Juice†

2005 ◽  
Vol 68 (1) ◽  
pp. 146-149 ◽  
Author(s):  
ANDREW G. GEHRING ◽  
SHU-I TU
Keyword(s):  
Escherichia Coli ◽  
Electrochemical Detection ◽  
Apple Juice ◽  
Magnetic Beads ◽  
Matrix Effects ◽  
Escherichia Coli O157 ◽  
Bacterial Cells ◽  
E Coli ◽  
Enzyme Substrate ◽  
Assay Time

We describe the application of enzyme-linked immunomagnetic electrochemistry (ELIME) for the rapid detection of Escherichia coli O157:H7 in buffered apple juice. The ELIME technique entails sandwiching bacterial analyte between antibody-coated magnetic beads and an alkaline phosphatase–conjugated antibody. The beads (with or without bound bacteria) were localized onto the surface of magnetized graphite ink electrodes in a multiwell plate format. The enzyme substrate, 1-naphthyl phosphate, was added, and conversion of substrate to an electroactive product was measured using electrochemical detection. With this technique, detection of whole, live E. coli O157:H7 bacterial cells was achieved with a minimum detectable level of ca. 5 × 103 cells per ml in Tris-buffered saline or buffered apple juice in an assay time of ca. 80 min. With adjustment of pH, the ELIME response for the bacteria in either sampling medium was similar, indicating that apple juice components did not contribute to any discernible sample matrix effects.

scholarly journals Action mechanism of Escherichia coli DNA photolyase. I. Formation of the enzyme-substrate complex.

1987 ◽  
Vol 262 (1) ◽  
pp. 478-485
Author(s):  
G B Sancar ◽  
F W Smith ◽  
R Reid ◽  
G Payne ◽  
M Levy ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Action Mechanism ◽  
Dna Photolyase ◽  
Substrate Complex ◽  
Enzyme Substrate

scholarly journals Identification of the faecal indicator Escherichia coli in wastewater through the β-D-glucuronidase activity: comparison between two enumeration methods, membrane filtration with TBX agar, and Colilert®-18

2016 ◽  
Vol 15 (2) ◽  
pp. 209-217 ◽  
Author(s):  
P. Vergine ◽  
C. Salerno ◽  
E. Barca ◽  
G. Berardi ◽  
A. Pollice
Keyword(s):  
Escherichia Coli ◽  
Membrane Filtration ◽  
Microbiological Quality ◽  
Treated Wastewater ◽  
E Coli ◽  
Glucuronidase Activity ◽  
Enzyme Substrate ◽  
Different Origins ◽  
Activity Comparison

Escherichia coli (E. coli) is one of the most commonly adopted indicators for the determination of the microbiological quality in water and treated wastewater. Two main types of methods are used for the enumeration of this faecal indicator: membrane filtration (MF) and enzyme substrate tests. For both types, several substrates based on the β-D-glucuronidase activity have been commercialized. The specificity of this enzyme for E. coli bacteria has generated considerable use of methods that identify the β-D-glucuronidase activity as a definite indication of the presence of E. coli, without any further confirmation. This approach has been recently questioned for the application to wastewater. The present study compares two methods belonging to the above-mentioned types for the enumeration of E. coli in wastewater: MF with Tryptone Bile X-glucuronide agar and the Colilert®-18 test. Confirmation tests showed low average percentages of false positives and false negatives for both enumeration methods (between 4 and 11%). Moreover, the counting capabilities of these two methods were compared for a set of 70 samples of wastewater having different origins and degrees of treatment. Statistical analysis showed that the Colilert®-18 test allowed on average for a significantly higher recovery of E. coli.

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