scholarly journals The Cyclodextrin Glycosyltransferase ofPaenibacillus pabuliUS132 Strain: Molecular Characterization and Overproduction of the Recombinant Enzyme

2008 ◽  
Vol 2008 ◽  
pp. 1-9 ◽  
Author(s):  
Sonia Jemli ◽  
Ezzedine Ben Messaoud ◽  
Sameh Ben Mabrouk ◽  
Samir Bejar
Keyword(s):  
Enzyme Production ◽  
Shake Flask ◽  
Active Form ◽  
Culture Conditions ◽  
Recombinant Enzyme ◽  
Cyclodextrin Glycosyltransferase ◽  
Gene Encoding ◽  
High Enzyme ◽  
Shake Flasks ◽  
Mature Enzyme

The gene encoding the cyclodextrin glycosyltransferase (CGTase) ofPaenibacillus pabuliUS132, previously described as efficient antistaling agent and good candidate for cyclodextrins production, was cloned, sequenced, and expressed inEscherichia coli. Sequence analysis showed that the mature enzyme (684 amino acids) was preceded by a signal peptide of 34 residues. The enzyme exhibited the highest identity (94%) to theβ-CGTase ofBacillus circulansno. 8. The production of the recombinant CGTase, as active form, was very low (about 1 U/mL) in shake flasks at37∘C. This production reached 22 U/mL after 22 hours of induction by mainly shifting the postinduction temperature from 37 to19∘Cand using 2TY instead of LB medium. High enzyme production (35 U/mL) was attained after 18 hours of induction in fermentor using the same culture conditions as in shake flask. The recombinant enzyme showedVmax⁡andKmvalues of253±36 μmol ofβ-cyclodextrin/mg/min and0.36±0.18 g/L, respectively.

scholarly journals Functional Cloning and Expression of the Schizophyllum commune Glucuronoyl Esterase Gene and Characterization of the Recombinant Enzyme

2012 ◽  
Vol 2012 ◽  
pp. 1-7 ◽  
Author(s):  
Dominic W. S. Wong ◽  
Victor J. Chan ◽  
Amanda A. McCormack ◽  
Ján Hirsch ◽  
Peter Biely
Keyword(s):  
Glucuronic Acid ◽  
Active Form ◽  
Schizophyllum Commune ◽  
Constitutive Expression ◽  
Recombinant Enzyme ◽  
Cloning And Expression ◽  
Gene Encoding ◽  
Glucuronoyl Esterase ◽  
Esterase Gene

The gene encoding Schizophyllum commune glucuronoyl esterase was identified in the scaffold 17 of the genome, containing two introns of 50 bp and 48 bp, with a transcript sequence of 1179 bp. The gene was synthesized and cloned into Pichia pastoris expression vector pGAPZα to achieve constitutive expression and secretion of the recombinant enzyme in soluble active form. The purified protein was 53 kD with glycosylation and had an acidic pI of 3.7. Activity analysis on several uronic acids and their derivatives suggests that the enzyme recognized only esters of 4-O-methyl-D-glucuronic acid derivatives, even with a 4-nitrophenyl aglycon but did not hydrolyze the ester of D-galacturonic acid. The kinetic values were Km 0.25 mM, Vmax 16.3 μM⋅min−1, and kcat 9.27 s−1 with 4-nitrophenyl 2-O-(methyl 4-O-methyl-α-D-glucopyranosyluronate)-β-D-xylopyranoside as the substrate.

scholarly journals A Novel Spore Peptidoglycan Hydrolase of Bacillus cereus: Biochemical Characterization and Nucleotide Sequence of the Corresponding Gene, sleL

2000 ◽  
Vol 182 (6) ◽  
pp. 1499-1506 ◽  
Author(s):  
Yinghua Chen ◽  
Satoshi Fukuoka ◽  
Shio Makino
Keyword(s):  
Nucleotide Sequence ◽  
Biochemical Characterization ◽  
Active Form ◽  
Potassium Phosphate ◽  
Peripheral Region ◽  
Lytic Enzyme ◽  
Gene Encoding ◽  
Dormant Spore ◽  
Mature Enzyme ◽  
Data Banks

ABSTRACT The exudate of germinated spores of B. cereus IFO 13597 in 0.15 M KCl–50 mM potassium phosphate (pH 7.0) contained a spore-lytic enzyme which has substrate specificity for fragmented spore cortex from wild-type organisms (cortical-fragment-lytic enzyme [CFLE]), in addition to a previously characterized germination-specific hydrolase which acts on intact spore cortex (spore cortex-lytic enzyme [SCLE]) (R. Moriyama, S. Kudoh, S. Miyata, S. Nonobe, A. Hattori, and S. Makino, J. Bacteriol. 178:5330–5332, 1996). CFLE was not capable of degrading isolated cortical fragments from spores of Bacillus subtilis ADD1, which lacks muramic acid δ-lactam. This suggests that CFLE cooperates with SCLE in cortex hydrolysis during germination. CFLE was purified in an active form and identified as a 48-kDa protein which functions as anN-acetylglucosaminidase. Immunochemical studies suggested that the mature enzyme is localized on a rather peripheral region of the dormant spore, probably the exterior of the cortex layer. A gene encoding the enzyme, sleL, was cloned in Escherichia coli, and the nucleotide sequence was determined. The gene encodes a protein of 430 amino acids with a deduced molecular weight of 48,136. The N-terminal region contains a repeated motif common to several peptidoglycan binding proteins. Inspection of the data banks showed no similarity of CFLE with N-acetylglucosaminidases found so far, suggesting that CFLE is a novel type ofN-acetylglucosaminidase. The B. subtilis genome sequence contains genes, yaaH and ydhD, which encode putative proteins showing similarity to SleL.

Excretory overexpression of Paenibacillus pabuli US132 cyclodextrin glucanotransferase (CGTase) in Escherichia coli: gene cloning and optimization of the culture conditions using experimental design

Biologia ◽  
2011 ◽  
Vol 66 (6) ◽  
Author(s):  
Dorra Ayadi ◽  
Radhouane Kammoun ◽  
Sonia Jemli ◽  
Samir Bejar
Keyword(s):  
Escherichia Coli ◽  
Bacillus Stearothermophilus ◽  
Active Form ◽  
Culture Conditions ◽  
Cyclodextrin Glucanotransferase ◽  
Gene Encoding ◽  
Box Behnken Design ◽  
Post Induction ◽  
Optimized Conditions ◽  
Maximum Expression

AbstractThe gene encoding the cyclodextrin glucanotransferase of Paenibacillus pabuli US132 was connected to the amylase signal peptide of Bacillus stearothermophilus. This leads to an efficient secretion of the recombinant enzyme into the culture medium of Escherichia coli as an active form contrasting with the native construction leading to a periplasmic production. The optimum cultivation conditions for the maximum expression were optimized, using a Box-Behnken design under the response surface methodology, and found to be a post-induction temperature of 24°C, an induction-starting A600 nm of 0.85, an isopropyl-β-D-thiogalactopyranoside level of 0.045 mM and a post-induction time of 3.9 h. The screening of media components and their concentration were achieved using a Plackett-Burman and a Box-Behnken designs sequentially. Under the optimized conditions selected and in agreement with the predicted model, an activity of 6.03 U/mL was attained. This CGTase production was three-times higher than that using the non-optimized culture conditions (2 U/mL).

scholarly journals Colorimetric determination of alpha and beta-cyclodextrins and studies on optimization of CGTase production from B. firmus using factorial designs

2004 ◽  
Vol 47 (6) ◽  
pp. 837-841 ◽  
Author(s):  
Ilma Hiroko Higuti ◽  
Priscila Anunciação da Silva ◽  
Juliana Papp ◽  
Vivian Mayumi de Eiróz Okiyama ◽  
Edicléia Alves de Andrade ◽  
...  
Keyword(s):  
Enzyme Production ◽  
Organic Nitrogen ◽  
Shake Flask ◽  
Incubation Temperature ◽  
Inorganic Nitrogen ◽  
Colorimetric Determination ◽  
Cyclodextrin Glycosyltransferase ◽  
Inorganic Nitrogen Source ◽  
Initial Ph

Cyclodextrin glycosyltransferase (EC 2.4.1.19, CGTase) production from B. firmus, isolated from soil of Curitiba, PR, was optimized in shake flask using an experimental design approach. The CGTase was produced when the carbon source was starch and beta-CD, but when simple sugars such as glucose, galactose, lactose, sucrose, and maltose were used, there was no enzyme production. CGTase production was the same with either organic nitrogen or inorganic nitrogen source. CGTase activity decreased 2-fold when incubation temperature was increased from 28 to 37 ° C, and decreased 2.1- fold when the initial pH was lowered from 10.3 to 7.4. The colorimetric determinations of alpha - and beta -CD were analyzed as a non-linear relationship and the equilibrium constant for alpha -CD/methyl orange and beta -CD/phenolphthalein complexes were 7.69 x 10³ L / mol and 2.33 x 10³ L/ mol, respectively.

scholarly journals Identification and characterization of YLR328W, theSaccharomyces cerevisiaestructural gene encoding NMN adenylyltransferase. Expression and characterization of the recombinant enzyme

FEBS Letters ◽  
1999 ◽  
Vol 455 (1-2) ◽  
pp. 13-17 ◽  
Author(s):  
Monica Emanuelli ◽  
Francesco Carnevali ◽  
Maria Lorenzi ◽  
Nadia Raffaelli ◽  
Adolfo Amici ◽  
...  
Keyword(s):  
Recombinant Enzyme ◽  
Gene Encoding ◽  
Identification And Characterization

scholarly journals Plasmid-Encoded Metallo-β-Lactamase (IMP-6) Conferring Resistance to Carbapenems, Especially Meropenem

2001 ◽  
Vol 45 (5) ◽  
pp. 1343-1348 ◽  
Author(s):  
Hisakazu Yano ◽  
Akio Kuga ◽  
Ryoichi Okamoto ◽  
Hidero Kitasato ◽  
Toshimitsu Kobayashi ◽  
...  
Keyword(s):  
Point Mutation ◽  
Antimicrobial Agents ◽  
Pcr Amplification ◽  
Penicillin G ◽  
Gene Encoding ◽  
Mature Enzyme ◽  
Dna Fragment ◽  
Lactamase Gene ◽  
Reduced Activity ◽  
Northern Japan

ABSTRACT In 1996, Serratia marcescens KU3838 was isolated from the urine of a patient with a urinary tract infection at a hospital in northern Japan and was found to contain the plasmid pKU501. Previously, we determined that pKU501 carries bla IMP and the genes for TEM-1-type β-lactamases as well as producing both types of β-lactamases (H. Yano, A. Kuga, K. Irinoda, R. Okamoto, T. Kobayashi, and M. Inoue, J. Antibiot. 52:1135–1139, 1999). pKU502 is a recombinant plasmid that contains a 1.5-kb DNA fragment, including the metallo-β-lactamase gene, and is obtained by PCR amplification of pKU501. The sequence of the metallo-β-lactamase gene in pKU502 was determined and revealed that this metallo-β-lactamase gene differed from the gene encoding IMP-1 by one point mutation, leading to one amino acid substitution: 640-A in the base sequence of the IMP-1 gene was replaced by G, and Ser-196 was replaced by Gly in the mature enzyme. This enzyme was designated IMP-6. The strains that produced IMP-6 were resistant to carbapenems. The MICs of panipenem and especially meropenem were higher than the MIC of imipenem for these strains. The k cat/Km value of IMP-6 was about sevenfold higher against meropenem than against imipenem, although the MIC of meropenem for KU1917, which produced IMP-1, was lower than that of imipenem, and the MIC of panipenem was equal to that of imipenem. These results support the hypothesis that IMP-6 has extended substrate profiles against carbapenems. However, the activity of IMP-6 was very low against penicillin G and piperacillin. These results suggest that IMP-6 acquired high activity against carbapenems, especially meropenem, via the point mutation but in the process lost activity against penicillins. Although IMP-6 has reduced activity against penicillins due to this point mutation, pKU501 confers resistance to a variety of antimicrobial agents because it also produces TEM-1-type enzyme.

scholarly journals A Novel Gene Encoding Xanthan Lyase of Paenibacillus alginolyticus Strain XL-1

2000 ◽  
Vol 66 (9) ◽  
pp. 3945-3950 ◽  
Author(s):  
Harald J. Ruijssenaars ◽  
Sybe Hartmans ◽  
Jan C. Verdoes
Keyword(s):  
Signal Sequence ◽  
Fold Increase ◽  
Side Chain ◽  
Cloning And Sequencing ◽  
Gene Encoding ◽  
E Coli ◽  
Mature Enzyme ◽  
Locust Bean ◽  
Encoding Gene ◽  
Amino Acid Signal

ABSTRACT Xanthan-modifying enzymes are powerful tools in studying structure-function relationships of this polysaccharide. One of these modifying enzymes is xanthan lyase, which removes the terminal side chain residue of xanthan. In this paper, the cloning and sequencing of the first xanthan lyase-encoding gene is described, i.e., thexalA gene, encoding pyruvated mannose-specific xanthan lyase of Paenibacillus alginolyticus XL-1. ThexalA gene encoded a 100,823-Da protein, including a 36-amino-acid signal sequence. The 96,887-Da mature enzyme could be expressed functionally in Escherichia coli. Like the native enzyme, the recombinant enzyme showed no activity on depyruvated xanthan. Compared to production by P. alginolyticus, a 30-fold increase in volumetric productivity of soluble xanthan lyase was achieved by heterologous production in E. coli. The recombinant xanthan lyase was used to produce modified xanthan, which showed a dramatic loss of the capacity to form gels with locust bean gum.

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