scholarly journals Optimization Of Expression Conditions Of The Acetylesterase CE16 From Hypocrea Jecorina Encoded By A Synthetic Gene And Expressed In Escherichia coli Cells

2015 ◽  
Vol 14 (2) ◽  
pp. 201-211
Author(s):  
Daniela Jamrichová ◽  
Andrej Godány ◽  
Ľubica Urbániková
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Molecular Mass ◽  
Trichoderma Reesei ◽  
Active Form ◽  
Synthetic Gene ◽  
Hypocrea Jecorina ◽  
Secretion Signal ◽  
The Difference ◽  
C 30

Abstract Acetylesterase CE16 was identified as a part of the enzymatic cocktail secreted by fungus Hypocrea jecorina (anamorph: Trichoderma reesei) during its growth on cellulose. Later it was classified as the first member of a newly organized carbohydrate esterase family CE16. Further studies showed that acetylesterase is crucial for complete deacetylation of naturally acetylated xylans enabling their saccharification by xylanases. To study the relationship between structure and function of acetylesterase, highly purified recombinant enzyme produced by Trichoderma reesei Rut C-30 was prepared. The enzyme was composed of 348 amino acid residues from which the 1 - 19 formed a secretion signal peptide. Determined molecular mass of purified recombinant acetylesterase (Aes1) was 45 kDa which was more than molecular mass calculated from amino acid sequence. As it has been proved later, the difference was caused by the enzyme glycosylation. Glycosylation of proteins increases their stability, but it can also be a source of heterogeneity, which might be a problem during crystallization. To make the future X-ray study of the enzyme easier, recombinant non-glycosylated enzyme needed to be prepared. For these purposes, a synthetic gene optimized for protein expression in Escherichia coli was designed and synthetized. The first nonglycosylated acetylesterase obtained by the expression of its synthetic gene in E. coli cells was mostly insoluble or aggregated. Conditions of cell cultivation, induction of gene expression and cells disruption were necessary to optimize. Presently, after optimization of all mentioned steps, the non-glycosylated recombinant CE16 acetylesterase was prepared in the soluble and active form, ready for further downstream procedures, involving protein purification and crystallization.

scholarly journals Molecular Characterization and Heterologous Expression of the Gene Encoding a Low-Molecular-Mass Endoglucanase from Trichoderma reesei QM9414

1998 ◽  
Vol 64 (2) ◽  
pp. 555-563 ◽  
Author(s):  
Hirofumi Okada ◽  
Kohji Tada ◽  
Tadashi Sekiya ◽  
Kengo Yokoyama ◽  
Akinori Takahashi ◽  
...  
Keyword(s):  
Amino Acid ◽  
Schizosaccharomyces Pombe ◽  
Molecular Mass ◽  
Trichoderma Reesei ◽  
Active Form ◽  
Leader Peptide ◽  
Cdna Clones ◽  
Gene Transcript ◽  
E Coli ◽  
Molecular Masses

ABSTRACT We have isolated the genomic and cDNA clones encoding EG III (a low-molecular-mass endo-β-1,4-glucanase) gene fromTrichoderma reesei QM9414. The nucleotide sequence of the cDNA fragment was verified to contain a 702-bp open reading frame that encodes a 234-amino-acid propeptide. The deduced protein sequence has significant homologies with family H endo-β-1,4-glucanases. The 16-amino-acid N-terminal sequence was shown to function as a leader peptide for possible secretion. Northern blot analysis showed that the EG III gene transcript, with a length of about 700 bp, was expressed markedly by cellulose but not by glucose. The protein has been expressed as a mature form in Escherichia coli and as secreted forms in Saccharomyces cerevisiae and Schizosaccharomyces pombe under the control of tac, alcohol dehydrogenase (ADH1), and human cytomegalovirus promoters, respectively. The S. cerevisiae and Schizosaccharomyces pombe recombinant strains showed strong cellulolytic activities on agar plates containing carboxymethyl cellulose. The E. coli strain expressed small amounts of EG III in an active form and large amounts of EG III in an inactive form. The molecular masses of the recombinant EG IIIs were estimated to be 25, 28, and 29 kDa for E. coli, S. cerevisiae, and Schizosaccharomyces pombe, respectively, by immunoblot analysis following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Parts of the yeast recombinant EG IIIs decreased their molecular masses to 25 kDa after treatment with endoglycosidase H and α-mannosidase, suggesting that they are N glycosylated at least partly.

scholarly journals The catalase-peroxidase of Synechococcus PCC 7942: purification, nucleotide sequence analysis and expression in Escherichia coli

1996 ◽  
Vol 316 (1) ◽  
pp. 251-257 ◽  
Author(s):  
Michinori MUTSUDA ◽  
Takahiro ISHIKAWA ◽  
Toru TAKEDA ◽  
Shigeru SHIGEOKA
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Molecular Mass ◽  
Peroxidase Activity ◽  
Catalase Activity ◽  
Native Enzyme ◽  
Nucleotide Sequence Analysis ◽  
Synechococcus Pcc 7942 ◽  
Catalase Peroxidase ◽  
Pcc 7942

Synechococcus PCC 7942, a cyanobacterium, possesses catalase–peroxidase as the sole hydrogen peroxide-scavenging system. The enzyme has been purified to electrophoretic homogenenity from the cells. The native enzyme had a molecular mass of 150 kDa and was composed of two identical subunits of molecular mass 79 kDa. The apparent Km value of the catalase activity for H2O2 was 4.2±0.27 mM and the kcat value was 2.6×104 s-1. The enzyme contained high catalase activity and an appreciable peroxidase activity with o-dianisidine and pyrogallol. The catalase activity was not inhibited by 3-amino-1,2,4-triazole but by KCN and NaN3 (apparent Ki values 19.3±0.84 and 20.2±0.95 μM respectively). The enzyme showed an absorption spectrum of typical protohaem and contained one protohaem molecule per dimer. The gene encoding catalase–peroxidase was cloned from the chromosomal DNA of Synechococcus PCC 7942. A 2160 bp open reading frame (ORF), coding a catalase–peroxidase of 720 amino acid residues (approx. 79.9 kDa), was observed. The deduced amino acid sequence coincided with that of the N-terminus of the purified enzyme and showed a remarkable similarity to those of a family of catalase–peroxidases of prokaryotic cells. Escherichia coli BL21(DE3)plysS, harbouring a recombinant plasmid containing the catalase–peroxidase gene, produced a large amount of proteins that co-migrated on SDS/PAGE with the native enzyme. The recombinant enzyme showed the same ratio of catalase activity to peroxidase activity with o-dianisidine and the same Km for H2O2 as the native enzyme.

scholarly journals Purification and Characterization of the Recombinant Thermus sp. Strain T2 α-Galactosidase Expressed in Escherichia coli

2001 ◽  
Vol 67 (4) ◽  
pp. 1601-1606 ◽  
Author(s):  
Mitsunori Ishiguro ◽  
Satoshi Kaneko ◽  
Atsushi Kuno ◽  
Yoshinori Koyama ◽  
Shigeki Yoshida ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Active Form ◽  
Gel Filtration ◽  
Amino Acid Sequences ◽  
Side Chain ◽  
Catalytic Function ◽  
Native Polyacrylamide Gel Electrophoresis

ABSTRACT The nucleotide sequence of the Thermus sp. strain T2 DNA coding for a thermostable α-galactosidase was determined. The deduced amino acid sequence of the enzyme predicts a polypeptide of 474 amino acids (M r, 53,514). The observed homology between the deduced amino acid sequences of the enzyme and α-galactosidase from Thermus brockianus was over 70%.Thermus sp. strain T2 α-galactosidase was expressed in its active form in Escherichia coli and purified. Native polyacrylamide gel electrophoresis and gel filtration chromatography data suggest that the enzyme is octameric. The enzyme was most active at 75°C forp-nitrophenyl-α-d-galactopyranoside hydrolysis, and it retained 50% of its initial activity after 1 h of incubation at 70°C. The enzyme was extremely stable over a broad range of pH (pH 6 to 13) after treatment at 40°C for 1 h. The enzyme acted on the terminal α-galactosyl residue, not on the side chain residue, of the galactomanno-oligosaccharides as well as those of yeasts and Mortierella vinacea α-galactosidase I. The enzyme has only one Cys residue in the molecule.para-Chloromercuribenzoic acid completely inhibited the enzyme but did not affect the mutant enzyme which contained Ala instead of Cys, indicating that this Cys residue is not responsible for its catalytic function.

scholarly journals Efficient production of active chicken avidin using a bacterial signal peptide in Escherichia coli

2004 ◽  
Vol 384 (2) ◽  
pp. 385-390 ◽  
Author(s):  
Vesa P. HYTÖNEN ◽  
Olli H. LAITINEN ◽  
Tomi T. AIRENNE ◽  
Heidi KIDRON ◽  
Niko J. MELTOLA ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Signal Sequence ◽  
Specific Binding ◽  
Affinity Purification ◽  
Active Form ◽  
Efficient Production ◽  
E Coli ◽  
Secretion Signal ◽  
Technology Applications ◽  
Biotin Binding

Chicken avidin is a highly popular tool with countless applications in the life sciences. In the present study, an efficient method for producing avidin protein in the periplasmic space of Escherichia coli in the active form is described. Avidin was produced by replacing the native signal sequence of the protein with a bacterial OmpA secretion signal. The yield after a single 2-iminobiotin–agarose affinity purification step was approx. 10 mg/l of virtually pure avidin. Purified avidin had 3.7 free biotin-binding sites per tetramer and showed the same biotin-binding affinity and thermal stability as egg-white avidin. Avidin crystallized under various conditions, which will enable X-ray crystallographic studies. Avidin produced in E. coli lacks the carbohydrate chains of chicken avidin and the absence of glycosylation should decrease the non-specific binding that avidin exhibits towards many materials [Rosebrough and Hartley (1996) J. Nucl. Med. 37, 1380–1384]. The present method provides a feasible and inexpensive alternative for the production of recombinant avidin, avidin mutants and avidin fusion proteins for novel avidin–biotin technology applications.

scholarly journals Spectroscopic studies on an oxygen-binding haemoglobin-like flavohaemoprotein from Escherichia coli

1992 ◽  
Vol 288 (2) ◽  
pp. 649-655 ◽  
Author(s):  
N Ioannidis ◽  
C E Cooper ◽  
R K Poole
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Molecular Mass ◽  
Spectroscopic Studies ◽  
Oxygen Binding ◽  
Histidine Residue ◽  
Nitrosyl Complex ◽  
Sds Page ◽  
Prosthetic Groups

The Escherichia coli haemoglobin-like flavohaemoprotein (Hmp) has been purified to near homogeneity using two chromatographic steps. The prosthetic groups are identified as FAD and protohaem IX. SDS/PAGE has indicated a molecular mass of 44 kDa for the monomeric protein consistent with the amino-acid sequence deduced from the hmp+ gene. The protein, as isolated, is in the Fe(III) state, exhibiting absorbance maxima at 403.5, 540 (shoulder) and 627 nm. The ferrous and carbonmonoxyferrous states resemble those of haemoglobin, showing maxima at 431.5 and 558 nm, and 421, 542 and 566 nm respectively. Upon aerobic addition of NAD(P)H, the ferric state is reduced to the oxygenated Fe(II) state, characterized by maxima at 413, 544 and 580 nm. This oxy form is not stable and slowly decays to the ferric state. Addition of dithionite and nitrite to the ferric protein results in the formation of a nitrosyl complex, whose e.p.r. characteristics indicate that the b-type haem is attached to the protein through a nitrogenous ligand, probably originating from a histidine residue.

scholarly journals Triphenylmethane Reductase from Citrobacter sp. Strain KCTC 18061P: Purification, Characterization, Gene Cloning, and Overexpression of a Functional Protein in Escherichia coli

2005 ◽  
Vol 71 (12) ◽  
pp. 7955-7960 ◽  
Author(s):  
Moon-Sun Jang ◽  
Young-Mi Lee ◽  
Cheorl-Ho Kim ◽  
Jai-Heon Lee ◽  
Dong-Woo Kang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Molecular Mass ◽  
Enzymatic Reaction ◽  
Amino Acid Sequences ◽  
Binding Motif ◽  
Triphenylmethane Dyes ◽  
Functional Protein ◽  
Triphenylmethane Reductase

ABSTRACT We purified to homogeneity an enzyme from Citrobacter sp. strain KCTC 18061P capable of decolorizing triphenylmethane dyes. The native form of the enzyme was identified as a homodimer with a subunit molecular mass of about 31 kDa. It catalyzes the NADH-dependent reduction of triphenylmethane dyes, with remarkable substrate specificity related to dye structure. Maximal enzyme activity occurred at pH 9.0 and 60°C. The enzymatic reaction product of the triphenylmethane dye crystal violet was identified as its leuco form by UV-visible spectral changes and thin-layer chromatography. A gene encoding this enzyme was isolated based on its N-terminal and internal amino acid sequences. The nucleotide sequence of the gene has a single open reading frame encoding 287 amino acids with a predicted molecular mass of 30,954 Da. Although the deduced amino acid sequence displays 99% identity to the hypothetical protein from Listeria monocytogenes strain 4b H7858, it shows no overall functional similarity to any known protein in the public databases. At the N terminus, the amino acid sequence has high homology to sequences of NAD(P)H-dependent enzymes containing the dinucleotide-binding motif GXXGXXG. The enzyme was heterologously expressed in Escherichia coli, and the purified recombinant enzyme showed characteristics similar to those of the native enzyme. This is the first report of a triphenylmethane reductase characterized from any organism.

scholarly journals Structure of the β-Galactosidase Gene fromThermus sp. Strain T2: Expression in Escherichia coli and Purification in a Single Step of an Active Fusion Protein

1998 ◽  
Vol 64 (6) ◽  
pp. 2187-2191 ◽  
Author(s):  
Alejandro Vian ◽  
Alfonso V. Carrascosa ◽  
José L. García ◽  
Estrella Cortés
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Bacillus Stearothermophilus ◽  
Specific Activity ◽  
Active Form ◽  
Deae Cellulose ◽  
Single Step ◽  
Amino Acid Sequences ◽  
Glycosyl Hydrolases ◽  
Flanking Regions

ABSTRACT The nucleotide sequence of both the bgaA gene, coding for a thermostable β-galactosidase of Thermus sp. strain T2, and its flanking regions was determined. The deduced amino acid sequence of the enzyme predicts a polypeptide of 645 amino acids (M r, 73,595). Comparative analysis of the open reading frames located in the flanking regions of the bgaAgene revealed that they might encode proteins involved in the transport and hydrolysis of sugars. The observed homology between the deduced amino acid sequences of BgaA and the β-galactosidase ofBacillus stearothermophilus allows us to classify the new enzyme within family 42 of glycosyl hydrolases. BgaA was overexpressed in its active form in Escherichia coli, but more interestingly, an active chimeric β-galactosidase was constructed by fusing the BgaA protein to the choline-binding domain of the major pneumococcal autolysin. This chimera illustrates a novel approach for producing an active and thermostable hybrid enzyme that can be purified in a single step by affinity chromatography on DEAE-cellulose, retaining the catalytic properties of the native enzyme. The chimeric enzyme showed a specific activity of 191,000 U/mg at 70°C and aKm value of 1.6 mM witho-nitrophenyl-β-d-galactopyranoside as a substrate, and it retained 50% of its initial activity after 1 h of incubation at 70°C.

scholarly journals N-acetyl-d-neuraminic acid lyase generates the sialic acid for colominic acid biosynthesis in Escherichia coli K1

1996 ◽  
Vol 317 (1) ◽  
pp. 157-165 ◽  
Author(s):  
Miguel A. FERRERO ◽  
Angel REGLERO ◽  
Manuel FERNANDEZ-LOPEZ ◽  
Roberto ORDAS ◽  
Leandro B. RODRIGUEZ-APARICIO
Keyword(s):  
Escherichia Coli ◽  
Sialic Acid ◽  
Amino Acid ◽  
Molecular Mass ◽  
Gel Filtration ◽  
Neuraminic Acid ◽  
Acid Biosynthesis ◽  
Colominic Acid ◽  
Apparent Homogeneity ◽  
Escherichia Coli K1

Colominic acid is a capsular homopolymer from Escherichia coli K1 composed of α(2-8)-linked N-acetyl-d-neuraminic acid (NeuAc) residues. Recently, we have described that NeuAc synthesis in this bacterium occurs through the action of NeuAc lyase (EC 4.1.3.3) [Rodríguez-Aparicio, Ferrero and Reglero (1995) Biochem. J. 308, 501–505]. In the present work we analysed and characterized this enzyme. E. coli K1 NeuAc lyase is detected from the early logarithmic phase of growth, is induced by NeuAc and is not repressed by glucose. The enzyme was purified to apparent homogeneity (312-fold) using two types of hydrophobic chromatographies (butyl-agarose and phenyl-Sepharose CL-4B), gel filtration on Sephacryl S-200, and anion-exchange chromatography on DEAE-FPLC. The pure enzyme, whose amino acid composition and N-terminal amino acid sequence are also established, has a native molecular mass, estimated by gel filtration, of 135±3 kDa, whereas its molecular mass in SDS/PAGE was 33±1 kDa. The enzyme was able to synthesize and cleave NeuAc in a reversible reaction. The maximal rate of catalysis was achieved in 125 mM Tris/HCl buffer, pH 7.8, at 37 °C. Under these conditions, the Km values calculated for N-acetyl-d-mannosamine and pyruvate (condensation direction), and NeuAc (hydrolysis direction) were 7.7, 8.3 and 4.8 mM respectively. NeuAc synthesis by the pure enzyme was activated by Ca2+ and inhibited by Mn2+ and NeuAc, whereas the enzyme cleavage direction was inhibited by Ca2+, Mn2+ and pyruvate. The reaction products, NeuAc and pyruvate, and Ca2+ are able to regulate the direction of this enzyme (synthesis or cleavage of sialic acid) and, accordingly, to modulate colominic acid biosynthesis.

scholarly journals Different Rifampin Sensitivities of Escherichia coli and Mycobacterium tuberculosis RNA Polymerases Are Not Explained by the Difference in the β-Subunit Rifampin Regions I and II

2005 ◽  
Vol 49 (4) ◽  
pp. 1587-1590 ◽  
Author(s):  
N. Zenkin ◽  
A. Kulbachinskiy ◽  
I. Bass ◽  
V. Nikiforov
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Mycobacterium Tuberculosis ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Rna Polymerase ◽  
Β Subunit ◽  
E Coli ◽  
Increased Sensitivity ◽  
The Difference

ABSTRACT Mycobacterium tuberculosis RNA polymerase is 1,000-fold more sensitive to rifampin than Escherichia coli RNA polymerase. Chimeric E. coli RNA polymerase in which the β-subunit segment encompassing rifampin regions I and II (amino acids [aa] 463 through 590) was replaced with the corresponding region from M. tuberculosis (aa 382 through 509) did not show an increased sensitivity to the antibiotic. Thus, the difference in amino acid sequence between the rifampin regions I and II of the two species does not account for the difference in rifampin sensitivity of the two polymerases.

Characterization of two forms of cationic peroxidase from cultured peanut cells

1992 ◽  
Vol 70 (2) ◽  
pp. 166-169 ◽  
Author(s):  
James P. O'Donnell ◽  
Lianglu Wan ◽  
R. B. van Huystee
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Molecular Mass ◽  
Concanavalin A ◽  
Specific Activity ◽  
Calcium Content ◽  
Molecular Forms ◽  
The Difference ◽  
Peanut Peroxidase

Two forms of cationic peroxidase from peanut cells were differentiated by concanavalin A affinity chromatography. They differed in molecular mass as well as concanavalin A binding, leading to the initial suggestion that they represented two isozymes of peroxidase. However, similar values for the specific activity, Soret absorption, calcium content, and peptide molecular mass were observed for each of the forms. Therefore, the binding and nonbinding fractions most likely represent two molecular forms of cationic peanut peroxidase, rather than two distinct cationic isozymes. The difference between these two forms is discussed in terms of glycosylation. Through the amino acid sequence analysis of the formic acid treated peptide, the cationic isozyme has been shown to be identical in amino acid sequence to the cDNA clone PNC1.Key words: peanut, peroxidase, glycan, characterization, sequence.

Sign in / Sign up

Export Citation Format

Share Document