scholarly journals A Novel Highly Thermostable Multifunctional Beta-Glycosidase from CrenarchaeonAcidilobus saccharovorans

Archaea ◽  
2015 ◽  
Vol 2015 ◽  
pp. 1-6 ◽  
Author(s):  
Vadim M. Gumerov ◽  
Andrey L. Rakitin ◽  
Andrey V. Mardanov ◽  
Nikolai V. Ravin
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Half Life ◽  
Sequence Similarity ◽  
Hydrolytic Activity ◽  
Maximum Activity ◽  
Glycoside Hydrolases ◽  
Amino Acid Residues ◽  
High Tolerance ◽  
High Sequence Similarity

We expressed a putativeβ-galactosidase Asac_1390 from hyperthermophilic crenarchaeonAcidilobus saccharovoransinEscherichia coliand purified the recombinant enzyme. Asac_1390 is composed of 490 amino acid residues and showed high sequence similarity to family 1 glycoside hydrolases from various thermophilic Crenarchaeota. The maximum activity was observed at pH 6.0 and 93°C. The half-life of the enzyme at 90°C was about 7 hours. Asac_1390 displayed high tolerance to glucose and exhibits hydrolytic activity towards cellobiose and various aryl glucosides. The hydrolytic activity withp-nitrophenyl (pNP) substrates followed the order pNP-β-D-galactopyranoside (328 U mg−1), pNP-β-D-glucopyranoside (246 U mg−1), pNP-β-D-xylopyranoside (72 U mg−1), and pNP-β-D-mannopyranoside (28 U mg−1). Thus the enzyme was actually a multifunctionalβ-glycosidase. Therefore, the utilization of Asac_1390 may contribute to facilitating the efficient degradation of lignocellulosic biomass and help enhance bioconversion processes.

scholarly journals Isomaltulose Synthase from Klebsiella sp. Strain LX3: Gene Cloning and Characterization and Engineering of Thermostability

2002 ◽  
Vol 68 (6) ◽  
pp. 2676-2682 ◽  
Author(s):  
Daohai Zhang ◽  
Xianzhen Li ◽  
Lian-Hui Zhang
Keyword(s):  
Amino Acid ◽  
Sequence Alignment ◽  
Half Life ◽  
Bacterial Isolate ◽  
Catalytic Efficiency ◽  
Fold Increase ◽  
Maximum Activity ◽  
Amino Acid Residues ◽  
Sequence Domain ◽  
Gene Cloning And Characterization

ABSTRACT The gene (palI) encoding isomaltulose synthase (PalI) from a soil bacterial isolate, Klebsiella sp. strain LX3, was cloned and characterized. PalI converts sucrose into isomaltulose, trehalulose, and trace amounts of glucose and fructose. Sequence domain analysis showed that PalI contains an α-amylase domain and (β/α)8-barrel structures, suggesting that it belongs to the α-amylase family. Sequence alignment indicated that the five amino acid residues of catalytic importance in α-amylases and glucosyltransferases (Asp241, Glu295, Asp369, His145, and His368) are conserved in PalI. Purified recombinant PalI displayed high catalytic efficiency, with a Km of 54.6 ± 1.7 mM for sucrose, and maximum activity (approximately 328.0 ± 2.5 U/mg) at pH 6.0 and 35°C. PalI activity was strongly inhibited by Fe3+ and Hg2+ and was enhanced by Mn2+ and Mg2+. The half-life of PalI was 1.8 min at 50°C. Replacement of selected amino acid residues by proline significantly increased the thermostability of PalI. Simultaneous replacement of Glu498 and Arg310 with proline resulted in an 11-fold increase in the half-life of PalI at 50°C.

scholarly journals Isolation and Characterization of a Novel Cold-Active, Halotolerant Endoxylanase from Echinicola rosea Sp. Nov. JL3085T

Marine Drugs ◽  
2020 ◽  
Vol 18 (5) ◽  
pp. 245
Author(s):  
Jianlong He ◽  
Le Liu ◽  
Xiaoyan Liu ◽  
Kai Tang
Keyword(s):  
Amino Acid ◽  
Marine Bacterium ◽  
Maximum Velocity ◽  
Maximum Activity ◽  
Glycoside Hydrolases ◽  
Amino Acid Residues ◽  
Xylanase Gene ◽  
Isolation And Characterization ◽  
Cold Active

We cloned a xylanase gene (xynT) from marine bacterium Echinicola rosea sp. nov. JL3085T and recombinantly expressed it in Escherichia coli BL21. This gene encoded a polypeptide with 379 amino acid residues and a molecular weight of ~43 kDa. Its amino acid sequence shared 45.3% similarity with an endoxylanase from Cellvibrio mixtus that belongs to glycoside hydrolases family 10 (GH10). The XynT showed maximum activity at 40 °C and pH 7.0, and a maximum velocity of 62 μmoL min−1 mg−1. The XynT retained its maximum activity by more than 69%, 51%, and 26% at 10 °C, 5 °C, and 0 °C, respectively. It also exhibited the highest activity of 135% in the presence of 4 M NaCl and retained 76% of its activity after 24 h incubation with 4 M NaCl. This novel xylanase, XynT, is a cold-active and halotolerant enzyme that may have promising applications in drug, food, feed, and bioremediation industries.

scholarly journals S-Methylmethionine Metabolism in Escherichia coli

1999 ◽  
Vol 181 (2) ◽  
pp. 662-665 ◽  
Author(s):  
Martin Thanbichler ◽  
Bernhard Neuhierl ◽  
August Böck
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Sequence Similarity ◽  
Physiological Role ◽  
High Sequence Similarity ◽  
Reading Frame ◽  
Amino Acid Permeases ◽  
Sequence Similarities ◽  
Homocysteine Methyltransferase

ABSTRACT Selenium-accumulating Astragalus spp. contain an enzyme which specifically transfers a methyl group fromS-methylmethionine to the selenol of selenocysteine, thus converting it to a nontoxic, since nonproteinogenic, amino acid. Analysis of the amino acid sequence of this enzyme revealed thatEscherichia coli possesses a protein (YagD) which shares high sequence similarity with the enzyme. The properties and physiological role of YagD were investigated. YagD is anS-methylmethionine: homocysteine methyltransferase which also accepts selenohomocysteine as a substrate. Mutants inyagD which also possess defects in metE andmetH are unable to utilize S-methylmethionine for growth, whereas a metE metH double mutant still grows on S-methylmethionine. Upstream of yagD and overlapping with its reading frame is a gene (ykfD) which, when inactivated, also blocks growth on methylmethionine in ametE metH genetic background. Since it displays sequence similarities with amino acid permeases it appears to be the transporter for S-methylmethionine. Methionine but notS-methylmethionine in the medium reduces the amount ofyagD protein. This and the existence of four MET box motifs upstream of yfkD indicate that the two genes are members of the methionine regulon. The physiological roles of the ykfDand yagD products appear to reside in the acquisition ofS-methylmethionine, which is an abundant plant product, and its utilization for methionine biosynthesis.

Expression and partial purification of human pro-opiomelanocortin in Escherichia coli

1989 ◽  
Vol 3 (2) ◽  
pp. 105-112 ◽  
Author(s):  
T. S. Grewal ◽  
P. J. Lowry ◽  
D. Savva
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Fusion Protein ◽  
Western Blot ◽  
Blot Analysis ◽  
Expression Vectors ◽  
Partial Purification ◽  
Amino Acid Residues ◽  
E Coli ◽  
Dna Fragment

ABSTRACT A large portion of the human pro-opiomelanocortin (POMC) peptide corresponding to amino acid residues 59–241 has been cloned and expressed in Escherichia coli. A 1·0 kb DNA fragment encoding this peptide was cloned into the expression vectors pUC8 and pUR291. Plasmid pJMBG51 (a pUC8 recombinant) was found to direct the expression of a 24 kDa peptide. The recombinant pUR291 (pJMBG52) was shown to produce a β-galactosidase fusion protein of 140 kDa. Western blot analysis showed that both the 24 kDa and 140 kDa peptides are recognized by antibodies raised against POMC-derived peptides. The β-galactosidase fusion protein has been partially purified from crude E. coli cell lysates using affinity chromatography on p-aminobenzyl-1-thio-β-d-galactopyranoside agarose.

Developing structure-based models to predict substrate specificity of D-group (Type II) molybdenum enzymes: application to a molybdo-enzyme of unknown function from Archaeoglobus fulgidus

2006 ◽  
Vol 34 (1) ◽  
pp. 118-121 ◽  
Author(s):  
E.J. Dridge ◽  
D.J. Richardson ◽  
R.J. Lewis ◽  
C.S. Butler
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Sequence Analysis ◽  
Archaeoglobus Fulgidus ◽  
Substrate Selectivity ◽  
Type Ii ◽  
Amino Acid Residues ◽  
Sequence Alignments ◽  
X Ray ◽  
Group Type

The AF0174–AF0176 gene cluster in Archaeoglobus fulgidus encodes a putative oxyanion reductase of the D-type (Type II) family of molybdo-enzymes. Sequence analysis reveals that the catalytic subunit AF0176 shares low identity (31–32%) and similarity (41–42%) to both NarG and SerA, the catalytic components of the respiratory nitrate and selenate reductases respectively. Consequently, predicting the oxyanion substrate selectivity of AF0176 has proved difficult based solely on sequence alignments. In the present study, we have modelled both AF0176 and SerA on the recently determined X-ray structure of the NAR (nitrate reductase) from Escherichia coli and have identified a number of key amino acid residues, conserved in all known NAR sequences, including AF0176, that we speculate may enhance selectivity towards trigonal planar (NO3−) rather than tetrahedral (SeO42− and ClO4−) substrates.

scholarly journals Molecular Cloning and Characterization of Bifidobacterium bifidum 1,2-α-l-Fucosidase (AfcA), a Novel Inverting Glycosidase (Glycoside Hydrolase Family 95)

2004 ◽  
Vol 186 (15) ◽  
pp. 4885-4893 ◽  
Author(s):  
Takane Katayama ◽  
Akiko Sakuma ◽  
Takatoshi Kimura ◽  
Yutaka Makimura ◽  
Jun Hiratake ◽  
...  
Keyword(s):  
Amino Acid ◽  
Genomic Library ◽  
Bifidobacterium Bifidum ◽  
Glycoside Hydrolases ◽  
Glycoside Hydrolase Family ◽  
Amino Acid Residues ◽  
Gene Encoding ◽  
Sugar Chain ◽  
Hydrolysis Of

ABSTRACT A genomic library of Bifidobacterium bifidum constructed in Escherichia coli was screened for the ability to hydrolyze the α-(1→2) linkage of 2′-fucosyllactose, and a gene encoding 1,2-α-l-fucosidase (AfcA) was isolated. The afcA gene was found to comprise 1,959 amino acid residues with a predicted molecular mass of 205 kDa and containing a signal peptide and a membrane anchor at the N and C termini, respectively. A domain responsible for fucosidase activity (the Fuc domain; amino acid residues 577 to 1474) was localized by deletion analysis and then purified as a hexahistidine-tagged protein. The recombinant Fuc domain specifically hydrolyzed the terminal α-(1→2)-fucosidic linkages of various oligosaccharides and a sugar chain of a glycoprotein. The stereochemical course of the hydrolysis of 2′-fucosyllactose was determined to be inversion by using 1H nuclear magnetic resonance. The primary structure of the Fuc domain exhibited no similarity to those of any glycoside hydrolases (GHs) but showed high similarity to those of several hypothetical proteins in a database. Thus, it was revealed that the AfcA protein constitutes a novel inverting GH family (GH family 95).

scholarly journals Cloning, post-translational modifications, heterologous expression and ligand-binding of boar salivary lipocalin

2000 ◽  
Vol 350 (2) ◽  
pp. 369-379 ◽  
Author(s):  
Dietrich LOEBEL ◽  
Andrea SCALONI ◽  
Sara PAOLINI ◽  
Carlo FINI ◽  
Lino FERRARA ◽  
...  
Keyword(s):  
Amino Acid ◽  
Sequence Similarity ◽  
Three Dimensional ◽  
Protein Isoforms ◽  
Cysteine Residues ◽  
Protein Chemistry ◽  
Single Individual ◽  
Amino Acid Residues ◽  
Three Dimensional Model ◽  
Submaxillary Glands

Boar submaxillary glands produce the sex-specific salivary lipocalin (SAL), which binds steroidal sex pheromones as endogenous ligands. The cDNA encoding SAL was cloned and sequenced. From a single individual, two protein isoforms, differing in three amino acid residues, were purified and structurally characterized by a combined Edman degradation/MS approach. These experiments ascertained that the mature polypeptide is composed of 168 amino acid residues, that one of the three putative glycosylation sites is post-translationally modified and the structure of the bound glycosidic moieties. Two of the cysteine residues are paired together in a disulphide bridge, whereas the remaining two occur as free thiols. SAL bears sequence similarity to other lipocalins; on this basis, a three-dimensional model of the protein has been built. A SAL isoform was expressed in Escherichiacoli in good yields. Protein chemistry and CD experiments verified that the recombinant product shows the same redox state at the cysteine residues and that the same conformation is observed as in the natural protein, thus suggesting similar folding. Binding experiments on natural and recombinant SAL were performed with the fluorescent probe 1-aminoanthracene, which was efficiently displaced by the steroidal sex pheromone, as well as by several odorants.

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