Purification, characterization, gene cloning and preliminary X-ray data of the exo-inulinase from Aspergillus awamori

2002 ◽  
Vol 362 (1) ◽  
pp. 131-135 ◽  
Author(s):  
Michael ARAND ◽  
Alexander M. GOLUBEV ◽  
J. R. Brandao NETO ◽  
Igor POLIKARPOV ◽  
R. WATTIEZ ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Solid Phase ◽  
Aspergillus Awamori ◽  
Glycoside Hydrolase Family ◽  
Orthorhombic Space Group ◽  
Ph Optimum ◽  
X Ray ◽  
Cell Parameters ◽  
Hydrolysis Of

Extracellular exo-inulinase has been isolated from a solid-phase culture of the filamentous fungus Aspergillus awamori var. 2250. The apparent molecular mass of the monomer enzyme was 69±1kDa, with a pI of 4.4 and a pH optimum of 4.5. The enzyme hydrolysed the β-(2 → 1)-fructan (inulin) and β-(2 → 6)-fructan (levan) via exo-cleavage, releasing fructose. The values for the Michaelis constants Km and Vmax in the hydrolysis of inulin were 0.003±0.0001mM and 175±5μmol·min−1·mg−1. The same parameters in the hydrolysis of levan were 2.08±0.04mg/ml and 1.2±0.02μmol/min per mg, respectively. The gene and cDNA encoding the A. awamori exo-inulinase were cloned and sequenced. The amino acid sequence indicated that the protein belongs to glycoside hydrolase family 32. A surprisingly high similarity was found to fructosyltransferase from Aspergillus foetidus (90.7% on the level of the amino acid sequence), despite the fact that the latter enzyme is unable to hydrolyse inulin and levan. Crystals of the native exo-inulinase were obtained and found to belong to the orthorhombic space group P212121 with cell parameters a = 64.726 Å (1Å = 0.1 nm), b = 82.041 Å and c = 136.075 Å. Crystals diffracted beyond 1.54 Å, and useful X-ray data were collected to a resolution of 1.73 Å.

scholarly journals Crystallization and preliminary X-ray crystallographic analysis of α-glucosidase HaG fromHalomonassp. strain H11

2014 ◽  
Vol 70 (4) ◽  
pp. 464-466 ◽  
Author(s):  
Xing Shen ◽  
Wataru Saburi ◽  
Zuo-Qi Gai ◽  
Keisuke Komoda ◽  
Jian Yu ◽  
...  
Keyword(s):  
Halophilic Bacterium ◽  
Crystallographic Analysis ◽  
Low Molecular Weight ◽  
Glycoside Hydrolase Family ◽  
Molecular Replacement ◽  
Data Set ◽  
X Ray ◽  
Cell Parameters ◽  
Hydrolysis Of ◽  
Hydrolase Family

The α-glucosidase HaG from the halophilic bacteriumHalomonassp. strain H11 catalyzes the hydrolysis of the glucosidic linkage at the nonreducing end of α-glucosides, such as maltose and sucrose, to release α-glucose. Based on its amino-acid sequence, this enzyme is classified as a member of glycoside hydrolase family 13. HaG has three unique characteristics: (i) a very narrow substrate specificity, almost exclusively hydrolyzing disaccharides; (ii) activation by monovalent cations, such as K+, Rb+, Cs+and NH4+; and (iii) high transfer activity of the glucose moiety to the OH group of low-molecular-weight compounds, including glycerol and 6-gingerol. Crystallographic studies have been performed in order to understand these special features. An expression vector was constructed and recombinant HaG protein was overexpressed, purified and crystallized. A data set to 2.15 Å resolution was collected and processed. The crystal belonged to space groupP212121, with unit-cell parametersa= 60.2,b= 119.2,c= 177.2 Å. The structure has been determined by molecular replacement using the isomaltulose synthase PalI as the search model (PDB entry 1m53).

scholarly journals Identification and Characterization of a Novel Intracellular Alkaline α-Amylase from the Hyperthermophilic Bacterium Thermotoga maritima MSB8

2006 ◽  
Vol 72 (3) ◽  
pp. 2206-2211 ◽  
Author(s):  
Meike Ballschmiter ◽  
Ole Fütterer ◽  
Wolfgang Liebl
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Thermotoga Maritima ◽  
Sequence Similarity ◽  
Hydrolytic Activity ◽  
Amino Acid Sequence Similarity ◽  
Glycoside Hydrolase Family ◽  
Ph Optimum ◽  
Intracellular Enzyme ◽  
Hyperthermophilic Bacterium

ABSTRACT The gene for a novel α-amylase, designated AmyC, from the hyperthermophilic bacterium Thermotoga maritima was cloned and heterologously overexpressed in Escherichia coli. The putative intracellular enzyme had no amino acid sequence similarity to glycoside hydrolase family (GHF) 13 α-amylases, yet the range of substrate hydrolysis and the product profile clearly define the protein as an α-amylase. Based on sequence similarity AmyC belongs to a subgroup within GHF 57. On the basis of amino acid sequence similarity, Glu185 and Asp349 could be identified as the catalytic residues of AmyC. Using a 60-min assay, the maximum hydrolytic activity of the purified enzyme, which was dithiothreitol dependent, was found to be at 90°C. AmyC displayed a remarkably high pH optimum of pH 8.5 and an unusual sensitivity towards both ATP and EDTA.

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).

Heklaite, KNaSiF6, a new fumarolic mineral from Hekla volcano, Iceland

2010 ◽  
Vol 74 (1) ◽  
pp. 147-157 ◽  
Author(s):  
A. Garavelli ◽  
T. Balić-Žunić ◽  
D. Mitolo ◽  
P. Acquafredda ◽  
E. Leonardsen ◽  
...  
Keyword(s):  
Powder Diffraction ◽  
Quantitative Chemical Analysis ◽  
Unit Cell Parameters ◽  
Orthorhombic Space Group ◽  
Synthetic Compound ◽  
Calculated Density ◽  
X Ray ◽  
Cell Parameters ◽  
Fine Grained ◽  
The Ideal

AbstractHeklaite, with the ideal formula KNaSiF6, was found among fumarolic encrustations collected in 1992 on the Hekla volcano, Iceland. Heklaite forms a fine-grained mass of micron- to sub-micron-sized crystals intimately associated with malladrite, hieratite and ralstonite. The mineral is colourless, transparent, non-fluorescent, has a vitreous lustre and a white streak. The calculated density is 2.69 g cm–3. An SEM-EDS quantitative chemical analysis shows the following range of concentrations (wt.%): Na 11.61–12.74 (average 11.98), K 17.02–18.97 (average 18.29), Si 13.48 –14.17 (average 13.91), F 54.88–56.19 (average 55.66). The empirical chemical formula, calculated on the basis of 9 a.p.f.u., is Na1.07K0.96Si1.01F5.97. X-ray powder diffraction indicates that heklaite is orthorhombic, space group Pnma, with the following unit-cell parameters: a = 9.3387(7) Å, b = 5.5032(4) Å, c = 9.7957(8) Å , V = 503.43(7) Å3, Z = 4. The eight strongest reflections in the powder diffraction pattern [d in Å (I/I0) (hkl)] are: 4.33 (53) (102); 4.26 (56) (111); 3.40 (49) (112); 3.37 (47) (202); 3.34 (100) (211); 2.251 (27) (303); 2.050 (52) (123); 2.016 (29) (321). On the basis of chemical analyses and X-ray data, heklaite corresponds to the synthetic compound KNaSiF6. The name is for the type locality, the Hekla volcano, Iceland.

scholarly journals Crystallization, optimization and preliminary X-ray characterization of a metal-dependent PI-PLC fromStreptomyces antibioticus

2012 ◽  
Vol 68 (11) ◽  
pp. 1378-1386 ◽  
Author(s):  
Michael R. Jackson ◽  
Thomas L. Selby
Keyword(s):  
X Ray Diffraction ◽  
Hanging Drop ◽  
Orthorhombic Space Group ◽  
X Ray ◽  
Streptomyces Antibioticus ◽  
Cell Parameters ◽  
Heavy Atoms ◽  
Hanging Drop Method

A recombinant metal-dependent phosphatidylinositol-specific phospholipase C (PI-PLC) fromStreptomyces antibioticushas been crystallized by the hanging-drop method with and without heavy metals. The native crystals belonged to the orthorhombic space groupP222, with unit-cell parametersa= 41.26,b= 51.86,c = 154.78 Å. The X-ray diffraction results showed significant differences in the crystal quality of samples soaked with heavy atoms. Additionally, drop pinning, which increases the surface area of the drops, was also used to improve crystal growth and quality. The combination of heavy-metal soaks and drop pinning was found to be critical for producing high-quality crystals that diffracted to 1.23 Å resolution.

The Amino Acid Sequence of Glucagon. IV. The Hydrolysis of Glucagon with Subtilisin

1957 ◽  
Vol 79 (11) ◽  
pp. 2805-2807 ◽  
Author(s):  
L. G. Sinn ◽  
Otto K. Behrens ◽  
W. W. Bromer
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Hydrolysis Of

Physicochemical properties of a novel α-L-arabinofuranosidase fromRhizomucor pusillusHHT-1

2001 ◽  
Vol 47 (8) ◽  
pp. 767-772 ◽  
Author(s):  
A KM Shofiqur Rahman ◽  
Shinya Kawamura ◽  
Masahiro Hatsu ◽  
M M Hoq ◽  
Kazuhiro Takamizawa
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Molecular Mass ◽  
Gel Filtration ◽  
Hydrolytic Activity ◽  
Exchange Chromatography ◽  
Ph Optimum ◽  
Rhizomucor Pusillus ◽  
Terminal Amino ◽  
Metal Cofactors

The zygomycete fungus Rhizomucor pusillus HHT-1, cultured on L(+)arabinose as a sole carbon source, produced extracellular α-L-arabinofuranosidase. The enzyme was purified by (NH4)2SO4fractionation, gel filtration, and ion exchange chromatography. The molecular mass of this monomeric enzyme was 88 kDa. The native enzyme had a pI of 4.2 and displayed a pH optimum and stability of 4.0 and 7.0–10.0, respectively. The temperature optimum was 65°C, and it was stable up to 70°C. The Kmand Vmaxfor p-nitrophenyl α-L-arabinofuranoside were 0.59 mM and 387 µmol·min–1·mg–1protein, respectively. Activity was not stimulated by metal cofactors. The N-terminal amino acid sequence did not show any similarity to other arabinofuranosidases. Higher hydrolytic activity was recorded with p-nitrophenyl α-L-arabinofuranoside, arabinotriose, and sugar beet arabinan; lower hydrolytic activity was recorded with oat–spelt xylan and arabinogalactan, indicating specificity for the low molecular mass L(+)-arabinose containing oligosaccharides with furanoside configuration.Key words: α-L-arabinofuranosidase, enzyme purification, amino acid sequence, Rhizomucor pusillus.

scholarly journals Crystallization and preliminary X-ray analysis of the FliH–FliI complex responsible for bacterial flagellar type III protein export

2012 ◽  
Vol 68 (11) ◽  
pp. 1311-1314 ◽  
Author(s):  
Yumiko Uchida ◽  
Tohru Minamino ◽  
Keiichi Namba ◽  
Katsumi Imada
Keyword(s):  
Self Assembly ◽  
Specific Protein ◽  
Protein Export ◽  
Orthorhombic Space Group ◽  
X Ray ◽  
Cell Parameters ◽  
Peg 400 ◽  
Diffusion Technique ◽  
Flagellar Proteins ◽  
Soluble Components

The bacterial flagellar proteins are translocated into the central channel of the flagellum by a specific protein-export apparatus for self-assembly at the distal growing end. FliH and FliI are soluble components of the export apparatus and form an FliH2–FliI heterotrimer in the cytoplasm. FliI is an ATPase and the FliH2–FliI complex delivers export substrates from the cytoplasm to an export gate made up of six integral membrane proteins of the export apparatus. In this study, an FliHCfragment consisting of residues 99–235 was co-purified with FliI and the FliHC2–FliI complex was crystallized. Crystals were obtained using the hanging-drop vapour-diffusion technique with PEG 400 as a precipitant. The crystals belonged to the orthorhombic space groupP212121, with unit-cell parametersa= 133.7,b= 147.3,c= 164.2 Å, and diffracted to 3.0 Å resolution.

The amino acid sequence of yeast phosphoglycerate mutase

1982 ◽  
Vol 215 (1198) ◽  
pp. 19-44 ◽  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Proteolytic Enzymes ◽  
Phosphoglycerate Mutase ◽  
Crystallographic Structure ◽  
X Ray ◽  
C Terminus ◽  
Detailed Interpretation ◽  
High Degree

The complete amino acid sequence of yeast phosphoglycerate mutase comprising 241 residues has been determined. The sequence was deduced from the two cyanogen bromide fragments, and from the peptides derived from these fragments after digestion by a number of proteolytic enzymes. Determination of this sequence now allows a detailed interpretation of the existing high-resolution X-ray crystallographic structure. A comparison of the sequence reported here with the sequences of peptides from phosphoglycerate mutases from other species, and with the sequence of erythrocyte diphosphoglycerate mutase, indicates that these enzymes have a high degree of structural homology. Autolysis of phosphoglycerate mutase by yeast extracts leads to the complete loss of mutase activity, and the formation of electrophoretically distinguishable forms (R. Sasaki, E. Sugimoto & H. Chiba, Archs Biochem. Biophys. 115, 53-61 (1966)). It is apparent from the amino acid sequence that these changes are due to the loss of an 8─12 residue peptide from the C-terminus.

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