Cloning and High-Level Expression of α-Galactosidase cDNA from Penicillium purpurogenum

ABSTRACT The cDNA coding for Penicillium purpurogenumα-galactosidase (αGal) was cloned and sequenced. The deduced amino acid sequence of the α-Gal cDNA showed that the mature enzyme consisted of 419 amino acid residues with a molecular mass of 46,334 Da. The derived amino acid sequence of the enzyme showed similarity to eukaryotic αGals from plants, animals, yeasts, and filamentous fungi. The highest similarity observed (57% identity) was toTrichoderma reesei AGLI. The cDNA was expressed inSaccharomyces cerevisiae under the control of the yeast GAL10 promoter. Almost all of the enzyme produced was secreted into the culture medium, and the expression level reached was approximately 0.2 g/liter. The recombinant enzyme purified to homogeneity was highly glycosylated, showed slightly higher specific activity, and exhibited properties almost identical to those of the native enzyme from P. purpurogenum in terms of the N-terminal amino acid sequence, thermoactivity, pH profile, and mode of action on galacto-oligosaccharides.

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The overexpression and complete amino acid sequence of Escherichia coli 3-dehydroquinase

Biochemical Journal ◽

10.1042/bj2380475 ◽

1986 ◽

Vol 238 (2) ◽

pp. 475-483 ◽

Cited By ~ 36

Author(s):

K Duncan ◽

S Chaudhuri ◽

M S Campbell ◽

J R Coggins

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Nucleotide Sequence ◽

Amino Acid Sequence ◽

Polypeptide Chain ◽

Amino Acid Residues ◽

Transcript Mapping ◽

Terminal Amino Acid ◽

Terminal Amino ◽

Terminal Amino Acid Sequence

The enzyme 3-dehydroquinase was purified in milligram quantities from an overproducing strain of Escherichia coli. The amino acid sequence was deduced from the nucleotide sequence of the aroD gene and confirmed by determining the amino acid composition of the overproduced enzyme and its N-terminal amino acid sequence. The complete polypeptide chain consists of 240 amino acid residues and has a calculated subunit Mr of 26,377. Transcript mapping revealed that aroD is a typical monocistronic gene.

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Isolation, properties and amino acid sequence of a long-chain neurotoxin, Acanthophis antarcticus b, from the venom of an Australian snake (the common death adder, Acanthophis antarcticus)

Biochemical Journal ◽

10.1042/bj1930899 ◽

1981 ◽

Vol 193 (3) ◽

pp. 899-906 ◽

Cited By ~ 24

Author(s):

H S Kim ◽

N Tamiya

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Intramuscular Injection ◽

Amino Acid Residues ◽

Methionine Residue ◽

N Terminus ◽

The Common ◽

Ld50 Value ◽

Almost All ◽

Long Chain

The venom of an Australian elapid snake, the common death adder (Acanthophis antarcticus), was chromatographed on a CM-cellulose CM52 column. One of the neurotoxic components, Acanthophis antarcticus b (toxin Aa b) was isolated in about 9.4% (A280) yield. The complete amino acid sequence of toxin Aa b was elucidated. Toxin Aa b is composed of 73 amino acid residues, with ten half-cystine residues, and has a formula weight of 8135. Toxin Aa b has no histidine or methionine residue in its sequence. The amino acid sequence of toxin Aa b is homologous with those of other neurotoxins with known sequences, although it is novel in having a valine residue at its N-terminus and an arginine residue at position-23, where a lysine residue is found in almost all the so-far-known neurotoxins. Irrespective of the latter replacement, the toxin Aa b is fully active, with an LD50 value (in mice) of 0.13 microgram/g body weight on intramuscular injection.

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Amino acid sequence of the N-terminal 108 amino acid residues of the B chain of subcomponent C1q of the first component of human complement

Biochemical Journal ◽

10.1042/bj1730863 ◽

1978 ◽

Vol 173 (3) ◽

pp. 863-868 ◽

Cited By ~ 19

Author(s):

K B Reid ◽

E O Thompson

Keyword(s):

Amino Acid ◽

Carboxylic Acid ◽

Amino Acid Sequence ◽

Disulphide Bond ◽

Amino Acid Residues ◽

Human Complement ◽

Terminal Amino Acid ◽

Exact Position ◽

Terminal Amino

The amino acid sequence of the N-terminal 108 residues of the B chain of subcomponent C1q of the first component of human complement was determined. The B chain has a blocked N-terminal amino acid, which was judged to be 5-oxopyrrolidine-2-carboxylic acid. A collagen-like region of 84 residues was found, which started at position B-6, and all of the six hydroxylysine residues and 12 hydroxyproline residues present in the chain were found in this region. Four of the six hydroxylysine residues may be glycosylated. The repeating nature of the collagen-like region is broken at position B-9, where alanine is found in a position where glycine would be expected. The exact position of the interchain disulphide bond joining the A and B chains of human subcomponent C1q was shown to be between residues A4 and B4.

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Isolation and properties of lysophospholipases from the venom of an Australian elapid snake, Pseudechis australis

Biochemical Journal ◽

10.1042/bj2030269 ◽

1982 ◽

Vol 203 (1) ◽

pp. 269-276 ◽

Cited By ~ 22

Author(s):

C Takasaki ◽

N Tamiya

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Phospholipase A2 ◽

Maximum Activity ◽

Haemolytic Activity ◽

Amino Acid Residues ◽

Terminal Amino Acid ◽

Optimum Ph ◽

Terminal Amino ◽

Terminal Amino Acid Sequence

Two lysophospholipases were isolated from the venom of an Australian elapid snake (subfamily Acanthophiinae), Pseudechis australis, by sequential chromatography on CM-52 cellulose, Sephadex G-75 and DE-52 cellulose columns. They were very similar to each other. One of them, lysophospholipase I, was obtained as a homodimer, the monomer of which consisted of 123 amino acid residues with seven disulphide bridges. The amino acid composition and the N-terminal amino acid sequence of the enzyme were similar to those of phospholipase A2, Ca2+ was required for its activity and the maximum activity was attained at 2 mM-CaCl2 in the presence of 1 mM-EDTA. The optimum pH was 7.5. Lysophospholipase I hydrolysed lysophosphatidylcholine more rapidly than lysophosphatidylethanolamine. It did not hydrolyse, however, phosphatidylcholine, 1-palmitoylglycerol, tripalmitoylglycerol or p-nitrophenyl acetate. Modification of the enzyme with p-bromophenacyl bromide or 2-nitrophenylsulphenyl chloride suppressed the activity. A strong direct haemolytic activity was exhibited when the lysophospholipase was present together with phospholipase A2.

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Characterization of the d-Xylulose 5-Phosphate/d-Fructose 6-Phosphate Phosphoketolase Gene (xfp) from Bifidobacterium lactis

Journal of Bacteriology ◽

10.1128/jb.183.9.2929-2936.2001 ◽

2001 ◽

Vol 183 (9) ◽

pp. 2929-2936 ◽

Cited By ~ 80

Author(s):

Leo Meile ◽

Lukas M. Rohr ◽

Thomas A. Geissmann ◽

Monique Herensperger ◽

Michael Teuber

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Specific Activity ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Guanosine Monophosphate ◽

Bifidobacterium Lactis ◽

Terminal Amino ◽

Cloned Gene

ABSTRACT A d-xylulose 5-phosphate/d-fructose 6-phosphate phosphoketolase (Xfp) from the probioticBifidobacterium lactis was purified to homogeneity. The specific activity of the purified enzyme with d-fructose 6-phosphate as a substrate is 4.28 Units per mg of enzyme.Km values for d-xylulose 5-phosphate and d-fructose 6-phosphate are 45 and 10 mM, respectively. The native enzyme has a molecular mass of 550,000 Da. The subunit size upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis (90,000 Da) corresponds with the size (92,529 Da) calculated from the amino acid sequence of the isolated gene (namedxfp) encoding 825 amino acids. The xfp gene was identified on the chromosome of B. lactis with the help of degenerated nucleotide probes deduced from the common N-terminal amino acid sequence of both the native and denatured enzyme. Comparison of the deduced amino acid sequence of the cloned gene with sequences in public databases revealed high homologies with hypothetical proteins (26 to 55% identity) in 20 microbial genomes. The amino acid sequence derived from the xfp gene contains typical thiamine diphosphate (ThDP) binding sites reported for other ThDP-dependent enzymes. Two truncated putative genes, pta andguaA, were localized adjacent to xfp on theB. lactis chromosome coding for a phosphotransacetylase and a guanosine monophosphate synthetase homologous to products of genes inMycobacterium tuberculosis. However, xfp is transcribed in B. lactis as a monocistronic operon. It is the first reported and sequenced gene of a phosphoketolase.

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Grazing Molecule Excitation as a Tool to Analyse the Amino Acid Sequence in Oligopeptides

Journal of Atomic Molecular and Optical Physics ◽

10.1155/2011/356589 ◽

2011 ◽

Vol 2011 ◽

pp. 1-6

Author(s):

H. Jungclas ◽

L. Schmidt ◽

V. V. Komarov ◽

A. M. Popova

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Specific Property ◽

Spectrometric Method ◽

Mass Spectrometric Method ◽

Amino Acid Residues ◽

Peptide Ions ◽

Peptide Molecule ◽

Atomic Distance ◽

Terminal Amino

A novel mass spectrometric method to analyse the sequence of amino acid residues in oligopeptides is proposed. Amino acid residues in peptide molecules contain chain-like structures of identical CH dipoles (IR antennas), which acquire IR energy quanta by interaction with periodic Coulomb fields and accumulate vibration excitation energy. This can subsequently lead to the dissociation of specific trap bonds inside the peptide molecule. Such excitation and dissociation processes are assumed to occur when peptide ions graze at atomic distance along a set of screened charges on a surface. These processes of grazing molecule excitation (GME) and dissociation (GMD) were applied to analyse sequences of oligopeptides by using TOF mass spectrometry. At specific grazing velocities the experimental fragment ion spectra of oligopeptides must contain a peak of high abundance corresponding to the N-terminal amino acid. This specific property of GMD offers the possibility to determine the amino acid sequence of oligopeptides.

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N-Terminal amino acid sequence of 55 residues of hog pepsin

Collection of Czechoslovak Chemical Communications ◽

10.1135/cccc19741933 ◽

1974 ◽

Vol 39 (7) ◽

pp. 1933-1939

Author(s):

L. Morávek

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Terminal Amino Acid ◽

Terminal Amino ◽

Terminal Amino Acid Sequence

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C-Terminal amino acid sequence of chicken pepsinogen and its homology with sequences of other acid proteases

Collection of Czechoslovak Chemical Communications ◽

10.1135/cccc19801144 ◽

1980 ◽

Vol 45 (4) ◽

pp. 1144-1154 ◽

Cited By ~ 3

Author(s):

Miroslav Baudyš ◽

Helena Keilová ◽

Vladimír Kostka

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

The Other ◽

Terminal Sequence ◽

Terminal Part ◽

Terminal Amino Acid ◽

Tryptic Peptides ◽

Terminal Amino ◽

High Degree ◽

Terminal Amino Acid Sequence

To determine the primary structure of the C-terminal part of the molecule of chicken pepsinogen the tryptic, chymotryptic and thermolytic digest of the protein were investigated and peptides derived from this region were sought. These peptides permitted the following 21-residue C-terminal sequence to be determined: ...Ile-Arg-Glu-Tyr-Tyr-Val-Ile-Phe-Asp-Arg-Ala-Asn-Asn-Lys-Val-Gly-Leu-Ser-Pro-Leu-Ser.COOH. A comparison of this structure with the C-terminal sequential regions of the other acid proteases shows a high degree of homology between chicken pepsinogen and these proteases (e.g., the degree of homology with respect to hog pepsinogen and calf prochymosin is about 66%). Additional tryptic peptides, derived from the N-terminal part of the zymogen molecule whose amino acid sequence has been reported before, were also obtained in this study. This sequence was extended by two residues using an overlapping peptide. An ancillary result of this study was the isolation of tryptic peptides derived from other regions of the zymogen molecule.

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Amino acid sequence of cyanogen bromide fragment CB5 of human hemopexin

Collection of Czechoslovak Chemical Communications ◽

10.1135/cccc19890803 ◽

1989 ◽

Vol 54 (3) ◽

pp. 803-810 ◽

Cited By ~ 1

Author(s):

Ivan Kluh ◽

Ladislav Morávek ◽

Manfred Pavlík

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Acid Hydrolysis ◽

Cyanogen Bromide ◽

Polypeptide Chain ◽

Amino Acid Residues ◽

Mild Acid Hydrolysis ◽

Methionine Residue ◽

Cyanogen Bromide Fragment ◽

Mild Acid

Cyanogen bromide fragment CB5 represents the region of the polypeptide chain of hemopexin between the fourth and fifth methionine residue (residues 232-352). It contains 120 amino acid residues in the following sequence: Arg-Cys-Ser-Pro-His-Leu-Val-Leu-Ser-Ala-Leu-Thr-Ser-Asp-Asn-His-Gly-Ala-Thr-Tyr-Ala-Phe-Ser-Gly-Thr-His-Tyr-Trp-Arg-Leu-Asp-Thr-Ser-Arg-Asp-Gly-Trp-His-Ser-Trp-Pro-Ile-Ala-His-Gln-Trp-Pro-Gln-Gly-Pro-Ser-Ala-Val-Asp-Ala-Ala-Phe-Ser-Trp-Glu-Glu-Lys-Leu-Tyr-Leu-Val-Gln-Gly-Thr-Gln-Val-Tyr-Val-Phe-Leu-Thr-Lys-Gly-Gly-Tyr-Thr-Leu-Val-Ser-Gly-Tyr-Pro-Lys-Arg-Leu-Glu-Lys-Glu-Val-Gly-Thr-Pro-His-Gly-Ile-Ile-Leu-Asp-Ser-Val-Asp-Ala-Ala-Phe-Ile-Cys-Pro-Gly-Ser-Ser-Arg-Leu-His-Ile-Met. The sequence was derived from the data on peptides prepared by cleavage of fragment CB5 by mild acid hydrolysis, by trypsin and chymotrypsin.

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