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 Structural features of the low-molecular-mass human salivary mucin

1992 ◽  
Vol 287 (2) ◽  
pp. 639-643 ◽  
Author(s):  
M S Reddy ◽  
L A Bobek ◽  
G G Haraszthy ◽  
A R Biesbrock ◽  
M J Levine
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Molecular Mass ◽  
Gel Filtration ◽  
Structural Features ◽  
Cdna Expression Library ◽  
Cdna Clones ◽  
Expression Library ◽  
Mrna Species ◽  
Salivary Mucin

The low-molecular-mass human salivary mucin has at least two isoforms, MG2a and MG2b, that differ primarily in their sialic acid and fucose content. In this study, we characterize further these isoforms, particularly their peptide moieties. Trypsin digests of MG2a and MG2b yielded high- and low-molecular-mass glycopeptides following gel filtration on Sephacryl S-300. The larger glycopeptides from MG2a and MG2b had similar amino acid compositions and identical N-terminal sequences, suggesting common structural features between their peptides. An oligonucleotide probe generated from the amino acid sequence of the smaller glycopeptide from MG2a was employed in Northern-blot analysis. This probe specifically hybridized to two mRNA species from human submandibular and sublingual glands. A cDNA clone selected from a human submandibular gland cDNA expression library with antibody generated against deglycosylated MG2a also hybridized to these two mRNA species. In both cases, the larger mRNA was polydisperse, and the hybridization signal was more intense in the sublingual gland. In addition, the N-terminal amino acid sequence of the larger glycopeptide was found to be part of one of the selected MG2 cDNA clones.

scholarly journals Isolation of four forms of acetone-induced cytochrome P-450 in chicken liver by h.p.l.c. and their enzymic characterization

1990 ◽  
Vol 269 (1) ◽  
pp. 85-91 ◽  
Author(s):  
J F Sinclair ◽  
S Wood ◽  
L Lambrecht ◽  
N Gorman ◽  
L Mende-Mueller ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Exchange Chromatography ◽  
Amino Acid Sequences ◽  
Chicken Liver ◽  
Terminal Amino Acid ◽  
Catalytic Activities ◽  
Terminal Amino ◽  
Cytochrome P 450 ◽  
Terminal Amino Acid Sequence

The purpose of this study was to purify and characterize the forms of cytochrome P-450 induced in chicken liver by acetone or ethanol. Using high performance liquid ion-exchange chromatography, we were able to isolate at least four different forms of cytochrome P-450 which were induced by acetone in chicken liver. All four forms of cytochrome P-450 proved to be distinct proteins, as indicated by their N-terminal amino acid sequences and their reconstituted catalytic activities. Two of these forms, also induced by glutethimide in chicken embryo liver, appeared to be cytochromes P450IIH1 and P450IIH2. Both of these cytochromes P-450 have identical catalytic activities towards benzphetamine demethylation. However, they differ in their abilities to hydroxylate p-nitrophenol and to convert acetaminophen into a metabolite that forms a covalent adduct with glutathione at the 3-position. Another form of cytochrome P-450 induced by acetone is highly active in the hydroxylation of p-nitrophenol and in the conversion of acetaminophen to a reactive metabolite, similar to reactions catalysed by mammalian cytochrome P450IIE. Yet the N-terminal amino acid sequence of this form has only 30-33% similarity with cytochrome P450IIE purified from rat, rabbit and human livers. A fourth form of cytochrome P-450 was identified whose N-terminal amino acid sequence and enzymic activities do not correspond to any mammalian cytochromes P-450 reported to be induced by acetone or ethanol.

Purification and characterization of the extracellular α-amylase activity of the yeast Schwanniomyces alluvius

1987 ◽  
Vol 65 (10) ◽  
pp. 899-908 ◽  
Author(s):  
F. Moranelli ◽  
M. Yaguchi ◽  
G. B. Calleja ◽  
A. Nasim
Keyword(s):  
Amino Acid ◽  
Amylase Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Exchange Chromatography ◽  
Proteinase K ◽  
Ph Optimum ◽  
Sds Page

The extracellular α-amylase activity of the yeast Schwanniomyces alluvius has been purified by anion-exchange chromatography on DEAE-cellulose and gel-filtration chromatography on Sephadex G-100. Sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS–PAGE) and N-terminal amino acid analysis of the purified sample indicated that the enzyme preparation was homogeneous. The enzyme is a glycoprotein having a molecular mass of 52 kilodaltons (kDa) estimated by SDS–PAGE and 39 kDa by gel filtration on Sephadex G-100. Chromatofocusing shows that it is an acidic protein. It is resistant to trypsin but sensitive to proteinase K. Its activity is inhibited by the divalent cation chelators EDTA and EGTA and it is insensitive to sulfhydryl-blocking agents. Exogenous divalent cations are inhibitory as are high concentrations of monovalent salts. The enzyme has a pH optimum between 3.75 and 5.5 and displays maximum stability in the pH range of 4.0–7.0. Under the conditions tested, the activity is maximal between 45 and 50 °C and is very thermolabile. Analysis of its amino acid composition supports its acidic nature.

scholarly journals Studies on Marsupial Protein. II. Amino Acid Sequence of the -Chain of Haemoglobin from the Grey Kangaroo, Macropus Giganteus

1969 ◽  
Vol 22 (6) ◽  
pp. 1437 ◽  
Author(s):  
GM Air ◽  
EOP Thompson
Keyword(s):  
Amino Acid ◽  
Ion Exchange ◽  
Amino Acid Sequence ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Amino Acid Sequences ◽  
Tryptic Digestion ◽  
Grey Kangaroo

The amino acid sequence of the jS-chain of haemoglobin from M. giganteus has been determined. The soluble peptides formed by tryptic digestion were isolated by gel filtration, ion-exchange chromatography, and paper ionophoresis, and amino acid sequences determined by the "dansyl"-Edman procedure. Special procedures were necessary for three peptides which were insoluble.

Purification, cloning, and DNA sequence analysis of a chitinase from an overproducing mutant of Streptomyces peucetius defective in daunorubicin biosynthesis

2001 ◽  
Vol 47 (3) ◽  
pp. 179-187 ◽  
Author(s):  
Kuzhandhaivel S Vetrivel ◽  
Shunmugiah K Pandian ◽  
Uma Chaudhary ◽  
Kuppamuthu Dharmalingam
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Molecular Mass ◽  
Catalytic Domain ◽  
Chitinase Gene ◽  
Wild Type ◽  
Terminal Amino Acid ◽  
Streptomyces Peucetius ◽  
Terminal Amino ◽  
Terminal Amino Acid Sequence

Extracellular chitinases of Streptomyces peucetius and a chitinase overproducing mutant, SPVI, were purified to homogeneity by ion exchange and gel filtration chromatography. The purified enzyme has a molecular mass of 42 kDa on SDS-PAGE, and the N-terminal amino acid sequence of the protein from the wild type showed homology to catalytic domains (Domain IV) of several other Streptomyces chitinases such as S. lividans 66, S. coelicolor A3(2), S. plicatus, and S. thermoviolaceus OPC-520. Purified SPVI chitinase cross-reacted to anti-chitinase antibodies of wild-type S. peucetius chitinase. A genomic library of SPVI constructed in E. coli using λ DASH II was probed with chiC of S. lividans 66 to screen for the chitinase gene. A 2.7 kb fragment containing the chitinase gene was subcloned from a λ DASH II clone, and sequenced. The deduced protein had a molecular mass of 68 kDa, and showed domain organization similar to that of S. lividans 66 chiC. The N-terminal amino acid sequence of the purified S. peucetius chitinase matched with the N-terminus of the catalytic domain, indicating the proteolytic processing of 68 kDa chitinase precursor protein to 42 kDa mature chitinase containing the catalytic domain only. A putative chiR sequence of a two-component regulatory system was found upstream of the chiC sequence.Key words: chitinase, chitinase purification, Streptomyces peucetius, daunorubicin, chiC.

scholarly journals Myoglobins of Cartilaginous Fishes III. Amino Acid Sequence of Myoglobin of the Shark Galeorhinus Australis

1981 ◽  
Vol 34 (1) ◽  
pp. 5 ◽  
Author(s):  
WK Fisher ◽  
DD Koureas ◽  
EOP Thompson
Keyword(s):  
Amino Acid ◽  
Ion Exchange ◽  
Amino Acid Sequence ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Red Muscle ◽  
Cartilaginous Fishes ◽  
Port Jackson

Myoglobin isolated from the red muscle of the school shark Galeorhinus australis was purified by gel filtration and ion-exchange chromatography. The amino acid sequence was determined following digestion with trypsin and purification of the peptides by paper ionophoresis and chromatography. Sequences of purified peptides were determined by the dansyl-Edman procedure and the peptides aligned by homology with the sequence of the myoglobin of the gummy shark Mustelus antarcticus. The two myoglobin sequences showed a marked similarity (16 differences), but both sequences showed approximately the same number of differences (68) from myoglobin of the Port Jackson shark Heterodontus portusjacksoni. There are 19 residues unique to the three shark myoglobin sequences.

scholarly journals Polysaccharide Lyase: Molecular Cloning, Sequencing, and Overexpression of the Xanthan Lyase Gene of Bacillus sp. Strain GL1

2001 ◽  
Vol 67 (2) ◽  
pp. 713-720 ◽  
Author(s):  
Wataru Hashimoto ◽  
Hikaru Miki ◽  
Noriaki Tsuchiya ◽  
Hirokazu Nankai ◽  
Kousaku Murata
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Molecular Mass ◽  
Signal Peptide ◽  
Streptomyces Griseus ◽  
Peptide Sequence ◽  
Terminal Amino Acid ◽  
Signal Peptide Sequence ◽  
Terminal Amino ◽  
Terminal Amino Acid Sequence

ABSTRACT When grown on xanthan as a carbon source, the bacteriumBacillus sp. strain GL1 produces extracellular xanthan lyase (75 kDa), catalyzing the first step of xanthan depolymerization (H. Nankai, W. Hashimoto, H. Miki, S. Kawai, and K. Murata, Appl. Environ. Microbiol. 65:2520–2526, 1999). A gene for the lyase was cloned, and its nucleotide sequence was determined. The gene contained an open reading frame consisting of 2,793 bp coding for a polypeptide with a molecular weight of 99,308. The polypeptide had a signal peptide (2 kDa) consisting of 25 amino acid residues preceding the N-terminal amino acid sequence of the enzyme and exhibited significant homology with hyaluronidase of Streptomyces griseus(identity score, 37.7%). Escherichia coli transformed with the gene without the signal peptide sequence showed a xanthan lyase activity and produced intracellularly a large amount of the enzyme (400 mg/liter of culture) with a molecular mass of 97 kDa. During storage at 4°C, the purified enzyme (97 kDa) from E. coli was converted to a low-molecular-mass (75-kDa) enzyme with properties closely similar to those of the enzyme (75 kDa) fromBacillus sp. strain GL1, specifically in optimum pH and temperature for activity, substrate specificity, and mode of action. Logarithmically growing cells of Bacillus sp. strain GL1 on the medium with xanthan were also found to secrete not only xanthan lyase (75 kDa) but also a 97-kDa protein with the same N-terminal amino acid sequence as that of xanthan lyase (75 kDa). These results suggest that, in Bacillus sp. strain GL1, xanthan lyase is first synthesized as a preproform (99 kDa), secreted as a precursor (97 kDa) by a signal peptide-dependent mechanism, and then processed into a mature form (75 kDa) through excision of a C-terminal protein fragment with a molecular mass of 22 kDa.

Thermolytic digest of chicken pepsin

1981 ◽  
Vol 46 (8) ◽  
pp. 1994-2004 ◽  
Author(s):  
Miroslav Baudyš ◽  
Vladimír Kostka ◽  
Helena Keilová
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Linear Structure ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Terminal Amino Acid ◽  
Terminal Amino ◽  
Terminal Amino Acid Sequence ◽  
High Voltage Electrophoresis ◽  
Final Purification

Chicken pepsin prepared by the activation of pepsinogen was digested with thermolysin. The thermolytic digest was fractionated by chromatography on Sephadex G-25 fine. Certain fractions were subsequently subjected to ion exchange chromatography on Dowex 50-X2. The final purification was effected by paper chromatography and high voltage electrophoresis. By these procedures a series of homogeneous peptides was obtained; of the latter 54 nonoverlapping (save for a few exceptions) peptides are described in this paper. These peptides in addition to the thermolytic peptides reported before represent 80% of the linear structure of the whole molecule. The N-terminal amino acid sequence of chicken pepsin is discussed from the viewpoint of the recent data obtained by the analysis of the thermolytic digest.

Human hemopexin. Preparation of the heme-rich protein

1982 ◽  
Vol 47 (2) ◽  
pp. 535-542 ◽  
Author(s):  
Ladislav Morávek ◽  
Josef Borvák ◽  
Karel Grüner ◽  
Bedřich Meloun ◽  
Petr Štrop ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Amino Acid Analysis ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Terminal Amino Acid ◽  
Terminal Amino ◽  
Simplified Procedure ◽  
Pooled Samples ◽  
Terminal Amino Acid Sequence

A simplified procedure was developed for the preparation of hemopexin from Cohn fraction IV obtained from partially hemolyzed pooled samples of serum. The method is based on precipitation with rivanol, chromatography on DEAE-cellulose, and gel filtration; it permits large quantities of the material to be treated on a laboratory scale. The preparation of heme-rich hemopexin obtained was characterized by amino acid analysis and the following N-terminal amino acid sequence: Thr-Pro-Leu-Pro-Arg-Gly-Ser-Ala-His-Gly-Asn-Val-Ala-Glu-Gly-Glu-Thr(Thr)Thr-Asn-Pro-Asp-Val-(Gly)(Leu).

scholarly journals Purification and Characterization of a Novel Antifungal Flagellin Protein from Endophyte Bacillus methylotrophicus NJ13 against Ilyonectria robusta

2019 ◽  
Vol 7 (12) ◽  
pp. 605 ◽  
Author(s):  
Yun Jiang ◽  
Chao Ran ◽  
Lin Chen ◽  
Wang Yin ◽  
Yang Liu ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Gene Sequence ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Inhibitory Effect ◽  
Exchange Chromatography ◽  
Antifungal Protein ◽  
Bacillus Methylotrophicus ◽  
Cloned Gene

Endophyte Bacillus methylotrophicus NJ13 was isolated from Panax ginseng. Its sterile fermentation liquid showed a significant inhibitory effect against Ilyonectria robusta, causing the rusty root rot of P. ginseng and P. quinquefolius. The antifungal protein was obtained after precipitation by 20% saturated ammonium sulfate, desalted by Sephadex G-25, weak anion exchange chromatography, and gel filtration chromatography. SDS-PAGE showed that the purified protein was approximately 29 KDa. The antifungal protein after desalting was not resistant to temperatures higher than 100 °C, resistant to acid conditions, and did not tolerate organic solvents and protease K. The amino acid sequence of purified antifungal protein had an identity of 76% to flagellin from Bacillus velezensis. The isoelectric point of the protein was 4.97 and its molecular mass was 27 KDa. Therefore, a specific primer G1 was designed based on the flagellin gene sequence, and a 770 bp gene sequence was cloned in NJ13 genomic DNA, which shared the same size of flagellin. There were ten base differences between the gene sequences of flagellin and the cloned gene, however, the amino acid sequence encoded by the cloned gene was identical to the flagellin. In conclusion, the antifungal protein produced by biocontrol agent NJ13 contained a flagellin protein.

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