scholarly journals Molecular Identification of Oligoalginate Lyase ofSphingomonas sp. Strain A1 as One of the Enzymes Required for Complete Depolymerization of Alginate

2000 ◽  
Vol 182 (16) ◽  
pp. 4572-4577 ◽  
Author(s):  
Wataru Hashimoto ◽  
Osamu Miyake ◽  
Keiko Momma ◽  
Shigeyuki Kawai ◽  
Kousaku Murata
Keyword(s):  
Molecular Weight ◽  
Open Reading Frame ◽  
Bacterial Cells ◽  
Reading Frame ◽  
Glycosidic Bonds ◽  
Atp Binding Cassette Transporter ◽  
Alginate Oligosaccharides ◽  
Its Gene ◽  
Single Precursor ◽  
Alginate Lyases

ABSTRACT A bacterium, Sphingomonas sp. strain A1, can incorporate alginate into cells through a novel ABC (ATP-binding cassette) transporter system specific to the macromolecule. The transported alginate is depolymerized to di- and trisaccharides by three kinds of cytoplasmic alginate lyases (A1-I [66 kDa], A1-II [25 kDa], and A1-III [40 kDa]) generated from a single precursor through posttranslational autoprocessing. The resultant alginate oligosaccharides were degraded to monosaccharides by cytoplasmic oligoalginate lyase. The enzyme and its gene were isolated from the bacterial cells grown in the presence of alginate. The purified enzyme was a monomer with a molecular mass of 85 kDa and cleaved glycosidic bonds not only in oligosaccharides produced from alginate by alginate lyases but also in polysaccharides (alginate, polymannuronate, and polyguluronate) most efficiently at pH 8.0 and 37°C. The reaction catalyzed by the oligoalginate lyase was exolytic and thought to play an important role in the complete depolymerization of alginate in Sphingomonas sp. strain A1. The gene for this novel enzyme consisted of an open reading frame of 2,286 bp encoding a polypeptide with a molecular weight of 86,543 and was located downstream of the genes coding for the precursor of alginate lyases (aly) and the ABC transporter (algS,algM1, and algM2). This result indicates that the genes for proteins required for the transport and complete depolymerization of alginate are assembled to form a cluster.

scholarly journals Biochemical Characterization and Elucidation of Action Pattern of a Novel Polysaccharide Lyase 6 Family Alginate Lyase from Marine Bacterium Flammeovirga sp. NJ-04

Marine Drugs ◽  
2019 ◽  
Vol 17 (6) ◽  
pp. 323 ◽  
Author(s):  
Qian Li ◽  
Fu Hu ◽  
Benwei Zhu ◽  
Yun Sun ◽  
Zhong Yao
Keyword(s):  
Biochemical Characterization ◽  
Alginate Lyase ◽  
Ionization Mass ◽  
Action Pattern ◽  
Polysaccharide Lyase ◽  
Glycosidic Bonds ◽  
Alginate Oligosaccharides ◽  
Degradation Pattern ◽  
Alginate Lyases ◽  
Layer Chromatography

Alginate lyases have been widely used to prepare alginate oligosaccharides in food, agricultural, and medical industries. Therefore, discovering and characterizing novel alginate lyases with excellent properties has drawn increasing attention. Herein, a novel alginate lyase FsAlyPL6 of Polysaccharide Lyase (PL) 6 family is identified and biochemically characterized from Flammeovirga sp. NJ-04. It shows highest activity at 45 °C and could retain 50% of activity after being incubated at 45 °C for 1 h. The Thin-Layer Chromatography (TLC) and Electrospray Ionization Mass Spectrometry (ESI-MS) analysis indicates that FsAlyPL6 endolytically degrades alginate polysaccharide into oligosaccharides ranging from monosaccharides to pentasaccharides. In addition, the action pattern of the enzyme is also elucidated and the result suggests that FsAlyPL6 could recognize tetrasaccharide as the minimal substrate and cleave the glycosidic bonds between the subsites of −1 and +3. The research provides extended insights into the substrate recognition and degradation pattern of PL6 alginate lyases, which may further expand the application of alginate lyases.

A second expressed kininogen gene in mice

2006 ◽  
Vol 26 (2) ◽  
pp. 152-157 ◽  
Author(s):  
Edward G. Shesely ◽  
Chun-Bo Hu ◽  
François Alhenc-Gelas ◽  
Pierre Meneton ◽  
Oscar A. Carretero
Keyword(s):  
Molecular Weight ◽  
Open Reading Frame ◽  
Low Molecular Weight ◽  
High Molecular Weight Kininogen ◽  
Rt Pcr ◽  
Reading Frame ◽  
Rna Ligase ◽  
Mouse Tissues ◽  
Rapid Amplification ◽  
Race Pcr

We isolated PCR, RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE-PCR)-, and RT-PCR-generated clones from mouse kininogen family transcripts. DNA sequencing indicated that the clones were from two distinct genes. One set (K1) is from the previously reported mouse kininogen gene. The second set (K2) has an open reading frame, is 93% identical to K1 in the overlapping nucleotide sequence, and, unlike T-kininogens in the rat, encodes a bradykinin motif identical to K1. We discovered that K2 exists with two different 5′ ends. We used RT-PCR to determine the distribution and relative abundance of K1 and K2 mRNA in mouse tissues. K2 is transcribed and K1 and K2 are generally both expressed in the same tissues; however, they differ in their regulation of the alternative splicing event that yields either low-molecular-weight kininogen (LMWK) or high-molecular-weight kininogen (HMWK). For example, in the liver K1 is expressed as both HMWK and LMWK, whereas K2 is only expressed as LMWK. Conversely, in the kidney K2 is strongly expressed as both HMWK and LMWK, whereas K1 is not expressed as HMWK and expressed only very weakly as LMWK.

scholarly journals Sequence of the Structural Gene for Xanthine Dehydrogenase (rosy Locus) in Drosophila melanogaster

Genetics ◽  
1987 ◽  
Vol 116 (1) ◽  
pp. 67-73
Author(s):  
Tim P Keith ◽  
Margaret A Riley ◽  
Martin Kreitman ◽  
R C Lewontin ◽  
Daniel Curtis ◽  
...  
Keyword(s):  
Amino Acids ◽  
Molecular Weight ◽  
Drosophila Melanogaster ◽  
Amino Acid ◽  
Nucleotide Sequence ◽  
Structural Gene ◽  
Xanthine Dehydrogenase ◽  
Open Reading Frame ◽  
Reading Frame ◽  
Eco Ri

ABSTRACT We determined the nucleotide sequence of a 4.6-kb Eco RI fragment containing 70% of the rosy locus. In combination with information on the 5′ sequence, the gene has been sequenced in entirety. rosy cDNAs have been isolated and intron/exon boundaries have been determined. We find an open reading frame which spans four exons and would encode a protein of 1335 amino acids. The molecular weight of the encoded protein (xanthine dehydrogenase), based on the amino acid translation, is 146,898 daltons which agrees well with earlier biophysical estimates. Characteristics of the protein are discussed.

scholarly journals BIOACTIVITY CHARACTERIZATION OF PURIFIED RECOMBINANT HYPOTHETICAL PROTEIN CODED BY OPEN READING FRAME-112 OF STREPTOMYCES.

2021 ◽  
Vol 52 (2) ◽  
pp. 502-511
Author(s):  
R. M. Al-Obaidi ◽  
G. F. Salih ◽  
B. F. Nore
Keyword(s):  
Protective Effect ◽  
Hypothetical Protein ◽  
Positive Control ◽  
Open Reading Frame ◽  
Bacterial Cells ◽  
Disk Diffusion Test ◽  
Reading Frame ◽  
Over Expression ◽  
Sds Page

This study was aimed to investigate the Open Reading Frame-112 (ORF-112) gene, which encoded for a hypothetical 218 amino acids protein in Streptomyces bacteria. A complete ORF-112 gene was synthesized, with addition of a 6xHis-Tag at the N-terminal location. The synthesized DNA nucleotides were sub-cloned into bacterial expression plasmid pBAT4. The pBAT4-ORF-112 plasmid transformed in bacterial cells BL21(De3)pLysS, intended for protein over expression, induced by isopropyl β- d-1-thiogalactopyranoside (IPTG). The IMAC affinity chromatography technique was deployed for protein purifications. Highly-purified fractions of ORF-112 were achieved by using affinity Ni++-columns. The purified ORF-112 protein was tested for possible biological activity.  The SDS-PAGE analysis exhibited a soluble 30 kDa size purified ORF-112 protein which showed a slight gel shifting from the predicted size. The virulent activity test on purified fractions of ORF-112 was measured using the Disk Diffusion Test and it disclosed a clear zone formed in response to fungi Candida albicans growth. The data implies that the ORF-112 protein has an acceptable protective effect against the fungus C. albicans as compared to positive control ketoconazole (KCZ) (P < 0.05) while the protein has a significantly lower protective effect against the fungus than Itraconazole (ICZ) (P > 0.05). The results clarify the hypothetical ORF-112 protein is a novel protein with protective response effect against fungi cells C. albicans on disk-diffusion.test.

scholarly journals Norwalk Virus Open Reading Frame 3 Encodes a Minor Structural Protein

2000 ◽  
Vol 74 (14) ◽  
pp. 6581-6591 ◽  
Author(s):  
Pamela J. Glass ◽  
Laura J. White ◽  
Judith M. Ball ◽  
Isabelle Leparc-Goffart ◽  
Michele E. Hardy ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Insect Cells ◽  
Open Reading Frame ◽  
Norwalk Virus ◽  
Structural Protein ◽  
Coding Sequences ◽  
Reading Frame ◽  
Single Protein ◽  
Minor Structural Protein ◽  
A Minor

ABSTRACT Norwalk virus (NV) is a causative agent of acute epidemic nonbacterial gastroenteritis in humans. The inability to cultivate NV has required the use of molecular techniques to examine the genome organization and functions of the viral proteins. The function of the NV protein encoded by open reading frame 3 (ORF 3) has been unknown. In this paper, we report the characterization of the NV ORF 3 protein expressed in a cell-free translation system and in insect cells and show its association with recombinant virus-like particles (VLPs) and NV virions. Expression of the ORF 3 coding region in rabbit reticulocyte lysates resulted in the production of a single protein with an apparent molecular weight of 23,000 (23K protein), which is not modified by N-linked glycosylation. The ORF 3 protein was expressed in insect cells by using two different baculovirus recombinants; one recombinant contained the entire 3′ end of the genome beginning with the ORF 2 coding sequences (ORFs 2+3), and the second recombinant contained ORF 3 alone. Expression from the construct containing both ORF 2 and ORF 3 resulted in the expression of a single protein (23K protein) detected by Western blot analysis with ORF 3-specific peptide antisera. However, expression from a construct containing only the ORF 3 coding sequences resulted in the production of multiple forms of the ORF 3 protein ranging in size from 23,000 to 35,000. Indirect-immunofluorescence studies using an ORF 3 peptide antiserum showed that the ORF 3 protein is localized to the cytoplasm of infected insect cells. The 23K ORF 3 protein was consistently associated with recombinant VLPs purified from the media of insect cells infected with a baculovirus recombinant containing the entire 3′ end of the NV genome. Western blot analysis of NV purified from the stools of NV-infected volunteers revealed the presence of a 35K protein as well as multiple higher-molecular-weight bands specifically recognized by an ORF 3 peptide antiserum. These results indicate that the ORF 3 protein is a minor structural protein of the virion.

scholarly journals Isolation and sequencing of cDNA clones encoding alpha and beta subunits of Drosophila melanogaster casein kinase II.

1987 ◽  
Vol 7 (10) ◽  
pp. 3409-3417 ◽  
Author(s):  
A Saxena ◽  
R Padmanabha ◽  
C V Glover
Keyword(s):  
Molecular Weight ◽  
Drosophila Melanogaster ◽  
Amino Acid ◽  
Protein Kinase ◽  
Casein Kinase Ii ◽  
Casein Kinase ◽  
Open Reading Frame ◽  
Beta Subunit ◽  
Amino Acid Residues ◽  
Reading Frame

Cloned cDNAs encoding both subunits of Drosophila melanogaster casein kinase II have been isolated by immunological screening of lambda gt11 expression libraries, and the complete amino acid sequence of both polypeptides has been deduced by DNA sequencing. The alpha cDNA contained an open reading frame of 336 amino acid residues, yielding a predicted molecular weight for the alpha polypeptide of 39,833. The alpha sequence contained the expected semi-invariant residues present in the catalytic domain of previously sequenced protein kinases, confirming that it is the catalytic subunit of the enzyme. Pairwise homology comparisons between the alpha sequence and the sequences of a variety of vertebrate protein kinase suggested that casein kinase II is a distantly related member of the protein kinase family. The beta subunit was derived from an open reading frame of 215 amino acid residues and was predicted to have a molecular weight of 24,700. The beta subunit exhibited no extensive homology to other proteins whose sequences are currently known.

scholarly journals Heterologous, Expression, and Characterization of Thermostable Glucoamylase Derived fromAspergillus flavusNSH9 inPichia pastoris

2016 ◽  
Vol 2016 ◽  
pp. 1-10 ◽  
Author(s):  
Kazi Muhammad Rezaul Karim ◽  
Ahmad Husaini ◽  
Md. Anowar Hossain ◽  
Ngieng Ngui Sing ◽  
Fazia Mohd Sinang ◽  
...  
Keyword(s):  
Amino Acids ◽  
Molecular Weight ◽  
Pichia Pastoris ◽  
Aspergillus Flavus ◽  
Residual Activity ◽  
Open Reading Frame ◽  
Reading Frame ◽  
Higher Temperature ◽  
Positive Effect

A novel thermostable glucoamylase cDNA without starch binding domain (SBD) ofAspergillus flavusNSH9 was successfully identified, isolated, and overexpressed inPichia pastorisGS115. The complete open reading frame of glucoamylase fromAspergillus flavusNSH9 was identified by employing PCR that encodes 493 amino acids lacking in the SBD. The first 17 amino acids were presumed to be a signal peptide. The cDNA was cloned intoPichia pastorisand the highest expression of recombinant glucoamylase (rGA) was observed after 8 days of incubation period with 1% methanol. The molecular weight of the purified rGA was about 78 kDa and exhibited optimum catalytic activity at pH 5.0 and temperature of 70°C. The enzyme was stable at higher temperature with 50% of residual activity observed after 20 min at 90°C and 100°C. Low concentration of metal (Mg++, Fe++, Zn++, Cu++, and Pb++) had positive effect on rGA activity. This rGA has the potential for use and application in the saccharification steps, due to its thermostability, in the starch processing industries.

Sign in / Sign up

Export Citation Format

Share Document