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Isolation, identification and in silico analysis of alpha-amylase gene of Aspergillus niger strain CSA35 obtained from cassava undergoing spoilage.

Oghenetega J Avwioroko | Akpovwehwee A Anigboro | Nnanna N Unachukwu | Nyerhovwo J Tonukari
Biochemistry and biophysics reports | 2018

In this investigation, a gene (CDF_Amyl) encoding extracellular α-amylase in Aspergillus niger strain CSA35 associated with cassava spoilage was amplified using specific primers and characterized in silico. The gene had a partial nucleotide sequence of 968 bp and encoded a protein of 222 aa residues with a molecular weight and isoelectric point of 25.13 kDa and 4.17, respectively. Its catalytic site was located in the active site domain. BLASTp analysis showed that the protein primary sequence of the α-amylase gene had 98% and 99% homologies with the α-amylase of A. niger and A. oryzae RIB40, respectively. The gene is more closely related to α-amylase genes from fungi than to bacterial, plant, or animal α-amylase genes. Restriction mapping of the gene showed it can be digested with restriction enzymes like NcoI, PstI, SmaI, and BcLI among others but not with EcoRI and EcoRV. Its protein product had a hydrophobicity score of - 0.43 but no transmembrane helix. The CDF_Amyl protein was subcellularly localized in the secretory pathway, an indication of its release into extracellular space after secretion. Also, the 3D structure of the CDF-Amyl protein was barrel-shaped with domains characteristic of α-amylases. The encoded α-amylase Vmax is 6.90 U/mg protein and Km is 6.70 mg/ml. It was concluded that the unique characteristics of the CDF_Amyl gene and its deduced protein could find applications in biotechnological, food and pharmaceutical industries where cloning and further modification of this gene would be required for product development and improvement.

Pubmed ID: 29872732

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European Molecular Biology Laboratory (tool)

RRID:SCR_004473

Intergovernmental organisation funded by public research money from its member states in Europe. Groups and laboratories perform basic research in molecular biology and molecular medicine, training for scientists, students and visitors. Provides development of services, new instruments and methods, data and technology in its member states.

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EMBOSS (tool)

RRID:SCR_008493

Software analysis package for molecular biology community. Automatically copes with data in variety of formats and allows transparent retrieval of sequence data from web. Libraries are provided with package. Provides toolkit for creating bioinformatics applications or workflows. Provides set of sequence analysis programs. Provided programs cover areas such as sequence alignment, rapid database searching with sequence patterns, protein motif identification, nucleotide sequence pattern analysis, codon usage analysis for small genomes, rapid identification of sequence patterns in large scale sequence sets, and presentation tools for publication.

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RRID:SCR_002872

The European Bioinformatics Institute (EBI) toolbox area provides a comprehensive range of tools for the field of bioinformatics. These are subdivided into categories in the left menu for convenience. EBI has developed a large number of very useful bioinformatics tools. A few examples include: - Similarity & Homology - the BLAST or FASTA programs can be used to look for sequence similarity and infer homology. - Protein Functional Analysis - InterProScan can be used to search for motifs in your protein sequence. - Proteomic Services NEW - UniProt DAS server allows researchers to show their research results in the context of UniProtKB/Swiss-Prot annotation. - Sequence Analysis - ClustalW2 a sequence alignment tool. - Structural Analysis - MSDfold can be used to query your protein structure and compare it to those in the Protein Data Bank (PDB). - Web Services - provide programmatic access to the various databases and retrieval/analysis services EBI provides. - Tools Miscellaneous - Expression Profiler a set of tools for clustering, analysis and visualization of gene expression and other genomic data. Sponsors: This resource is sponsored by EBI.

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RRID:SCR_003151

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RRID:SCR_004870

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RRID:SCR_010270

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RRID:SCR_001653

Web application to search protein databases using a translated nucleotide query. Translated BLAST services are useful when trying to find homologous proteins to a nucleotide coding region. Blastx compares translational products of the nucleotide query sequence to a protein database. Because blastx translates the query sequence in all six reading frames and provides combined significance statistics for hits to different frames, it is particularly useful when the reading frame of the query sequence is unknown or it contains errors that may lead to frame shifts or other coding errors. Thus blastx is often the first analysis performed with a newly determined nucleotide sequence and is used extensively in analyzing EST sequences. This search is more sensitive than nucleotide blast since the comparison is performed at the protein level.

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Clustal W2 (tool)

RRID:SCR_002909

THIS RESOURCE IS NO LONGER IN SERVICE, documented on January 19, 2022. Command line version of multiple sequence alignment program Clustal for DNA or proteins. Alignment is progressive and considers sequence redundancy. No longer being maintained. Please consider using Clustal Omega instead which accepts nucleic acid or protein sequences in multiple sequence formats NBRF/PIR, EMBL/UniProt, Pearson (FASTA), GDE, ALN/ClustalW, GCG/MSF, RSF.

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ExPASy Bioinformatics Resource Portal (tool)

RRID:SCR_012880

Portal which provides access to scientific databases and software tools (i.e., resources) in different areas of life sciences including proteomics, genomics, phylogeny, systems biology, population genetics, transcriptomics etc. It contains resources from many different SIB groups as well as external institutions.

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