Molecular and biochemical characterization of endo-beta-mannanases from germinating coffee (Coffea arabica) grains. 2001

P Marraccini, and W J Rogers, and C Allard, and M L André, and V Caillet, and N Lacoste, and F Lausanne, and S Michaux
Department of Plant Science, Nestlé Research Centre, Tours, France. pierre.marraccini@rdto.nestle.com

The activity of endo-beta-mannanase ([1-->4]-beta-mannan endohydrolase EC 3.2.1.78) is likely to be central to the metabolism of cell wall mannans during the germination of grains of coffee (Coffea spp.). In the present paper, we report the cloning and sequencing of two endo-beta-mannanase cDNAs (manA and manB) by different strategies from Coffea arabica L.. The manA cDNA was obtained by the use of oligonucleotides homologous to published sequences of other endo-beta-mannanases and manB by the use of oligonucleotides deduced from a purified enzyme from coffee. ManA and B proteins share about 56% sequence homology and include highly conserved regions found in other mannan endohydrolases. Purification of the activity by chromatography followed by separation by two-dimensional electrophoresis and amino acid sequencing demonstrated the existence of at least seven isomers of the ManB form. The existence of multiple manB genes was also indicated by Southern analysis, whereas only one or two gene copies were detected for manA. Northern hybridizations with manA- and manB-specific probes showed that mRNA transcripts for both cDNAs were present at the same periods of bean germination with transcript peaks at 20 days after imbibition of water (DAI). Transcripts were not detected during grain maturation or in the other tissues such as roots, stems, flowers and leaves. The peak endo-beta-mannanase activity occurred at approximately 28 DAI and was not detected in grains prior to imbibition. Activity and mRNA levels appeared to be tightly co-ordinated. Tests of substrate specificity with the purified ManB enzyme showed that activity required a minimum of five mannose units to function efficiently.

UI MeSH Term Description Entries
D007526 Isoelectric Point The pH in solutions of proteins and related compounds at which the dipolar ions are at a maximum. Isoelectric Points,Point, Isoelectric,Points, Isoelectric
D008361 Mannosidases Glycoside hydrolases that catalyze the hydrolysis of alpha or beta linked MANNOSE. Mannosidase
D008969 Molecular Sequence Data Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories. Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D010940 Plant Proteins Proteins found in plants (flowers, herbs, shrubs, trees, etc.). The concept does not include proteins found in vegetables for which PLANT PROTEINS, DIETARY is available. Plant Protein,Protein, Plant,Proteins, Plant
D003001 Cloning, Molecular The insertion of recombinant DNA molecules from prokaryotic and/or eukaryotic sources into a replicating vehicle, such as a plasmid or virus vector, and the introduction of the resultant hybrid molecules into recipient cells without altering the viability of those cells. Molecular Cloning
D003069 Coffee A beverage made from ground COFFEA beans (SEEDS) infused in hot water. It generally contains CAFFEINE and THEOPHYLLINE unless it is decaffeinated.
D000595 Amino Acid Sequence The order of amino acids as they occur in a polypeptide chain. This is referred to as the primary structure of proteins. It is of fundamental importance in determining PROTEIN CONFORMATION. Protein Structure, Primary,Amino Acid Sequences,Sequence, Amino Acid,Sequences, Amino Acid,Primary Protein Structure,Primary Protein Structures,Protein Structures, Primary,Structure, Primary Protein,Structures, Primary Protein
D001483 Base Sequence The sequence of PURINES and PYRIMIDINES in nucleic acids and polynucleotides. It is also called nucleotide sequence. DNA Sequence,Nucleotide Sequence,RNA Sequence,DNA Sequences,Base Sequences,Nucleotide Sequences,RNA Sequences,Sequence, Base,Sequence, DNA,Sequence, Nucleotide,Sequence, RNA,Sequences, Base,Sequences, DNA,Sequences, Nucleotide,Sequences, RNA
D012639 Seeds The encapsulated embryos of flowering plants. They are used as is or for animal feed because of the high content of concentrated nutrients like starches, proteins, and fats. Rapeseed, cottonseed, and sunflower seed are also produced for the oils (fats) they yield. Diaspores,Elaiosomes,Embryos, Plant,Plant Embryos,Plant Zygotes,Zygotes, Plant,Diaspore,Elaiosome,Embryo, Plant,Plant Embryo,Plant Zygote,Seed,Zygote, Plant
D015180 Electrophoresis, Gel, Two-Dimensional Electrophoresis in which a second perpendicular electrophoretic transport is performed on the separate components resulting from the first electrophoresis. This technique is usually performed on polyacrylamide gels. Gel Electrophoresis, Two-Dimensional,Polyacrylamide Gel Electrophoresis, Two-Dimensional,2-D Gel Electrophoresis,2-D Polyacrylamide Gel Electrophoresis,2D Gel Electrophoresis,2D PAGE,2D Polyacrylamide Gel Electrophoresis,Electrophoresis, Gel, 2-D,Electrophoresis, Gel, 2D,Electrophoresis, Gel, Two Dimensional,Polyacrylamide Gel Electrophoresis, 2-D,Polyacrylamide Gel Electrophoresis, 2D,Two Dimensional Gel Electrophoresis,2 D Gel Electrophoresis,2 D Polyacrylamide Gel Electrophoresis,Electrophoresis, 2-D Gel,Electrophoresis, 2D Gel,Electrophoresis, Two-Dimensional Gel,Gel Electrophoresis, 2-D,Gel Electrophoresis, 2D,Gel Electrophoresis, Two Dimensional,PAGE, 2D,Polyacrylamide Gel Electrophoresis, 2 D,Polyacrylamide Gel Electrophoresis, Two Dimensional,Two-Dimensional Gel Electrophoresis

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