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

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 dur ing the germination of grains of coffee (Coffea spp.). In the present paper , we report the cloning and sequencing of two endo-beta -mannanase cDNAs (m anA and manB) by different strategies from Coffea arabica L.. The manA cDNA was obtained by the use of oligonucleotides homologous to published sequen ces 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 follo wed by separation by two-dimensional electrophoresis and amino acid sequenc ing demonstrated the existence of at least seven isomers. of the ManB form. The existence of multiple manB genes was also indicated by Southern analys is, whereas only one or two gene copies were detected for manA. Northern hy bridizations with manA- and manB-specific probes showed that mRNA transcrip ts for both cDNAs were present at the same periods of bean germination with transcript peaks at 20 days after imbibition of water (DAI). Transcripts w ere not detected during grain maturation or in the other tissues such as ro ots, stems, flowers and leaves. The peak endo-beta -mannanase activity occu rred at approximately 28 DAI and was not detected in grains prior to imbibi tion. Activity and mRNA levels appeared to be tightly co-ordinated. Tests o f substrate specificity with the purified ManB enzyme showed that activity required a minimum of five mannose units to function efficiently.