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

Abstract

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.