Two glycoside hydrolases encoded by the mannan utilization gene cluster of alkaliphilic Bacillus sp. N16-5 were studied. The recombinant Gal27A (rGal27A) hydrolyzed both galactomannans and oligo-galactomannans to release galactose, while the recombinant Man113A (rMan113A) showed poor activity toward galactomannans, but it hydrolyzed mannooligosaccharides to release mannose and mannobiose. rGal27A showed synergistic interactions with rMan113A and recombinant β-mannanase ManA (rManA), which is also from Bacillus sp. N16-5, in galactomannan degradation. The synergy degree of rGal27A and rManA on hydrolysis of locust bean gum and guar gum was 1.13 and 2.21, respectively, and that of rGal27A and rMan113A reached 2.00 and 2.68. The main products of galactomannan hydrolyzed by rGal27A and rManA simultaneously were galactose, mannose, mannobiose, and mannotriose, while those of galactomannan hydrolyzed by rGal27A and rMan113A were galactose and mannose. The yields of mannose, mannobiose, and mannotriose dramatically increased compared with the hydrolysis in the presence of rManA or rMan113A alone.
Bacillus sp. N16-5 is an alkaliphile with a great ability to utilize mannan. Its mannan utilization gene cluster has been identified in a previous study. The ManR protein encoded by the cluster was predicted to be a LacI family regulator, and the transcription level of the mannan utilization gene cluster was upregulated after the manR gene was deleted, indicating that ManR is the repressor of this cluster. The transcription of the related genes was downregulated when manH, encoding the extracellular substrate-binding domain of the manno-oligosaccharide transporter, was deleted. Furthermore, isothermal titration calorimetry revealed that mannotetraose and mannopentose are ligands of ManR. These results all corroborate the hypothesis that the mannan utilization gene cluster is repressed by the transcription regulator ManR, and that the repression is removed when it binds to manno-oligosaccharides, which are generated by mannan degradation and transported into the cell by a specific transporter.
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