Cloning,Expression And Characterization Of ?-mannanase From Thermoanerobacterium Aotearoense

Sustainable development is studied more and more deeply into all areas of society.Hemicellulose is distributed as an important renewable energy source in every corner on the earth.Traditional physical and chemical methods of hemicellulose degradation are not friendly to the environment.It is necessary to find a biological approach to hydrolyze hemicellulose to solve environmental and energy problems.?-Mannanase??-mannanase,EC 3.2.1.78?belongs to the hemicellulase and can digest?-1,4-D-glycosidic bond from the main chain of?-1,4-D-mannan molecules and resulting in different lengths of manno-oligosaccharides.Manno-oligosaccharides has a wide range of applications in animal feed,paper industry,medicine,pharmacy and oil drlling.?-Mannanase was extracellularly expressed in Thermoanaerobacterium aotearoense P8G3#4 induced by locust bean gum.A cDNA encoding the?-mannanase was identified through genome sequence analysis.The open reading frame is composed of 1548 bp and codes for a preprotein of 515 amino acids with a putative signal peptide.Amino acid sequence blast results suggested that there are two domain in the order:the carbohydrate binding module?CBM35?and glycosyl hydrolase domain?GH26?.Its corresponding enzyme without signal sequence?Man25?was successfully overexpressed in Escherichia coli BL21?DE3?induced by 0.1 mM IPTG at 25°C for 4 h.Good purity of the mature Man25 was achieved through Ni-affinity chromatography and desalting with 37.73%yield.The optimum pH and temperature toward locust bean gum were 5.5 and 55°C.Metal ions depletion by EDTA strongly inhibited the activity of Man25.However,its activity was almost totally restored by the addition of Ca²⁺,Fe²⁺,Mg²⁺,Mn²⁺in EDTA pre-treated reaction mixture.Ca²⁺was benefit for thermostability of the purified Man25 by increasing T_m to 57°C.In addition,a facile and reagentless?-mannanase activity assay method on agar plates containing trypan blue was developed by taking full advantage of characteristics of E.coli BL21?DE3?strain.