Expression In Aspergillus Niger And Characterization Of ?-mannanases

Cellulose and hemicellulose reserves are the largest regenerative resource storage of nature at present.The utilization of hemicellulose draws more and more attentions now.?-mannase is one of the most important enzymes in hemicellulose degradation.?-mannase(EC 3.2.1.78)is a kind of hemicellulase,belonging to the endo-hydrolytic enzyme,working on the ?-1,4-mannose glycosidic bond in main chain of mannan molecular,?-mannase has very wide sources,but mainly comes from microorganisms.Because of ?-mannase's increasing applicable fields,extensive prospects,and the current ?-mannases' failure of meeting industrial application,developing new ?-mannases is getting more and more importance.This study successfully heterologous expresses 2 kinds of ?-mannases by genetic engineering means.This study was using Aspergillus niger as host to expresse ?-mannanases from Stachybotrys chartarum.Through sequence analysis of the Stachybotrys chartarum genome,two ?-mannanase genes(s16942 and s331)were identified.The primers were designed based on the DNA sequence and the ?-mannanase genes(s16942 and s331)were obtained,and then inserted to the vector pGm.Firstly,the expression plasmids would transfer into Escherichia coli.,then confirmed by single colony PCR and fragment sequencing.Secondly,the expression plasmids were transferred into Aspergillus niger strain.The?-mannanase producing strains(G1-pGm-s16942 and G1-pGm-s331)were isolated after screening several transformants using amdS selection plates and confirmed by fermentation and PCR fragment sequencing.Results showed that the molecular weight of the enzymes from G1-pGm-s 16942 and G1-pGm-s331 were about 48 kDa and 60 kDa respectively by SDS-PAGE gel analysis,and the recombinant proteins did not present in the negative control.Assays of enzymatic property using the crude enzyme preparations indicated that the crude enzyme from G1-pGm-s 16942 exhibited maximum activity(521 U/mL)under the optimum conditions(pH 7 and 60 ?);the crude enzyme from G1-pGm-s331 exhibited maximum activity(84 U/mL)under the optimum conditions(pH 7 and 50 ?).These two kinds of recombinant enzymes showed a worse toleration for higher temperatures than lowers,Especially the crude enzyme from G1-pGm-s16942 could have 85%residue activity when incubated 180 minutes in 40?;Both of these two kinds of recombinant enzymes showed a good toleration in pH,and they both could have 90%residue activity when incubated 400 hours in pH 6-pH 8.This was the first study of the heterologous expression of the ?-mannanase genes from Stachybotrys chartarum in Aspergillus niger host and the ?-mannanase genes could be expressed successfully with high activities and protein titers.