A beta-mannanase from Bacillus subtilis B36: purification, properties, sequencing, gene cloning and expression in Escherichia coli.
MANB36, a secrete endo-beta-1,4-D-mannanase produced by Bacillus subtilis B36, was purified to homogeneity from a culture supernatant and characterized. The optimum pH value for the mannanase activity of MANB36 is 6.4 and the optimum temperature is 50 degrees C. The enzyme activity of MANB36 is remarkably thermostable at 60 degrees C and the specific activity of MANB36 is 927.84 U/mg. Metal cations (except Hg2+ and Ag+), EDTA and 2-mercaptoethanol (2-ME) have no effects on enzyme activity. This enzyme exhibits high specificity with the substituted galactomannan locust bean gum (LBG). The gene encoding for MANB36, manB36, was cloned by PCR and sequenced. manB36 contains a single open reading frame (ORF) consisting of 1104 bp that encodes a protein of 367 amino acids. The predicted molecular weight of 38.13 kDa, calculated by the deduced protein of the gene manB36 without signal peptide, coincides with the apparent molecular weight of 38.0 kDa of the purified MANB36 estimated by SDS-PAGE. The mature protein of MANB36 has been expressed in Escherichia coli BL21 and the expressed mannanase has normal bioactivity.
Li
Ya Nan
YN
Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China.
Meng
Kun
K
Wang
Ya Ru
YR
Yao
Bin
B
D001412:Bacillus subtilis / Q000201:enzymology* / Q000254:growth & development; D001483:Base Sequence; D003001:Cloning, Molecular; D003470:Culture Media; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011994:Recombinant Proteins / Q000378:metabolism; D044902:beta-Mannosidase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism
D003470:Culture Media; D017931:DNA Primers; D011994:Recombinant Proteins; D044902:beta-Mannosidase