[Cloning and expression in Pichia pastoris of an alkaline mannanase gene].
A strain containing alkaline mannanase gene was isolated from soil by functional plates and the genome library was constructed. From it a mannanase gene TM1 was acquired and was sequenced. The BLAST analysis showed a lower-than-60% similarity of the amino acid sequence to those in GenBank and proved TM1 to be a new mannanase gene (GenBank accession number AY623903). The new gene without signal peptide was cloned into the Pichia pastoris expression vector pHBM905C. The recombinant plasmid pHBM1201 was digested by Sal I and transformed into Pichia pastoris KM71, GS115, SMD1168, respectively. All of the recombinant Pichia pastroris strains containing pHBM1201 secreted functional beta-mannanase. Because of its high mass of expression, the recombinant Pichia pastoris SMD1168-3 containing pHBM1201 was induced at shake flasks. The optimal temperature and pH of the beta-mannanase produced by the recombinant strains were 55 degrees C and 7.5, respectively. The enzymatic activity for konjak powder reached 41.8 with a half life of one hour. After keeping at 80 degrees C for 5 min, the enzymatic activity declined from 77% to 11% and the enzymatic activity could recover up to more than 60% when the temperature descended to 55 degrees C.
Tan
Xiu-Hua
XH
Laboratory of Molecular Microbiology & Gene Engineering, College of Life Science, Hubei University, Wuhan 430062, China. tanxh1997@hotmail.com
Wu
Yu-Yong
YY
Ma
Li-Xin
LX
Jiang
Si-Jing
SJ
D003001:Cloning, Molecular; D006863:Hydrogen-Ion Concentration; D010843:Pichia / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D012988:Soil Microbiology; D013696:Temperature; D044902:beta-Mannosidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D011994:Recombinant Proteins; D044902:beta-Mannosidase