[Cloning, expression and characterization of mannanase from Armillariella tabescens EJLY2098 in Pichia pastoris].
We used reverse transcriptase polymerase chain reaction (RT-PCR) and rapid amplification of cDNA end (RACE) techniques to obtain the full-length cDNA of beta-mannanase (EC 3.2.1.78) from Armillariella tabescens EJLY2098 (an edible fungus). Sequence analysis of the 1481 bp full-length cDNA encoding 445 amino acid residues indicated that the gene contained two structural domains, cellulose-binding domains (CBD) and glycoside hydrolase family 5 (GHF5) domains, other than the conserved beta-mannanase domain. Thus, we classified this gene as a member of glycoside hydrolase family 5. Next, we cloned a 1308 bp fragment encoding the beta-mannanase mature peptide (re-atMAN47) into the expression vector pPICZalphaA and expressed it in Pichia pastoris. The yield was 440 mg/L. Enzyme activity reached a maximum of 1.067 IU/mL after 72 h of methanol induction. The re-atMAN47 had an optimal temperature of 60 degrees C and an optimal pH of 5.5. It manifested broad thermostability from 30 degrees C-65 degrees C, and was stable between pH 4.5-7.0. This study represents the first record of a beta-mannanase from Armillariella tabescens EJLY2098 and provides a new source of carbohydrate hydrolysis enzyme with good biosafety, thermostability and wide pH stability. It is a good approach for the industrial needs of feed, food and pharmaceutical manufacturers.
Institute ofMicrobial Biotechnology, Jinan University, Guangzhou 510632, China.
Yanfeng
Y
Wang
AdaHangHeng
A
Wong
Xiaokui
X
Huang
Daling
D
Liu
Dongsheng
D
Yao
D055392:Armillaria / Q000145:classification / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D004795:Enzyme Stability; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D017422:Sequence Analysis, DNA; D044902:beta-Mannosidase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*
D011994:Recombinant Proteins; D044902:beta-Mannosidase