Cloning, expression and characterization of mannanase from Armillariella tabescens EJLY2098 in Pichia pastoris.
- Author:
Yanfeng WANG
1
;
AdaHangHeng WONG
;
Xiaokui HUANG
;
Daling LIU
;
Dongsheng YAO
Author Information
1. Institute ofMicrobial Biotechnology, Jinan University, Guangzhou 510632, China.
- Publication Type:Journal Article
- MeSH:
Armillaria;
classification;
enzymology;
genetics;
Cloning, Molecular;
Enzyme Stability;
Pichia;
genetics;
metabolism;
Recombinant Proteins;
biosynthesis;
genetics;
Sequence Analysis, DNA;
beta-Mannosidase;
biosynthesis;
chemistry;
genetics
- From:
Chinese Journal of Biotechnology
2009;25(6):920-926
- CountryChina
- Language:Chinese
-
Abstract:
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.
